College Level Microbiology by AudioLearn

College Level™ Microbiology

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TABLE OF CONTENTS Preface........................................................................................................ 1 Chapter One: Observing Microorganisms ................................................... 5 Types of Microorganisms................................................................................................. 5 Fundamentals of Microscopy .......................................................................................... 8 Staining of Microorganisms........................................................................................... 15 Key Takeaways ............................................................................................................... 19 Quiz ................................................................................................................................ 20 Chapter Two: Cell Basics ........................................................................... 24 Modern Cell Theory ....................................................................................................... 24 The Prokaryotic Cell....................................................................................................... 26 The Eukaryotic Cell ........................................................................................................ 32 Key Takeaways ............................................................................................................... 40 Quiz ................................................................................................................................ 41 Chapter Three: Acellular Pathogens .......................................................... 45 Viruses ............................................................................................................................ 45 Viral Life Cycle ............................................................................................................... 48 Isolation and Identification of Viruses .......................................................................... 51 Viroids and Prions ......................................................................................................... 53 Key Takeaways ............................................................................................................... 54 Quiz ................................................................................................................................ 55

Chapter Four: Types of Prokaryotic Cells .................................................. 59 Prokaryotes and Microbiomes ....................................................................................... 59 Proteobacteria ................................................................................................................ 62 Gram-negative Bacteria ................................................................................................. 63 Phototrophic Bacteria .................................................................................................... 65 Gram-positive Bacteria .................................................................................................. 65 Archaea........................................................................................................................... 67 Deeply Branching Bacteria ............................................................................................ 67 Key Takeaways ............................................................................................................... 68 Quiz ................................................................................................................................ 69 Chapter Five: Types of Eukaryotic Cells..................................................... 73 Unicellular Eukaryotic Parasites ................................................................................... 73 Fungi .............................................................................................................................. 79 Helminths ...................................................................................................................... 81 Algae ............................................................................................................................... 83 Lichens ........................................................................................................................... 84 Key Takeaways ............................................................................................................... 85 Quiz ................................................................................................................................ 86 Chapter Six: The Biochemistry of Microbiology ........................................ 90 Organic Molecules ......................................................................................................... 90 Lipids .............................................................................................................................. 94 Proteins .......................................................................................................................... 98 Carbohydrates .............................................................................................................. 100 Biochemical Principles in Microbiology ...................................................................... 102

Key Takeaways ............................................................................................................. 103 Quiz .............................................................................................................................. 104 Chapter Seven: Metabolic Processes in Microbiology .............................. 108 Enzymes and Cellular Energy ...................................................................................... 108 Catabolism of Carbohydrates ....................................................................................... 113 Fermentation ................................................................................................................ 116 Cellular Respiration ...................................................................................................... 117 Catabolism of Proteins and Lipids................................................................................ 118 Photosynthesis .............................................................................................................. 119 Biogeochemical Cycles .................................................................................................. 121 Key Takeaways ............................................................................................................. 123 Quiz .............................................................................................................................. 124 Chapter Eight: The Genome in Microbiology ........................................... 128 DNA Structure and Function ....................................................................................... 128 RNA Structure and Function ........................................................................................ 131 Cellular Genomes ......................................................................................................... 133 Key Takeaways ............................................................................................................. 135 Quiz .............................................................................................................................. 136 Chapter Nine: Microbial Genetics ........................................................... 140 DNA Replication .......................................................................................................... 140 RNA Transcription ....................................................................................................... 143 Translation and Protein Synthesis .............................................................................. 145 Mutations ..................................................................................................................... 148 Operons and Gene Regulation ..................................................................................... 149

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Genetic Diversity in Prokaryotes .................................................................................. 151 Key Takeaways ............................................................................................................. 154 Quiz ...............................................................................................................................155 Chapter Ten: Microbial Growth .............................................................. 159 Microbial Growth ......................................................................................................... 159 Effects of the Environment on Microbial Growth ....................................................... 163 Media and Microbial Growth....................................................................................... 165 Controlling Microbial Growth ..................................................................................... 166 Antiseptics .................................................................................................................... 168 Key Takeaways .............................................................................................................. 171 Quiz ...............................................................................................................................172 Chapter Eleven: Antimicrobial Agents ..................................................... 176 Antimicrobial Therapy ................................................................................................. 176 Antibacterial Therapy .................................................................................................. 178 Other Antimicrobial Therapies ..................................................................................... 181 Drug Resistance ........................................................................................................... 183 Identifying New Antimicrobials and Drug Sensitivities ............................................. 184 Key Takeaways ............................................................................................................. 186 Quiz .............................................................................................................................. 187 Chapter Twelve: Pathogenicity and Disease ............................................. 191 What is an Infectious Disease? ..................................................................................... 191 Pathogens ..................................................................................................................... 193 Virulence Factors for Viruses and Prokaryotes ........................................................... 196 Virulence Factors for Eukaryotic Pathogens ............................................................... 199

Tracking Infectious Diseases ...................................................................................... 200 Key Takeaways ............................................................................................................. 203 Quiz ..............................................................................................................................204 Chapter Thirteen: Innate Immune System .............................................. 208 Physical Defenses ........................................................................................................ 208 Chemical Defense Systems .......................................................................................... 210 Cellular Defense ........................................................................................................... 213 Inflammatory Processes .............................................................................................. 215 Pathogen Recognition and Phagocytosis ......................................................................217 Key Takeaways ............................................................................................................. 219 Quiz ..............................................................................................................................220 Chapter Fourteen: Adaptive Immune System .......................................... 224 Adaptive Immunity ...................................................................................................... 224 Major Histocompatibility Complexes and Antigen Presentation ............................... 228 T Lymphocyte Function ............................................................................................... 229 B Lymphocyte Function ............................................................................................... 231 Vaccinations ................................................................................................................. 232 Quiz .............................................................................................................................. 235 Chapter Fifteen: Advanced Laboratory Methods ..................................... 239 Polyclonal and Monoclonal Antibodies ....................................................................... 239 Detection of Antigen-Antibody Complexes ................................................................. 241 Agglutination Assays .................................................................................................... 243 EIAs and ELISA Testing .............................................................................................. 245 Using Fluorescent Antibody Methods ......................................................................... 246

Key Takeaways ............................................................................................................. 248 Quiz .............................................................................................................................. 249 Summary ................................................................................................ 253 Course Questions and Answers ............................................................... 256

PREFACE The purpose of this course is to introduce the college-level student to the science of living things on a small scale or the field of microbiology. Microbiology touches on many related topics, including the biochemistry of living things, the different features of cells, pathogens, and the immune system. It is important to understand the biochemistry involved in living structures and to know how organic molecules combine to make living things. Molecular genetics involves the structure and function of DNA and RNA, as well as how they are made and participate in protein synthesis. Pathogens, such as bacteria, viruses, and fungal organisms, cause human diseases and activate the immune system, which is also covered in detail during this course. Chapter one in the course introduces microbiology by first covering the different types of microorganism you might uncover in your quest to understand the fundamentals of this subject. In the laboratory setting, you may have to learn the different staining techniques involved in the identification of microbes so this is discussed in the chapter. There are different types of microscopy used to study pathogens and other microorganisms, including light microscopy, dark field microscopy, and electron microscopy—each of which is covered as part of the chapter. Chapter two opens up with a discussion of the origins of cell theory as well as the different historical aspects of how cells are viewed today. The two types of cells are introduced in chapter one and are further expanded upon in this chapter. Features that make prokaryotic cells unique and things that define what results in a cell being called eukaryotic are also covered in this chapter. Chapter three in the course involves the study of acellular pathogens, which mainly involves viruses. Viruses may or may not be pathogenic and do not have the capability of surviving outside of a cell. There are viruses that can infect all forms of life. The life cycle of viruses is discussed in the chapter along with the ways that viruses are cultured and isolated. There are other acellular pathogens less complex than viruses that are talked about in the chapter, including viroids, virusoids, and prions.

The topic of chapter four is prokaryotic cells. There are features of prokaryotic habitats and their microbiomes you need to know about. In addition, prokaryotes are divided into bacteria and archaea. The different type of bacteria, such as proteobacteria, Gramnegative bacteria, Gram-positive bacteria, and photobacteria are discussed in this chapter. The different features that describe the Archaea domain are also covered as part of the chapter. While there are many different kinds of eukaryotes, including plant and animal species, chapter five in the course focuses mainly on eukaryotic cells that qualify as microorganisms. These include unicellular pathogens that are also eukaryotic like protists, helminths, and fungi. Algae and lichens are not pathogenic but are still important microbiological organisms covered in this chapter. Chapter six teaches you the biochemistry you need to know in order to study microbiology. All living things are basically made structurally of organic molecules and the interactions between the molecules is strictly biochemical in nature. For this reason, you need to understand what the different organic molecules are in living things. Nucleic acids are studied in another chapter but carbohydrates, lipids, and proteins are part of this chapter. The way biochemistry helps in understanding microbiology is also covered in the chapter. Chapter seven in the course talks about cellular metabolism, which is how microbial organisms get their cellular energy. Most of this involves catabolism, which is the breakdown of certain molecules. How cells catabolize carbohydrates, lipids, and proteins is discussed in this chapter. Some organisms derive their energy from the sun. This is called photosynthesis, which is a part of this chapter. Finally, biogeochemical cycles are important to the environment so these are explained in the chapter. The focus of chapter eight is the genome of the cell. Cellular organisms generally have DNA making up their genome. Both DNA and RNA are nucleic acids, which are important in the genetic functioning of the cell. The structure and function of DNA and RNA are covered as part of the chapter. The totality of the DNA in a cell is referred to as the genome. The different characteristics of a cell’s genome are discussed in this chapter.

Chapter nine in the course expands on the study of DNA by looking into microbial genetics. The ways in which DNA is replicated, the transcription process, and the processes involved in protein synthesis are covered in the chapter. Other things discussed are genetic mutations and the different ways genes are regulated. How each of these things leads to genetic diversity in prokaryotes is also discussed in the chapter. Chapter ten touches on aspects of laboratory microbiology by looking into microbial growth. The different patterns of microbial binary fission and bacterial growth in cultures is important to understand as a laboratory microbiologist. There are certain factors that increase or decrease microbial growth, which are covered in the chapter, along with the different physical and chemical methods of controlling microbial growth both in culture and in the environment. Chapter eleven in the course focuses on the different antimicrobial agents used to treat infectious diseases. There are different classifications of antimicrobial agents, some being bacteriostatic and some being bactericidal. There are antibiotics, antifungals, and antivirals, which are covered in this chapter. The public and medical professionals face serious challenges with regard to antibiotic and drug resistances. Some of these challenges are discussed as part of this chapter. The focuses of chapter twelve are pathogenicity, infectious diseases, and epidemiology of infections. The basic definition of an infectious disease is explained as well as what defines a pathogen. There are specific virulence factors that identify viruses, prokaryotes, and some eukaryotes as being pathogenic in nature, which are discussed. The study of epidemiology as it applies to tracking infectious diseases is also covered in this chapter. Chapter thirteen in the chapter is about the innate immune system. It starts with physical mechanisms in the host used to prevent infection as well as chemical protective mechanisms. The innate immune system involves a nonspecific host response, including inflammation, which is explained in the chapter. The process of phagocytosis is crucial to the innate immune response; how this works is discussed in the chapter. The topic of chapter fourteen is the adaptive immune system, which is far more specific against certain pathogens than the innate immune system. There are B cells that are

responsible for making antibodies and T cells that participate in killing infected cells that have been marked with specific antibodies. Both the T cell line and the B cell line have memory cells that retain the memory of a past infection. Vaccines are given to provide immunity to individuals before they get an infection. Chapter fifteen in the course explains several different advance laboratory techniques used in the laboratory in the making of certain drugs and in the detection of diseases that cannot be identified with cultures. Antibodies are specific to a certain pathogen so there are techniques used to identify infectious diseases, which are generally viral in nature or involve fastidious pathogens for which antibodies have been made in the affected patient. Enzyme-linked immunosorbent assays or ELISA testing and fluorescent antibody testing are two of the techniques discussed in the chapter.

CHAPTER ONE: OBSERVING MICROORGANISMS This chapter introduces microbiology by first covering the different types of microorganism you might uncover in your quest to understand the fundamentals of this subject. In the laboratory setting, you may have to learn the different staining techniques involved in the identification of microbes. There are different types of microscopy used to study pathogens and other microorganisms, including light microscopy, dark field microscopy, and electron microscopy— each of which is covered as part of the chapter.

TYPES OF MICROORGANISMS In studying microorganisms, you will need to know the different types of microbes. The vast majority of microbes are too small to be seen with the naked eye but require some type of microscopy to be visible. The smallest microbes are viruses, which cannot be seen with a light microscopy. Bacteria are about one micrometer in size and are the smallest microbes that can be seen with a light microscope. Plant and animal cells are about 10 times or more larger than the average bacterium. Microorganisms can be viruses, which are acellular in nature, or can be part of one of the three major domains of life, which are Bacteria, Archaea, and Eukarya. Both bacteria and species in the Archaea domain are considered prokaryotes because their cells lack a nucleus. Those in the Eukarya domain are eukaryotes because they have a nucleus. Viruses do not belong to any of the domains of life. Bacteria are prokaryotic and are found in practically every habitat in our environment. Surprisingly, most bacteria are either helpful to humans or harmless. There are some, though, that are pathogenic and cause disease. Bacteria are noted for having cell walls that consist of a molecule called peptidoglycan. Bacteria can be of several shapes. Cocci are round bacteria; bacilli are rod-shaped bacteria; spirochetes are spiral and vibrio are curved bacterial species. Figure 1 shows the different bacterial shapes:

Figure 1.

Bacteria have a number of different types of metabolisms and can grow just about anywhere on earth, living off of many different nutrients. Some will be photosynthetic, even though they are not plants. Others do not participate in photosynthesis but live off of inorganic or organic compounds taken up from their surroundings. Archaea are also unicellular and prokaryotic. They are similar to bacteria but have a different evolutionary origin and different metabolisms. They do have a cell wall but these are made from what’s called pseudopeptidoglycan. They can be found in many environments, include those that are very extreme. There are no human pathogenic species in the archaea domain but there are those that reside within the human body. There are several types of eukaryotic microorganisms. By definition, these will have a nucleus and several organelles. One classification of eukaryotes are protists, which have their own category separate from animals, fungi, and plants.

Algae are examples of protists that may be unicellular or multicellular. These organisms also have a cell wall but it is made from cellulose. Algae are photosynthetic and use sunlight as their major source of energy. Algae are what agar used in a microbiology laboratory is made of. Because algae or self-sustaining, they are being investigated as a possible fuel source to be used instead of petroleum products. Algae come in many shapes and include diatoms, found in water sources throughout the world. Figure 2 describes what different diatoms look like:

Figure 2.

Protozoa are also protists, considered extremely important in the environmental food chain. Like algae, they are diverse. Some are motile because they have cilia or flagellae. Others use pseudopods, such as amoeba, in order to move. A few protozoa are photosynthetic, while others are not. Some are only parasitic, while others are freeliving and not pathogenic. Other protozoa are giardia, which are pathogenic to humans. Fungi are eukaryotic organisms, which can be unicellular or multicellular. Mushrooms are an example of multicellular fungi. These organisms have a cell wall that is made

from chitin, which is a unique property of fungal organisms. Single-celled fungi are typically yeast organisms, of which there are at least a thousand species known. They live in many different environments; only a few are considered pathogenic to humans and animals. Molds are multicellular fungal organisms that form colonies that can be seen by the human eye. You can see molds in damp environments, such as bathrooms and rotting plants. Molds participate in the decompensation process and are known allergens to humans. Some molds make mycotoxins that cause diseases. Molds can be helpful too, particularly in the making of certain antibiotics, including penicillin. Helminths are multicellular pathogens and are visible with the naked eye. The are not truly microorganisms but they do have microscopic larvae and eggs. There are multiple types of helminths that cause disease, such as tapeworms and pinworms, which reside in the gastrointestinal system. Viruses are acellular so they do not technically qualify as living things. Viruses are basically genetic material covered in a protein coat and sometimes wrapped in an envelope. They do not function independently but must incorporate their genetic material into a host cell in order to replicate their genome. There are viruses that infect bacteria, called bacteriophages, and viruses that attack other types of organisms. Surprisingly, not all viruses are strictly pathogenic.

FUNDAMENTALS OF MICROSCOPY As you will learn in this section, there are several different types of microscopy. The concept of invisible species being the cause of disease was first developed in the 1500s. At the time, there were no scientific techniques for actually seeing them, however. The first microscope was made by Antonie van Leeuwenhoek, who is believed to be the “father of microbiology”. He first observed single-celled organisms in the late 1600s. Galileo also pioneered the field of microscopy. Galileo used a compound microscope to evaluate insects, while van Leeuwenhoek developed light microscopy that could see bacteria.

Robert Hooke also lived in the late 1600s and contributed to microscopy by first evaluating cells. He studied cork, which led him to believe that cells were empty. The actual inventors of the microscope, however, were Hans and Zaccharias Janssen, who were lens makers. Their work predates the others; however, they did not publish their findings. The term, light microscope, actually applies to several different types of microscope. The typical type of light microscope you will use in the laboratory is a brightfield microscope but there are other types as well that are used primarily in research settings. Brightfield microscopy involves a microscope with at least two lenses that will reveal a darker image on a lighter background. Some will be monocular, while others are binocular, having two eyepieces. There is an ocular lens in the eyepiece that offers a 10 times magnification, and a number of objective lenses that have different levels of magnification. The total magnification seen is the product of both types of magnifications provided. Figure 3 shows a monocular light microscope:

Figure 3.

When you see an item under the microscope, it is referred to as a specimen. The specimen is fix onto the slide and clipped on the platform or stage of the microscope. There are knobs that adjust the ocular focusing coarsely and finely. There is usually a light source beneath the specimen to improve visualization. Light is focused on the specimen with a condenser lens. There Is also a diaphragm that will monitor the amount of light in reaching the specimen and a rheostat, that dims or brightens the light source. Light is differentially transmitted, reflected refracted, or absorbed by the different parts of the specimen, allowing for different features being seen. Chromophores or pigments will provide the different colors seen. Many chromophores actually come from the staining of the item. Magnifications up to 1000 times can be seen with a light microscope. Oil immersion is used at this magnification in order to decrease the scattering of light rays at higher-order magnitudes, improving resolution. Darkfield microscopy is related to brightfield microscopy with modification of the condenser. There is a tiny opaque disc that has been placed between the condenser lens and the illuminator. This is called the opaque light stop. The result is a brighter image on a dark background. The advantage is that it can be used on living specimens that are not stained. Figure 4 shows an image on darkfield microscopy:

Figure 4.

Phase contrast microscopy makes use of interference and refraction within a specimen to create better resolution without having to stain the specimen. The trick is to alter the wavelengths of light that pass through the specimen. The condenser contains an annular stop that makes a hollow cone of light that focuses on the specimen. There is a phase plate and a phase ring that cause the light waves to be out of phase from those that pass through the plate. There are peaks and troughs in the waves that can either augment or cancel each other out. The end result is a dark background and a lighter specimen that can be living and unstained. It can particularly outline cellular features. Differential interference contrast microscopy is related to phase contrast microscopy because interference patterns are created. There are two beams of light involved in polarization of the light waves. The images are high contrast and living organisms that appear three-dimensional. There is no staining required. Figure 5 shows an image seen with DIC microscopy:

Figure 5.

A fluorescence microscope makes use of fluorochromes, which can absorb light energy, revealing parts of the specimen as a fluorescent substance. Chlorophyll is naturally fluorescent and there are dyes also that will fluoresce under the microscope. The chromophores will emit light that has a longer wavelength than the UV light or shortwavelength light that is emitted from the light source. Bright colors can be seen against a dark background. This type of microscopy can be used in order to identify pathogens. Multiple fluorochromes can be used to distinguish between structures. Fluorescence microscopy can be used in immunofluorescence, which involves the use of fluorescent antibodies that tag parts of the specimen so they fluoresce under the microscope. There is direct immunofluorescence assays and indirect immunofluorescent assays. The difference is the actual target of the fluorescence. With indirect or IFA assays, the fluorescence tags antibodies that themselves do not fluoresce. This allows for a wider range of organisms to be identified because many different antibodies can be used to detect pathogens. Figure 6 shows the two types of immunofluorescence:

Figure 6.

A confocal microscope uses a laser to create a two-dimensional image of the specimen at different levels. The different levels of images can be reconstructed with a computer to create a three-dimensional image. It can be used to examine and unfixed images of things like biofilms of bacteria. Figure 7 shows a fluorescent image that has been creates with a confocal microscope:

Figure 7.

The confocal lens is not very good at looking at thicker images. This problem has led to the development of what’s called a two-photon microscopy, which uses fluorochromes, scanning, and infrared light in order to see the specimen. Because it involves low energy levels, it takes two photons to highlight the specimen. It can be used to see living tissues, such as the brain, whole organs, and embryos. Because it is costly to purchase and use, it is used mainly in research settings. Electron microscopy will allow for much better magnification than any form of light microscopy. It uses very short-wavelength electron beams to visualize the specimen. 13

The image that can be gotten can be magnified by 100,000 times magnification. This allows it to see things as small as a strand of DNA. It cannot visualize a living thing because of staining and preparation. There are two types of electron microscopy: these are the transmission electron microscope or TEM and the scanning electron microscope or SEM. The TEM shows a sharp image not unlike that of a light microscope. The electrons pass through the specimen. The technique requires a very thin specimen and staining is done using a heavy metal stain. The SEM will look at the surfaces of specimens in three-dimensional detail. The specimen is coated with gold in order to stain it. There are some SEMs that can magnify up to 2 million times magnification. Figure 8 shows a scanning electron microscopy image:

Figure 8.

Scanning probe microscopy doesn’t use electrons or light but passes sharp probes over the surface of the specimen. The images are the best magnification that can be seen, up to 100 million times magnification. It can look at atoms on certain surfaces.

There are two types of scanning probe microscopy. The first is called scanning tunneling microscopy or STM and the second is atomic force microscopy or AFM. The STM technique passes a probe above the specimen, creating an electric current between the specimen and the probe. The intensity of the current is measured. The AFM also passes a probe across the specimen and it moves up and down because of the different forces between the atoms and the probe. The amount of deflection is measured and an image is constructed.

STAINING OF MICROORGANISMS Without staining, it would be difficult to see most specimens because they do not have a lot of contrast between the different structures. For this reason, staining is used in many microscopy settings in order to see the structures better. In preparing the specimen, there are wet mounts and dry mounts available. Wet mounts involve suspending the specimen in a liquid medium. Water and sometimes stains are used after which a coverslip is applied to the glass slide. Fixation actually involves attaching the cells to the slide. It involves heat fixation or chemical treatment to kill the specimen. The organisms do not move and can easily be stained. Chemical fixating agents include acetic acid, methanol, ethanol, glutaraldehyde, and formaldehyde. Stains are also added to cause coloration of the specimen. All stains are salts that have both a negative and positive ion. One ion will be the chromophore and the other will be uncolored. If the chromophore belongs to the positive ion, it will be called a basic dye. If it belongs to the negative ion, it will be called an acidic dye. There will be a positive stain that is absorbed by what you want to see and a negative stain, which is absorbed by the background. Most positive chromophores are basic dyes that stick to bacterial cell walls. These include crystal violent, basic fuchsin, malachite green, safranin, and methylene blue. Negatively charged chromophores made up of acidic dyes are not taken up by the cell wall; these include rose Bengal, eosin, and acid fuchsin. Simple staining involves a single dye, while differential staining uses multiple dyes in order to show different structures as different colors. Gram staining, endospore 15

staining, acid-fast staining, and capsule staining are all techniques that require differential staining. You will likely be involved in Gram staining, which was invented by Hans Christian Gram in 1884. It is a differential stain that will tell the difference between certain types of bacteria based on their cell wall structure. There are several steps to this that you should know about: •

The sample is fixed with heat.

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Crystal violet is used to give all cells a dark purple color.

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Gram’s iodine is a mordant used to trap the crystal violent within the peptidoglycan cell wall.

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Ethanol or acetone and ethanol are used to decolorize cells with a thinner cell wall.

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Safranin is used as a secondary counterstain to make Gram-negative organisms red.

Figure 9 will show the Gram-stain procedure in the order it is done:

Figure 9.

Gram staining is used only on bacteria and can help define what bacterial species is the pathogen in a clinical situation. There will be Gram-positive and Gram-negative bacterial organisms. Acid-fast staining is also used to define different types of bacteria and can be used to define two separate types of gram-positive cells. There are two techniques used: the Ziehl-Neelsen technique and the Kinyoun technique. The main stain used in both is called carbolfuchsin. It is preferentially retained by cells that have mycolic acids in the cell wall, which will retain the stain after decolorization. Methylene blue is the counterstain. The main difference between the two techniques is that the Ziehl-Neelsen technique uses heat, while the other does not. Acid-fast bacteria will be red or pink against a blue background. Figure 10 shows what an acid-fast organism looks like:

Figure 10.

Some yeasts and bacteria will be surrounded by a capsule, which can affect their virulence. Because capsules do not absorb basic dyes, a negative staining technique is used to show up the background with capsule staining. The capsule looks like a halo on a colored background. Heat fixation Is not necessary. Dyes used for this purpose are nigrosin and India Ink. Positive staining can be used to highlight inner cell structures but these will not reveal the actual capsule. Endospore staining will show bacterial endospores. Endospores are structures that can survive harsh environments. There are two stains used in this type of staining. There is the Schaeffer-Fulton method that uses heat to infuse malachite green into the endospore. A safranin counterstain is used to stain the rest of the cell. The endospore will resist decolorization and will remain green. Flagella staining will reveal flagella structures. These are not otherwise easily seen with light microscopy. The mordant, usually tannic acid, is applied, which will show the flagellum. Then, a counterstain with pararosaniline or basic fuchsin will reveal the rest of the bacterium. Because flagella are delicate, the specimen must be carefully handled during the staining process. A very thin specimen slice is necessary for transmission electron microscopy. Because of this, the cells need to be imbedded in a plastic resin and later dehydrated. Ethanol is used instead of water inside the cells, and the resin solidifies the cells. The sections are sustained using an ultramicrotome. The dyes are not colored but include heavy metalbased stains, such as uranyl acetate and osmium tetroxide. With scanning electron microscopy, the specimen also needs to be dehydrated using ethanol. They need to be even drier than the TEM specimens and must be dried under liquid carbon dioxide. Gold or palladium are sputtered onto the surface of the specimen so it can more easily be seen.

KEY TAKEAWAYS •

There are several kinds of microorganisms that range from viruses to bacteria to multicellular pathogenic helminths.

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The first microscopes were invented in the 1600s in Europe.

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There are different types of light microscopy plus two different types of electron microscopy.

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Scanning probe microscopy does not use light waves or electron waves.

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There are positive stains and negative stains in light microscopy.

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Electron microscopy stains usually involve heavy metals.

QUIZ 1. Which type of microbe does not belong to any of the domains of life? a. Bacterium b. Helminth c. Parasite d. Virus Answer: d. Viruses do not belong to any of the domains of life, mostly because they are completely acellular. Some researchers do not believe that viruses are actually living things. 2. Which type of bacterial species is curved in shape? a. Vibrio b. Bacillus c. Coccus d. Spirochete Answer: a. The Vibrio organism is considered a curved-shaped bacterium. 3. What pathogen in humans is always multicellular? a. Fungi b. Protozoa c. Helminths d. Bacteria Answer: c. Helminths are always multicellular and are rarely microscopic; however, their eggs and larvae are generally microscopic in nature.

4. Which type of pathogen is most likely to infect a bacterium? a. Fungi b. Virus c. Archaea d. Protozoa Answer: b. Because bacteria are so small themselves, only viruses have the ability to infect them. Viruses that infect bacteria are called bacteriophages. 5. What is the advantage of using darkfield microscopy to look at an image under the microscope? a. The resolution is greater b. The image can be living and unstained c. It can see viral particles d. The magnification available is greater Answer: b. The main advantage of darkfield microscopy is that the image seen can be living and unstained so that the characteristics of a living organism can be understood. 6. Which type of microscopy will require staining in order to see the specimen? a. Electron microscopy b. Phase contrast microscopy c. Darkfield microscopy d. Differential interference contrast microscopy Answer: a. An electron microscope specimen requires a stain but none of the others need staining and can be used to visualize a living specimen.

7. What microscopy technique delivers the highest magnification possible in the field of microscopy? a. Scanning electron microscopy b. Scanning probe microscopy c. Transmission electron microscopy d. Two-photon microscopy Answer: b. Scanning probe microscopy offers the greatest magnification possible in a microscope. It does not use light or electron waves so it can see things at up to 100 million times magnification. 8. What chemical is least likely to be used to chemically fix a specimen when preparing a microscope slide? a. Acetic acid b. Methanol c. Sodium hydroxide d. Formaldehyde Answer: c. Each of these can be used to chemically fix a specimen; however, sodium hydroxide is a strong base that would more likely destroy the specimen. 9. Which type of staining technique makes use of India ink to negatively stain the background but nothing to stain the items of interest? a. Gram staining b. Endospore staining c. Capsule staining d. Acid-fast staining Answer: c. Capsules do not take up any stain so that a negative stain like India ink is used to highlight the background.

10. What stain is not used with electron microscopy? a. Gold b. Uranyl acetate c. Osmium tetroxide d. Pararosaniline Answer: d. Each of these is used in electron microscopy, except for pararosaniline, which is used for flagella staining.

CHAPTER TWO: CELL BASICS This chapter opens up with a discussion of the origins of cell theory as well as the different historical aspects of how cells are viewed today. The two types of cells are introduced in chapter one and further expanded upon in this chapter. Features that make prokaryotic cells unique and things that define what results in a cell being called eukaryotic are also covered in this chapter.

MODERN CELL THEORY Modern cell theory is based on two things. The first is that the cell is the fundamental unit that defines living organisms. The second is that cells can only come from other cells. They do not come through any type of spontaneous generation. The father of the cell was Robert Hooke. He was the first to describe cells in 1665 after seeing cork through a microscope. Because cork is not a living thing, he was unaware of the internal structures that make up cells. It took nearly two hundred more years until Mathias Schleiden was able to identify the living plant cells. Animal tissues were also looked at in the 1800s by Theodor Schwann. Modern cell theory came in the 1850s, when Robert Remak and Rudolf Virchow laid down the fact that cells were derived from other cells through division. Both scientists separately determined that there was no such thing as spontaneous generation and that cells comprised the basic structure of living things. By the early 1800s, Robert Brown and Andreas Schimper were studying the major inner structures within cells. Brown first described plant cell nuclei, while Schimper described chloroplasts for the first time. Schimper recognized that chloroplasts divided separately from the cell nuclei. Other’s suggested that chloroplasts were once photosynthetic bacteria that now grow inside the cell. This is the first evidence of the endosymbiotic theory, which exists today with regard to chloroplasts and mitochondria.

The endosymbiotic theory was further elaborated on in the 1900s. Ivan Wallin, an anatomist, claimed he could independently grow mitochondria outside of the cell. While this was refuted, it was later discovered that mitochondria and chloroplasts have their own DNA, which is something unique to these organelles. While these structures cannot exist independently from the cell, the endosymbiotic theory has been substantiated and is largely believed to be valid. The things that support this theory are the separate DNA found in these organelles, the fact that their ribosomes most resemble prokaryotic ribosomes, and the fact that they reproduce through binary fission, which is similar to the way prokaryotes divide. Another theory that needed to be shown was that of the origins of disease. In ancient Greece, there was the miasma theory, which stated that particles in decomposing matter caused disease. A person needed to be in close contact with this matter in order to have disease. It was believed that the Black Death of the middle ages originated because of miasma. The first to develop the germ theory of disease was Girolamo Fracastoro. In 1546, it was proposed that there were seed-like spores that could be transmitted through the air or from person to person. The germ theory was not completely clear initially as to what a microbe was so the theory was abandoned until the 1800s. The next breakthrough came in 1847 by Ignaz Semmelweis, who noted a much higher rate of postpartum infections or puerperal fever and deaths in those women who gave birth in the hospital with doctors or medical students in attendance versus those who were attended to by midwives. He believed that the doctors passed the infection on after doing autopsies and then examining women without washing hands in between. It was proposed that diseases were transferred this way and it was suggested that chlorinated lime water be used to wash hands between patients. This solved the problem. At about the same time, John Snow was studying cholera outbreaks, which was traced to two sources of water in London that had been contaminated. From this, it was proposed that the contamination was the cause of the disease. This was the first epidemiological study known to have been done and it changed public health practices in London.

There were several other contributors to the germ theory of disease. Louis Pasteur studied fermentation and developed the swan-neck flask experiment, which proved that some organisms causing bacterial contamination were airborne. Joseph Lister studied postoperative infections and insisted on strict handwashing during surgery. He also developed the concept of using antiseptics for surgical procedures. Robert Koch developed Koch’s postulates, which attributed certain diseases to a specific pathogen. Several infectious diseases, such as cholera, anthrax, and tuberculosis. This was the first incidence of the idea of “one microorganism, one disease”. This completely did away with the miasma theory and supported the germ theory of disease.

THE PROKARYOTIC CELL There are two different types of cells, which are very different from one another. All cells will have a cell membrane, regardless of the type. All will have cytoplasm, which is the watery component of the cell; all will have some type of nucleic acid or genetic material and all will have ribosomes. These components will be there whether the cell is prokaryotic or eukaryotic. Beyond this, however, prokaryotic cells and eukaryotic cells are very different from one another. In general, eukaryotic cells are larger than prokaryotic cells. Prokaryotic cells, compared to eukaryotic cells, are relatively simple and they do not have membrane-bound organelles. There are some inclusions that help to compartmentalize the cells. Figure 11 depicts what a typical prokaryotic cell looks like:

Figure 11.

We have already talked about bacterial shapes but not about their arrangement. Remember, there are cocci that are round, bacilli that are rod-shaped, Vibrio that are curved, and spirochetes and spirillum that are spiral. Cocci can be single or can occur in pairs, called a Diplococcus. Tetrads involve cells in a square shape and Streptococcus involves a chain of cocci. Staphylococcus will occur in a cluster and Streptobacillus, which is a chain of bacilli. Most of the prokaryotes will have a cell wall, which helps to make up the structure of the cell. The advantage of the cell wall is that it protects the cell from any change in osmotic pressure. This becomes important, too, in the structure of plant cells. Osmotic pressure involves the concentration of solutes in a given body of water. If there is a high concentration of solutes or dissolved substances, water will preferentially enter that space in order to dilute it out more. The reverse is true in very dilute solutions separated by a membrane with another space that is more concentrated. Water will leave the dilute solution and enter the concentrated space. The terms isotonic, hypertonic, and hypotonic refer to the relative concentrations of solutes in solution. A cell inside a hypotonic medium is in a dilute medium and the cell may burst. A cell inside a hypertonic medium is in a concentrated medium and the cell will shrink. A cell in an isotonic medium will neither burst or shrink. The cell wall helps

plant cells and bacterial or prokaryotic cells so they are not destroyed by changes in the tonicity of the surrounding environment. Prokaryotic cells have nucleoids rather than nuclei. There is usually one circular chromosome in the prokaryotic cell and it is circular and haploid, meaning it does not have a pair. There is no nucleus but there are nucleoid-associated proteins or NAPs that help to package and organize the chromosome. NAPs are like the histone proteins in eukaryotes that organize the genetic material. Plasmids are common in prokaryotic cells. These are considered extrachromosomal DNA arranged in small circles. Some cells have several hundreds of plasmids in one cell. Plasmids are not completely unique to bacteria and can be seen in both archaea and eukaryotic species. Many will be crucial to cell survival because they confer some type of advantage, such as improved antibiotic resistance. As mentioned, all cells have ribosomes to help make proteins. The ribosomes of each domain of life are unique to the domain. Ribosomes are largely made from ribosomal RNA and, in prokaryote, they are found in the cytoplasm. They are smaller than the ribosomes found in eukaryotic cells. There are two subunits, called the small subunit and the large subunit. Prokaryotic cells have inclusions, the purpose of which is to store excess nutrients. Because they are condensed in an inclusion, the nutrients in these structures do not contribute to the osmotic pressure of the cell. Glycogen and starches, which are clusters of glucose molecules, are often found in inclusions. Volutin granules also can be found in inclusions and contain in organic phosphate in some bacteria. A few bacterial species store elemental sulfur molecules for use in metabolic processes. Gas vacuoles are inclusions that assist in buoyancy. One species has iron oxide in the inclusions. Some bacterial species can form endospores, which will protect the bacterial DNA during periods of dormancy when there are no nutrients available or a satisfactory environment. Bacterial cells can survive for a long time as just an endospore. Endospores can be resistant to radiation and temperature changes and are dehydrated. They do not exhibit metabolic activity and require specialized endospore staining to be

seen microscopically. Spores form when the cell divides and buds off a spore that has been surrounded by a thick cortex. When conditions improve, the endospore germinates and reenters a vegetative state. Metabolic activity begins and the cell becomes vegetative again. Only some bacteria will form endospores. Most commonly, Bacillus and Clostridium species will form spores, leading to diseases like tetanus, anthrax, pseudomembranous colitis, botulism, and gas gangrene. The spores are hard to kill but can be killed with extreme sterilization. All cells are characterized by the presence of a plasma membrane. These membranes are selectively permeable, meaning they allow some substances but not others to pass through them. According to the fluid mosaic model, the cell membrane components are not rigid but “swim” in relation to one anther like rafts on a lake. Most of the membrane is a phospholipid bilayer, which is water-loving on the outside but water-hating on the inside. Figure 12 shows a typical plasma membrane:

Figure 12.

Archaea of unique cell membranes. The phospholipids are connected with ether bonds, rather than the ester bonds found in eukaryotes and bacterial cells. There are branched chains in archaea but straight chains in the other cell types. Some archaea have a lipid bilayer, while others have a lipid monolayer. Proteins are crucial to the function of the cell membrane. They are important in cell-cell communications, the ability to sense the environment, and help to confer pathogenic virulence. Sugars and proteins together are called glycoproteins that help identify the cell. The different cell types will have very different glycoproteins. You should know a little bit about membrane transport. Remember, the cell membranes to not just allow every molecule to get through. Osmosis involves just the transport of water across the membrane. Simple diffusion happens with small lipid-soluble or gaseous molecules and goes from an area of high concentration to an area of low concentration. Facilitated diffusion makes use of a carrier molecule to get a molecule through the membrane but no energy is required. Active transport involves the input of cellular energy in order to move things from an area of low concentration to an area of high concentration. ATP or adenosine triphosphate is the main energy currency of the cell. While there are no true membrane structures in prokaryotes, the plasma membrane will infold in order to enclose the necessary photosynthetic pigments so that the cell can participate in photosynthesis. These can be called thylakoids in cyanobacteria but in other photosynthetic bacteria, these are referred to as lamellae, chromatophores, or chlorosomes. Bacteria, archaea, fungi, and plants have cell walls but animal cells do not. The cell wall contains peptidoglycan in bacteria, which helps to define them. The cell walls of gramnegative cell walls are different from those in gram positive cell walls. Many antibiotics are directed at peptidoglycan because they are specific to the bacterial species. Peptidoglycan can also be recognized by the human immune system in order to define them as being pathogenic. Gram positive organisms have thick layers of teichoic acid as part of the cell wall, while this is a thin layer in gram-negative organisms. Gram-negative cell walls have an outer

membrane above the teichoic acid layer. This membrane contains lipoproteins, lipopolysaccharides, and porins. Acid-fast bacteria contain waxy mycolic acids as part of the cell wall. The outer cell membrane of gram-negative bacteria is part of the toxic nature of these bacterial species. Some bacteria have a glycocalyx or sugar-coating. There are two types. One is a capsule, which is more organized and made from polysaccharides or sugars and protein. The other is a slime layer, which is less organized but is loosely attached to the underlying cell wall. Washing can remove the slime layer. The glycocalyx helps in cell adherence to surfaces and help to make biofilms. Biofilms protect the cells by forming a protective colony that resists disinfection. Capsules can prevent the uptake of the bacteria by the immune cells. Bacteria are noted for their filamentous appendages that contribute to several functions in the cell. The three appendages are fimbriae, pili, and flagella. Fimbriae and pili are so similar that they are often terms that are not distinguished from one another. There are hundreds of fimbriae that are relatively short. The aid in the attachment of cells to other cells and to surfaces. This is importance in the virulence process. Pili, on the other hand, are not as numerous and are longer than fimbriae. The F pilus or sex pilus is crucial in the transfer of cellular DNA between two bacterial cells. It does not mean that bacterial cells engage in reproduction or sex. It is just the exchange of genetic information. Flagella are used in cell movement. They move like propellers that allow the motility of the cell. There is a basal body at the base of the flagellum that motorizes the whip-like flagella. Flagella can be arranged in four different ways. Monotrichous arrangement is a singular flagellum. Amphitrichous flagella have one flagellum at one or both ends of the cell. Lophotrichous flagella involves a tuft of flagella at each end. Peritrichous arrangement has flagella surrounding them.

THE EUKARYOTIC CELL Eukaryotes come from the domain Eukarya and are represented by plants, animals, protozoa, fungi, and algae. Many of those we will study in this course are unicellular but some are multicellular. The defining feature of these cells is the presence of membrane bound organelles. These are relatively fixed in place by a cell cytoskeleton, which helps to maintain the shape of the cell. The genetic material is usually not circular but is arranged linearly in one or more chromosomes. Figure 13 describes a eukaryotic cell:

Figure 13.

Eukaryotic cells tend to be larger than prokaryotes. They divide not by binary fission but through meiosis or mitosis, which will be covered later. Some have cell walls that can be made from chitin, cellulose, or silica, which is a component of some algae cell walls. Most, however, do not have a cell wall. Motility happens with flagella or cilia that are made from microtubules. The shapes of eukaryotes can vary widely from species to species.

The nucleus is membrane bound and contains the genetic material of the cell. The DNA inside the nucleus is tightly packed and is accompanied by histone proteins that help to wrap and organize the genetic material. A few microbes will have two nuclei that perform reproductive and metabolic functions. There are cells that have nuclei that divide without expansion or division of the cytoplasm. These are called coenocytes. The nuclear membrane is called a nuclear envelope. It has two separate lipid bilayers to form an inner and outer membrane. Each bilayer is unique. There are pores in the nuclear envelope that allow molecules like RNA to pass. The nuclear lamina consists of intermediate filaments that form a mesh inside the membranes in order to give the nucleus its shape. There are other intermediate filaments on the outside of the envelope that keep the nucleus in one place within the cell. The nucleolus is located inside the nucleus. It is the site for ribosomal RNA or rRNA. As this is the major component of ribosomes, it is also the site for the start of the assembly of ribosomes. The partial ribosomes are transported outside to the cytoplasm, where assembly is finished. Ribosomes in eukaryotic cells are larger than that found in prokaryotic cells. There is both a large and small subunit making up the total structure. There are free ribosomes and ribosomes associated with a membrane. The free ribosomes will synthesize watersoluble proteins, while membrane-bound ribosomes, bound to the rough endoplasmic reticulum, make proteins for export or for insertion into membranes. Some antibiotic drugs specifically make use of the fact that there are differences in prokaryotic and eukaryotic ribosomes. The endomembrane system involves a number of sacs, flattened disks, and membranous tubules. The endomembrane system is what makes up many organelles, such as the endoplasmic reticulum, Golgi apparatus, vesicles, and lysosomes. The endoplasmic reticulum is made from cisternae, which are flattened sacs, and tubules that are connected to one another and located just outside the nucleus. The rough endoplasmic reticulum is called rough because they are studded with ribosomes, while the smooth endoplasmic reticulum does not contain ribosomes. The rough endoplasmic reticulum is linked to protein synthesis and helps to make vesicles to

transport these proteins. The smooth endoplasmic reticulum makes lipids, detoxifies toxic molecules, and participates in carbohydrate metabolism. Figure 14 shows the endoplasmic reticulum:

Figure 14.

The Golgi apparatus consists of multiple stacked discs that contain enzymes. These enzymes will modify proteins and lipids to make proteoglycans, glycoproteins, and glycolipids that then can be transported out into the cytoplasm or outside the cell. There are two sides to the Golgi apparatus. The cis face is the receiving end of the apparatus, while the trans face is the exiting end of the apparatus. The Golgi apparatus, basically the post office system of the cell, contains cisternae. It makes secretory vesicles that leave the cell in the process of exocytosis, in which the

vesicles fuse with the plasma membrane in order to extrude the contents to the outside of the cell. Figure 15 shows what the Golgi apparatus looks like:

Figure 15.

Lysosomes are small membrane-bound structure in the cell that are responsible for digestion of unwanted larger particles. They contain multiple digestive enzymes that break down debris, nutrients, immune complexes, and even small microorganisms. These need to be compartmentalized in order to protect the cell from digesting itself.

Peroxisomes are also membrane-bound organelles that contain hydrogen peroxide, necessary for the breakdown of amino acids, uric acid, and fatty acids. They also contain catalase, which has the capacity to degrade the hydrogen peroxide. In some circumstances, peroxisomes also participate in lipid synthesis. The hydrogen peroxide needs to be compartmentalized because hydrogen peroxide will degrade the cell. The cytoskeleton of the eukaryotic cell is important in the internal cell structure. There are three types of fibers within the cytoskeleton, which are microtubules, intermediate filaments, and microfilaments. They are also important in the movement of structures within the cell. Things like mitosis, meiosis, and exocytosis all depend on the cytoskeleton. Microfilaments are made from two strands of actin wound together. These will work with motor proteins, such as myosin, in human muscle contraction as well as in the movement of the pseudopodia of amoeba. Microfilaments can build up and break down rapidly so as to allow for cellular movement. Intermediate filaments are the cables of the cell that form the nuclear lamina inside the nuclear envelope. They anchor adjacent cells in multicellular animals. Some will contain desmin, which is a protein important for the making of desmosomes that hold adjacent cells together. Others contain keratin found in skin, hair, and nails. Microtubules are made from two different types of the tubulin protein. The core of the microtubule is hollow. They form part of the cytoskeleton and work with two motor proteins called dynein and kinesin, which act to move organelles within the cell structure. Both the flagella and cilia of eukaryotic cells are made from microtubules that can rapidly break down and reassemble themselves. Microtubules form the mitotic spindle that can separate chromosomes during cell division. Centrosomes make microtubules that separate the chromosomes in mitosis. Mitochondria are considered the powerhouses of the cell because they participate in cellular respiration that ultimately makes ATP energy for the cell. They contain DNA and have a double membrane system. The inner membrane is highly convoluted and are where the metabolic enzymes are located. These invaginations are referred to as cristae. Figure 16 shows the internal structure of the mitochondria:

Figure 16.

Plants and algae contain chloroplasts, which are organelles that participate in photosynthesis. There are three membrane systems associated with them. There is an outer membrane, an inner membrane, and what’s called the thylakoid membrane system. The stroma is between the inner and outer membrane. The thylakoids are folded sacs containing chlorophyll pigment, where the photosynthesis takes place. The stacks of thylakoids contain grana in plant organisms. Figure 17 describes chloroplasts:

Figure 17.

The plasma membrane of eukaryotes is similar to that of prokaryotes. One major difference is the presence of cholesterol and other sterols in eukaryotes but not prokaryotes. Sphingolipids are specialized lipids in some eukaryotes that participate in cell to cell communication. Eukaryotes are different from prokaryotes because they can participate in endocytosis, which further specializes into phagocytosis or “cell eating” that applies mainly to immune cells. Pinocytosis is called “cell drinking” and involves taking in solutes and water by the cell. Exocytosis involves the release of vesicles from the inside of the cell to the outside of the cell. Those cells that do not have a cell wall will make an extracellular matrix, which is a mass of proteins and carbohydrates that exists between the adjacent cells. The basement membrane beneath epithelial cells in animals is made from the extracellular matrix. Collagen is one of the proteins made that can provide tissue strength. This matrix is responsible for protecting the cell from external stressors. As mentioned, eukaryotes contain flagella and cilia for movement. The flagella in eukaryotes are different from those found in prokaryotes. The eukaryotic flagella are

flexible rather than rigid and are made from microtubules. Dynein proteins provide the movement of the flagella. Cilia are similar to flagella but are shorter and cover the entire cell surface. They are also made from microtubules and have a basal body on the base of the cilium as well as the flagellum. Cilia have rapid, wave-like movements that can sweep particles past the cell or can sweep nutrients into the mouths of some cells, such as protozoa. Cilia are found in the respiratory tract of humans and other mammals.

KEY TAKEAWAYS •

Modern cell theory began with Robert Hooke’s identification of cells.

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The germ theory of disease replaced the miasma theory, which differ in their explanation of what causes disease.

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There are some features, such as a cell membrane, ribosomes, genetic material, and cytoplasm, that are similar in eukaryotic and prokaryotic cells.

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Prokaryotes lack membrane-bound organelles but do have some degree of compartmentalization.

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Eukaryotes have a number of membrane-bound organelles and a cytoskeleton, which is different than prokaryotic cells.

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Prokaryotes have nucleoids, while eukaryotes have membrane-bound nuclei containing DNA.

QUIZ 1. Who was considered to be the father of the cell for his observations of dead cork cells? a. Robert Remak b. Robert Hooke c. Rudolf Virchow d. Mathias Schleiden Answer: b. Robert Hooke first discovered cells, although his work was done on dead cork cells, which did not reveal any of the inside details of cellular structures. 2. What structure within cells first originated the endosymbiotic theory regarding cells? a. Mitochondria b. Nucleus c. Chloroplasts d. Golgi apparatus Answer: c. Chloroplasts were first discovered to divide independently from the cell nucleus, which gave rise to the endosymbiotic theory that regarded chloroplasts as once being primordial bacteria. 3. What cell structure is not found in both prokaryotic cells and eukaryotic cells? a. Nucleus b. Ribosomes c. Cell membrane d. Cytoplasm Answer: a. Each of these is found in both prokaryotic cells and eukaryotic cells; however, nuclei are only found in eukaryotic cells and not prokaryotic cells.

4. What best describes round bacterial organisms arranged in pairs? a. Streptococcus b. Staphylococcus c. Streptobacillus d. Diplococcus Answer: d. Diplococcus is what the organisms are called when they are round and are arranged in pairs with one another. 5. What is not a key feature of an endospore? a. Have metabolic activity b. Contain the cell’s genome c. Survives extreme environments d. Largely dehydrated Answer: a. Each of these is a feature of an endospore except that they do not have metabolic activity so they can survive without the presence of nutrients. 6. What membrane transport mechanism requires ATP energy input to make it happen? a. Osmosis b. Active transport c. Simple diffusion d. Facilitated diffusion Answer: b. Active transport is the only transport mechanism that requires ATP energy input in order to have the substance cross from an area of low concentration to an area of high concentration.

7. What is characteristic of a eukaryotic cell’s nuclear envelope? a. It is a single lipid monolayer b. It is a single lipid bilayer c. It is a double lipid bilayer d. It is a double lipid monolayer Answer: c. The nuclear envelope consists of a double lipid bilayer with two complete bilayers, an inner and outer bilayer. 8. What eukaryotic cell structure makes ribosomal RNA for the cell? a. Nucleus b. Rough endoplasmic reticulum c. Golgi apparatus d. Nucleolus Answer: d. The nucleolus is where the ribosomal RNA is located and where the start of the synthesis of ribosomes themselves begins. 9. Which two proteins are important in the movement of organelles within the cell? a. Tubulin and myosin b. Dynein and kinesin c. Keratin and tubulin d. Dynein and actin Answer: b. Dynein and kinesin are motor proteins that together work with microtubules in order to move organelles within the interior of the cell.

10. Inside what structure does chlorophyll inside the cell exist? a. Thylakoids b. Cristae c. Cisternae d. Microtubules Answer: a. Chlorophyll exists within the thylakoids, which are flattened membranous sacs that are found inside chloroplasts.

CHAPTER THREE: ACELLULAR PATHOGENS This chapter involves the study of acellular pathogens, which mainly involves viruses. Viruses may or may not be pathogenic and do not have the capability of surviving outside of a cell. There are viruses that can infect all forms of life. The life cycle of viruses is discussed in the chapter along with the ways that viruses are cultured and isolated. There are other acellular pathogens less complex than viruses that are talked about in the chapter, including viroids, virusoids, and prions.

VIRUSES Viruses are extremely small and cannot be seen with a light microscope. The first indication of the presence of something smaller than a bacterium dates back to the late 1800s when the causative organism for tobacco mosaic disease was discovered. In the beginning, the disease was felt to be caused by a chemical. This is why the word “virus” means “poison”. Viruses can now be seen with electron microscopy. Their exact evolutionary origin is unknown. They are not considered part of the domains of life because they are acellular. They must infect another cell in order to survive. New virus particles are called virions. These new virions pass the genetic material of the virus to another cell that has not yet been infected. Viruses contain either DNA or RNA but never have both within the same cell. Genetic material for reproduction of the virus particle is largely lacking. All cellular organisms can be infected by a virus but there is a host range, which involves those cells that are susceptible to a particular virus. Bacteriophages only infect bacterial cells, while other virus can only infect certain animal or plant cells. Not all viruses will kill the cell but will instead affect cell growth or will not appreciably affect the cell. There are different ways for a virus to infect a cell. This can involve direct contact between cells, contact derived through fomites, which are environmental things like dust particles, or through a vector. The vector can be a mechanical vector, in which there is external physical contact with the vector and the host, or a biological vector, 45

which involves biting the host and transmitting the virus existing within the body of the vector. Zoonoses are infections in humans that once existed in animals. Reverse zoonoses are infections in animals that once existed in humans. Viruses can be seen with electron microscopy and are between 20 nanometers and 900 nanometers in diameter. There are some giant species, however, that have recently been discovered. Viruses are of different shapes. Figure 18 shows what a bacteriophage looks like:

Figure 18.

Viruses are now known to contain some type of nucleic acid and a capsid, which is what the protein coat is called. There is no cytosol or cytoplasm, but there are some enzymes necessary to make new virions. Capsids are made of protein subunits known as 46

capsomeres that interlock to make the capsid. Viruses can be naked and will not contain an envelope; other viruses are called enveloped viruses that contain a viral phospholipid-containing viral envelope. Some viruses have spikes made of proteins. These spikes allow for attachment of the virus to the cell. Influenza, for example, contains H spikes and N spikes, depending on what they attach to in human cells. This is what names the influenza virus. The H1N1 virus caused the 1918 pandemic and the H3N2 virus caused the 1968 pandemic. The spikes are made from glycoproteins. Viruses have complex, polyhedral, or helical shapes. Figure 19 shows the different viral shapes:

Figure 19.

An icosahedral capsid is 20-sided, while there are also helical viruses like Ebola and the tobacco mosaic virus. Polyhedral viruses include rhinovirus and poliovirus subtypes. Bacteriophages have tail fibers, a polyhedral head, along with tail pins that attach the virus to the host cell. Poxviruses are shaped like bricks and have unique surface features. Viruses are also classified according to the viral genome they have. There can be positively stranded or negatively stranded RNA viruses, DNA viruses, and further distinction based on whether or not the virus is double-stranded or single-stranded.

VIRAL LIFE CYCLE As mentioned, viruses do not have an adequate genome to sustain themselves unless they infect a host cell and capture the host cell’s reproductive capacities. DNA viruses in most eukaryotic cells will need to be present in the nucleus for replication, while bacteriophages replicate in the cytoplasm. Large poxviruses are DNA viruses that can replicate in the cytoplasm. RNA viruses replicate primarily in the cytoplasm. Bacteriophage life cycles have been studied extensively. There are virulent phages that automatically kill the cell and temperate phages that become part of the host genome. These lead to latent infections that ultimately get activated to make progeny viruses or virions that have been newly assembled. Virulent phages basically take over the cell, reproduce to make new phages, and destroy the cell. There are five stages associated with the bacteriophage lytic cycle. The first stage is attachment, which involves association of the bacterial surface receptors with the virus particle. The second stage is penetration, with injection of the nucleic acids into the host. The virus itself remains outside. Then comes biosynthesis, which is replication of the viral proteins. After this is maturation, in which new virus particles are assembled. Lysis only happens with virulent phages that kill the cell. In what’s called the lysogenic cycle, the phage genome participates in attachment and penetration, just as with the lytic cycle. The difference is that the phage genome instead is integrated into the host genome and does not immediately kill the cell. In such cases, the integrated genome is referred to as the prophage and the bacterial host along with the prophage is called a lysogen. The entire process is known as lysogeny.

Certain prophages can contribute to the pathogenicity of the bacterial organism. Some toxic viral genes will make a bacterium more pathogenic. When this happens, it is called lysogenic conversion or phage conversion. An increase in pathogenicity does not necessarily have to happen. Some viruses will decrease the pathogenicity of the bacterial organism. In the case of Clostridium botulinum and Vibrio cholerae, the prophage increases virulence. At the time of induction, the viral genome is excised from the host genome and a lytic phase occurs, killing the cell. Transduction is the process of transferring bacterial DNA from one bacterium to another bacterium because of an infection with a bacteriophage. Sometimes, when the viral genome is being made and replicated, certain mistakes are made in which the host DNA gets spliced off in part and enters the new virion at the time of viral packaging. When the virus particle enters a new cell, the bacterial DNA segment goes into the new host. This can change the metabolic properties or antibiotic resistance of the new host. There are two kinds of transduction. In generalized transduction, the random DNA piece gets transferred as part of the lytic cycle. With specialized transduction, the transfer occurs at the end of the lysogenic cycle, after the prophage DNA is excised from the bacterial genome. There are certain situations of increased stress on the bacterium that stimulates induction and later transduction. Things like UV light or chemical exposures can all trigger the induction process. This is called specialized transduction because only the DNA next to where the prophage was inserted can get transferred from one cell to another. Transduction is important to the evolution of bacterial organisms. When viruses infect an animal host, there is attachment, penetration, viral biosynthesis, maturation of viral particles, and particle release. There are differences, though, in penetration, biosynthesis, and release stages. There isn’t injection of the viral genome but endocytosis of the viral particle. The fact that viruses only infect certain types of tissues is called tissue tropism. For example, the influenza virus only infects the respiratory tract. There are different ways to replicate the viral genome, depending on the type of nucleic acid making up the virus. The different RNA genomes can be translated directly into viral proteins. It cannot happen with a negative single-stranded RNA virus until it gets

turned into a positive-sense RNA molecule. Often, the enzyme to do this comes from the virus itself. Retroviruses are what Positive single-strand RNA viruses are called. HIV is called a retrovirus. The virus must have an enzyme called reverse transcriptase to make a piece of double-stranded DNA that can be incorporated into the host cell. This integrated virus particle is called a provirus. Persistent viral infections happen when the virus has not been cleared by the host but instead remain infective or silent without killing the host cell. There are two types of persistent infections. One of these is the latent infection, while the other is a chronic infection. Latent infections are asymptomatic and include oral and genital herpes as well as the chickenpox virus, which leads to herpes zoster. Chronic infections usually have some symptoms and include HIV and hepatitis C infections. Latent infections come from certain latent viruses that initially cause disease before they become dormant. Chickenpox causes acute disease but lives dormant in the host’s nerve cells for many years. When it becomes reactivated, shingles can occur, involving pustules that are only found in the distribution of the nerve ganglion near the spinal cord. Some latent viruses are separate circular genomes, while others become integrated into the genome of the host. Chronic infections are usually both persistent and recurrent. There will be symptoms. The problem is that the body cannot eliminate the specific virus. The person with HIV has a chronic infection that might not actually be symptomatic throughout the person’s life. If the virus is not held in check with antiviral agents or the person’s immune system, the patient ultimately develops AIDS, which is lethal to the patient. Plant viral infections are similar to animal viral infections. Plant viruses may be singlestranded or double-stranded and they may consist of RNA or DNA. Most, however, are positive single-stranded RNA viruses that get directly translated into proteins. Some viruses are highly host-specific, while others are not. They can be transmitted via arthropods, nematodes, fungi, or direct contact. Sometimes the vector itself is specific. Viruses that cause plant diseases are referred to as biotrophic parasites because they rarely kill the host completely. There is penetration and uncoating of the genome, in which the capsid is removed. Then the viral genome gets replicated to make new

virions. The infection can become systemic if it gets into the plant’s vascular system. The cycle starts over when the virus infects a new plant host. There are certain features of the viral growth curve. It starts with an inoculation phase, prior to the penetration of virus particles. During the eclipse phase, viruses have penetrated the cells and there are no free virions. Then there is a burst phase, when virus particles are released. The burst size becomes the number of virions released after the burst phase. The viral titer is the concentration of viral particles. The culture declines if there are no bacteria to penetrate anymore.

ISOLATION AND IDENTIFICATION OF VIRUSES Because viruses require a host, it can be difficult to culture them without a host cell. They are allowed to infect the host and are harvested after separating them from host cells after they’ve been infected and after the virus particles have been allowed to replicate. A filter is used to allow viruses but not bacteria to filter into the filtrate. Viruses can be grown within a living organism, in which it is called in vivo. If they are grown in vitro, it is done inside a test tube or on an agar plate. Bacteriophages are cultured in a culture or plate of bacteria, called a bacterial lawn. There will be a clear zone or plaque where the bacteria have died off on the agar plate. Animals will grow viruses in vivo. It can be done to identify certain pathogenic viruses, to help produce vaccines, and in research settings. An embryo from a chicken is often used for these types of settings. Remember that viruses have tissue tropism so they must be grown in the presence of specific tissues. Tissue cultures are used to grow certain viruses. A commonly used immortal cell line tissue culture is the HeLa cell line, which was isolated in the middle of the twentieth century from a woman who had cervical cancer. This cell line provides consistency in growing human cells in a culture medium. These are used to grow certain animal viruses. Samples of viruses can be prepared in an infected cell line, embryo, or whole host. There will be certain cytopathic effects from the infection, which involve changes in the

cells’ features or adherence to the culture medium surface. There can also be cell lysis as a result of the infection. The kind of cytopathic effect depends on what virus is being studied. Researchers look for specific cytopathic effects in the cell culture to identify the virus being studied. The hemagglutinin assay is one way of determining if a virus is present in a patient’s blood or serum. The serum or the liquid component of blood is isolated and mixed with blood. If the virus is present in the serum, there will be agglutination or clumping of red blood cells after the serum and test blood cells are mixed. It can be seen with the naked eye. In most cases, indirect hemagglutination is used, which involves the ability not to detect the viruses themselves but to detect antibodies that are directed at the viruses. Antibodies are mixed with the serum, binding to virus particles. When this happens, no hemagglutination can occur. This is called a hemagglutinin inhibition assay. In the nucleic acid amplification test or NAAT, specific nucleic acid sequences of the viral particles are detected. An example of this is the polymerase chain reaction. Many copies of the viral DNA segments are gotten and the nucleic acid segments are detected. Another similar test is the reverse transcriptase PCR test. Reverse transcriptase is an enzyme used to make a DNA sequence from the RNA present in the virus particle. This allows for amplification of the DNA segments and analysis of the presence of the virus. In an enzyme immunoassay or EIA, antibodies will attach to antigens. We will talk more about this process later. There is an enzyme attached to the antibody that can cause a specific reaction to occur. The enzymatic reaction that will then happen after the antigen and antibody have attached leads to a colored end-product. The color will then be detected.

VIROIDS AND PRIONS There are infectious particles that are even simpler than viruses. They will contain only a nucleic acid like RNA or only a protein that can cause disease. The three different types of these infectious particles include viroids, virusoids, and prions. Viroids are simply segments of circular RNA that can self-replicate. The viroids can hijack the host’s replication machinery in order to replicate the RNA segments. There is no protein coat involved. An example of a disease that can cause this is the potato tuber spindle disease. There are several severe plant-related viroid diseases that can damage crops and kill the host plant. These will be passed in similar ways as happens with viruses. Virusoids are also sub-viral particles. These are single-stranded RNA molecules that are related to viroids but differ in that they require a coinfection with another helper virus. These types of infections are much less common than viral or viroid infections and mainly infect plants. There are no proteins associated with virusoids themselves. Hepatitis D or hepatitis delta is one of these satellite RNA segments that can only infect a human that has the helper virus hepatitis B in order to result in the hepatitis D infection. Prions are not nucleic acids but are proteins that can be infective. Prions are a misfolded protein that resembles a natural protein but instead of being active, it leads to plaques in the tissues. The main prion that has been studied is one that causes transmissible spongiform encephalopathy or mad cow disease. The end result is the killing of brain cells, which becomes sponge-like in appearance. Infected humans will have dementia and the rapid onset of death within months. Creutzfeldt-Jakob disease is an example of this type of infection, although there are others. There is no cure for these diseases as they cannot be killed off.

KEY TAKEAWAYS •

Viruses have different shapes and sizes. They must replicate within a host cell.

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Viruses that infect bacteria are called bacteriophages.

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Viruses can cause lysis of a cell or can alter the cell in different ways without killing them.

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Viral life cycles start with attachment and penetration, followed by biosynthesis, maturation, and release or lysis of the cell.

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Transduction involves the passage of host DNA through a viral vector.

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There are several ways of detecting viruses in a human serum sample.

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Viroids, virusoids, and prions are simpler than viruses but are still infectious diseases.

QUIZ 1. What is a new virus particle called? a. Viroid b. Virion c. Virusoid d. Prion Answer: b. A virion is what a new virus particle is called. The viroid, virusoid, and prion are other types of acellular pathogens. 2. Transmission of a virus through dust particles is called what? a. Transmission through a biological vector b. Transmission through a mechanical vector c. Direct transmission d. Transmission through a fomite Answer: d. Transmission through a fomite involves contact with something in the environment, such as a dust particle, that passes on the virus particle. 3. Where in the eukaryotic cell do DNA viruses replicate? a. Cytoplasm b. Rough endoplasmic reticulum c. Nucleus d. Near the cell membrane Answer: c. DNA viruses enter the nucleus and replicate. The same is not true of bacteriophages and most RNA viruses.

4. What stage of the viral life cycle only happens with virulent phages when they infect a virus particle? a. Penetration b. Maturation c. Biosynthesis d. Lysis Answer: d. Lysis only happens with virulent phages because only these phages will lyse and destroy the cell they have infected. 5. What is it called when there is a trigger that removes the viral genome from the host cell, causing a reaction that goes on to killing the cell? a. Induction b. Lysogenic conversion c. Maturation d. Replication Answer: a. Induction is when the prophage DNA is excised out of the host genome, ultimately leading to the death and lysis of the cell. 6. What causes the lysogenic cycle of viral infections to lead specifically to what’s called specialized transduction? a. Only certain types of bacterial species can take up the transduced DNA. b. This type of infection and DNA transfer only occurs in bacteriophage infections. c. Only DNA segments that are near the site of the prophage get transduced. d. It can happen in the lytic cycle or lysogenic cycle of bacterial infections. Answer: c. This is called specialized transduction because only the DNA segments that are near the site of the prophage get transduced from one bacterial organism to another.

7. The virus growth cycle in a culture medium of bacteria goes in which order? a. Burst phase, eclipse phase, inoculation phase b. Inoculation phase, burst phase, eclipse phase c. Inoculation phase, eclipse phase, burst phase d. Eclipse phase, inoculation phase, burst phase Answer: c. The viral titer goes from the inoculation phase to the eclipse phase, which is during the acute infection, followed by the burst phase, in which virus particles are released up to a certain burst size. 8. In virology, what is tissue tropism? a. The tendency for viruses to kill the host cell or organism b. The tendency for viruses to become latent for a period of time c. The tendency for viruses to cause a chronic infection rather than kill the host d. The tendency for viruses to infect certain cells but not others Answer: d. Tissue tropism is the tendency for viruses to infect certain cells but not others. 9. What type of infectious disease is Creutzfeldt-Jakob disease? a. Viral b. Bacterial c. Viroid d. Prion Answer: d. Creutzfeldt-Jakob disease is a prion disease of the brain that causes dementia and the rapid onset of death from brain disease over several months.

10. What type of genome is seen in a prion disease? a. It does not have a genome b. It is generally single-stranded RNA c. It is a loop of double-stranded DNA d. It is often a single-stranded DNA segment Answer: a. Prions are strictly infectious abnormal proteins that do not themselves have any type of genome associated with them.

CHAPTER FOUR: TYPES OF PROKARYOTIC CELLS The topic of this chapter is prokaryotic cells. There are features of prokaryotic habitats and their microbiomes you need to know about. In addition, prokaryotes are divided into bacteria and archaea. The different type of bacteria, such as proteobacteria, Gramnegative bacteria, Gram-positive bacteria, and photobacteria are discussed in this chapter. The different features that describe the Archaea domain are also covered as part of the chapter.

PROKARYOTES AND MICROBIOMES Prokaryotes can survive in very diverse habitats, including those that are very cold, those that are very hot, and under extreme pressure. There are ten times more bacteria in the human body than there are human cells. Bacteria exist in the GI tract, skin, ears, respiratory tract, and vagina. Very few of these prokaryotic organisms are pathogenic. In fact, many bacterial species in soil are commensal with the plants and animals in the environment. Prokaryotes can also be found in the air. One of the advantages of prokaryotic life is that they can easily adapt their metabolism to the environment. Some will store carbohydrates, for example, under certain circumstances, but will metabolize these same carbohydrates when necessary. Some prokaryotes will fix and recycle the basic elements, such as nitrogen or carbon from carbon dioxide, which is something animals cannot do. This ability to capture carbon from CO2 is called carbon fixation. Other bacteria can fix the nitrogen in the air to make organic nitrogenous compounds. The Rhizobium species can turn nitrogen gas into ammonia, which is turned further into nucleic acids by plants associated with the bacterial species. Fixed nitrogen can be further used by those animals that eat the plants. Bacteria can be helpful in cleaning up toxic chemicals in the environment. There are certain species that will exist in manmade environments that are polluted. They can

clean the pollutants out of the soil and/or the water. This makes them possibly beneficial to man when it comes to environmental pollution. Prokaryotes will play a role in climate change. As the temperature of frozen areas rises, the bacteria that have been trapped in a frozen environment begin to proliferate. The trapped carbon sources in the permafrost are metabolized by bacteria and other prokaryotes. This allows for the release of methane and carbon dioxide into the environment, which adds to the greenhouse gas concentrations in the atmosphere, further warming the environment. Many bacterial species are symbiotic with other animals, plants, and prokaryotes. A symbiotic relationship implies that both parts of the relationship get something positive out of the relationship but this is not always the case. Populations or communities of prokaryotes can have cooperative interactions with the environment, which benefit the prokaryotes, or competitive interactions with the environment, which involves one population have an advantage over another. The study of these phenomena is referred to as microbial ecology. There are different types of symbiosis along a spectrum of harm versus benefit. These can be described as follows: •

Mutualism—this involves both populations benefiting from the relationship.

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Amensalism—involves harm to one population and no affect on the other population.

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Commensalism—this involves one population benefiting and one population unaffected.

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Neutralism—this involves neither population affected by the relationship.

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Parasitism—this involves one population benefiting and the other population harmed by the relationship.

Examples of mutualism is the one humans have with Bacteroides species. The bacterium can digest complex carbohydrates from plants that does not happen with human enzymes. The result is a breakdown of the carbohydrates into simple carbs that can be absorbed in the human GI tract. Humans also receive vitamin K from certain species of E. coli in the gut. 60

Amensalism can involve certain bacterial species that make toxic substances that kill other bacteria. The organism that secretes the toxin is not affected but the other population is killed. This is the case with certain skin bacteria that reside on human skin. With commensalism, humans help the Staphylococcus epidermidis species by allowing them to feed off of dead skin cells. This is not harmful to humans but certainly benefits the bacteria. In some cases, the relationship is mutualistic because the microorganism can secrete toxins that kill pathogenic bacteria for the human host. The relationship between pathogenic bacteria and humans is generally referred to as parasitism. The pathogen in this case receives a benefit, while the human host is harmed in the process. Most human infectious diseases are basically parasitic relationships between ourselves and the pathogen. The definition of microbiome involves those organisms, which can be eukaryotic or prokaryotic, that are linked to a particular organism or a given environment. With regard to humans, we will have both resident microbiota and transient microbiota. Resident microbiota will always live with the host, while transient microbiota are temporary residents. Pathogens are generally transient microbiota. People will vary with regard to their microbiota and the same person will have differences in their own microbiota over time. Prokaryotes are generally classified according to their patterns of staining and overall shape. Bacteria will be gram-negative, gram-positive, or atypical. We have already talked about how gram staining works and you have seen that the thick peptidoglycan wall in gram-positive organisms allow for their unique staining pattern. Atypical bacteria include things like Rickettsia, Chlamydia, and Mycoplasma because they either do not stain at all or because they are too small to be seen under normal circumstances. There has been a further breakdown of gram-negative bacteria into things like deeply branching bacteria and proteobacteria. Gram-positive bacteria can be further classified as well into those that have a lot of guanine and cytosine in their nucleic acid sequences and those that do not. You should know that there is ongoing reclassification of bacterial species occurring all the time.

PROTEOBACTERIA Proteobacteria are a phylum or subset of gram-negative bacteria. These can be found everywhere. There are five classifications of these bacteria, called alphaproteobacteria, betaprotobacteria, gammaproteobacteria, deltaproteobacteria, and epsilonproteobacteria. The alpha-proteobacteria are what are called oligotrophs. These are organisms that can live in environments that are very low in nutrients. They live deeply underground, deep in the ocean, or within glacial ice. Chlamydia and Rickettsia are alpha-proteobacteria that are obligate intracellular pathogens. They are inactive when not within a cell because they cannot make ATP energy by themselves. Rickettsia species can cause Rocky Mountain spotted fever, which is transmitted through infected tick bites. Another species causes epidemic typhus, while still another causes endemic typhus. Chlamydia is another type of alpha-proteobacteria. These are also obligate intracellular pathogens. They are spread via inactive elementary bodies, which are like endospores to become activated within an epithelial cell. Chlamydia trachomatis will be the causative agent in a common sexually-transmitted disease. There are other alpha-proteobacteria that are also obligate intracellular pathogens. The class called beta-proteobacteria are different from the alpha-proteobacteria because they require a great deal of nutrients in order to grow. They grow between aerobic and anaerobic areas of the human intestine and some are pathogenic to humans. Neisseria gonorrhoeae is one of these that causes the sexually-transmitted disease called gonorrhea. Another species will cause certain types of bacterial meningitis. They do not tolerate a great deal of oxygen and form pairs called diplococci. Bordetella pertussis causes whooping cough and is also difficult to grow in culture. Organisms that are gamma-proteobacteria are diverse and can cause human diseases. Pseudomonas is one of these. It can infect burns and wounds and can be seen in people who are immunosuppressed. Antibiotic resistance is common with these organisms. Pasteurella is another type of these organisms. These are passed from animal bites to humans. Haemophilus species also cause human diseases, including respiratory

infections and chancroid. Vibrio cholerae cause cholera. Legionella is a respiratory pathogen of this classification. Other species and genuses in the category of gamma-proteobacteria include Enterobacter, which thrives in the mammalian intestinal tract. There are two subcategories. One is Escherichia coli, which is a coliform bacterial organism, and noncoliforms, which include Shigella, Salmonella, and Yersinia pestis. E. coli is generally mutualistic but it can be pathogenic because of strains that produce certain dangerous toxins. One of these is the Shiga toxin, which leads to serious disease in humans. The delta-proteobacteria is a small classification of organisms that use sulfate as part of their electron transport chain. Very few of these are pathogenic to humans. The subclassification includes myxobacteria, which is a soil organism that scavenges on inorganic compounds. They have the capacity to create metabolically inactive spores called myxospores, which look like fruiting bodies and contain other organisms. The epsilon-proteobacteria are an extremely small group that are considered microaerophilic, meaning that they do not require much in the way of oxygen for their survival. The two bacteria most clinically important are Helicobacter and Campylobacter species. Campylobacter can cause food poisoning and Helicobacter causes chronic gastritis and stomach ulcers. Helicobacter pylori can survive in the acidic environment of the stomach and will make the stomach less acidic.

GRAM-NEGATIVE BACTERIA Most gram-negative bacteria are also proteobacteria. There are those, however, that do not belong to this category. One of these is the spirochetes, which are long, thin, and spiral. They are not easy to see, even after staining. For this reason, they are often observed through darkfield fluorescent technologies. They are extremely difficult to grow in cultures and move using an axial filament, which wraps around the cell itself, unlike a flagellum. Figure 20 shows what spirochetes look like under darkfield microscopy:

Figure 20.

There are some genera that cause human diseases. These include Treponema, which can cause syphilis and other diseases. Another is Borrelia, which has a species that causes Lyme disease. There is one phylum called the CFB group, which stands for Cytophaga, Fusobacterium, and Bacteroides. They are similar because they share similar genetic characteristics. Each of these is rod-shaped and lives in an anaerobic environment, being called fermenters for their ability to process cellulose in ruminant animals. Cytophaga are aquatic and glide through the water. Fusobacteria normally live in the mouth and can be highly pathogenic. Bacteroides species mainly live in the intestinal tract and make up a third of the gut microbiome. Bacteroides are generally mutualistic with humans as they are competitive with pathogenic bacteria. The organisms of the Planctomycetes are mainly aquatic. These are considered interesting to study because they do not reproduce through binary fission but send out buds that detach from the larger mother cell. Also of interest is the presence of an

appendage called a holdfast in some of the organisms that allow them to attach specifically to underwater surfaces. These sessile cells are the only type that reproduces.

PHOTOTROPHIC BACTERIA Bacteria considered to be phototrophic are not necessarily related to one another except for the fact that they are photosynthetic. There are proteobacteria and nonprotobacteria that are part of this group. Some produce oxygen, while others do not. This is called either oxygenic or anoxygenic photosynthesis. There are bacteriochlorophylls which are pigments related to plant-based chlorophyll. They come in a variety of colors. Some are considered sulfur bacteria, while others are not. The sulfur bacteria do not produce oxygen but produce elemental sulfur instead. Purple sulfur bacteria are anaerobes that are also aquatic. They make use of carbon dioxide but also use sulfites in order to make elemental sulfur. Green sulfur bacteria are similar but can also produce methane. Purple non-sulfur bacteria can use hydrogen rather than hydrogen sulfide. Green non-sulfur bacteria do not use sulfide for oxidative purposes but use organic sulfites. There are also the Cyanobacteria, which are oxygenic and make oxygen gas. They are believed to have helped throughout evolution to populate the earth with oxygen. Cyanobacteria live in extreme environments and many will fix nitrogen. There are some that harm the environment by causing toxic blooms that can harm people and animals.

GRAM-POSITIVE BACTERIA Gram-positive bacteria stain easily with crystal violet. Nowadays, gram-positive bacteria are further subdivided into those that have high levels of guanine and cytosine and those that don’t. This involves genetic testing of the different bacterial species. High levels of guanine and cytosine in bacteria are called high GC bacteria, while others are low GC bacteria. Actinobacteria and, in particular, actinomyces, are a diverse group of gram-positive bacteria that share the fact that they are high GC bacteria. Most are soil organisms,

although some live in water. There are a few that reside in the human mouth and cause things like oral abscesses and periodontitis. Most of these bacteria are aerobic. Actinobacteria include Mycobacteria. Mycobacteria are gram-positive organisms that are specifically acid-fast because of their mycolic acid coating. Mycobacterial organisms are causative of tuberculosis and leprosy or Hansen’s disease. Mycobacterium tuberculosis infects up to a third of the population of the world. Corynebacterium is a species of Actinobacteria that have diaminopimelic acid as part of their cell walls. The form palisades microscopically that look like the letter V. Most are not pathogenic; however, Corynebacterium diphtheria makes a toxin that results in the development of diphtheria. Bifidobacterium are filamentous and live in the gut and vagina. These are found in probiotics and are part of yogurt-making. Gardnerella vaginalis is inconsistently Gram-positive and is found in the vagina in humans. Other Actinobacteria of clinical significance are Nocardia and Propionibacterium. The low GC bacteria have less than half of their genome containing guanine and cytosine. The largest class of these is Clostridia of which the one most studied is Clostridium. These are rod-shaped anaerobes that make endospores. They are found in soil and cause several human diseases. These include gas gangrene from C. perfringens, botulism from C. botulinum, and pseudomembranous colitis from C. difficile. These are similar in that they make toxins leading to their disease states. Another classification is called Lactobacillales, which includes Streptococcus, Enterococcus, and Lactobacillus. Streptococcus causes many diseases, including pneumonia and strep throat. Bacilli are generally rod-shaped but a few are round. The two that are the most important to human diseases are Bacillus and Staphylococcus. Bacillus organisms make endospores. Bacillus anthracis causes anthrax, while Bacillus cereus causes food poisoning. Staphylococcus can be found on the skin as S. epidermidis, while S. aureus causes several diseases, including abscesses. Mycoplasma is a low GC bacterium that does not have a cell wall so, of course, it cannot be gram-positive. These are very small and pleomorphic so they can be a variety of shapes. The most notable species in human disease is M. pneumoniae, which causes atypical or “walking” pneumonia.

ARCHAEA Archaea are prokaryotic but distinct from bacteria in several ways. Like bacteria, they are unicellular. They have different linkages in their cell membranes than bacteria, and their cell wall makes pseudopeptidoglycan and not peptidoglycan. They have more complex and larger genomes than bacteria. Some are mesophiles and live in temperate habitats, while others are extremophiles that live in extreme habitats. As a rule, this is an extremely diverse group of organisms. None of them are pathogenic. The Crenarchaeota is a classification of archaea that live in aquatic environments. In fact, they are the predominant organisms in the ocean. Most will grow in high temperatures. Some will grow in acidic environments. Others will oxidize sulfur in order to make sulfuric acid granules. These are likely very ancient organisms evolutionarily speaking. Euryarchaeota have several types of methanogens that make methane out of carbon dioxide. They will live in extreme environments of both high and low temperatures. Others will make hydrogen sulfide or marsh gas. There are those that prefer extremely salty environments called Halobacteria, even though they are not bacteria. A few will undergo photosynthesis.

DEEPLY BRANCHING BACTERIA There are organisms referred to as deeply branching bacteria, not because they are actually branching but because they are extremely ancient from an evolutionary aspect. Some genera are felt to be one of the common roots of living things that exist today. All other organisms—bacteria, archaea, and Eukarya have originated from species considered to be deeply branching bacteria. It is believed that some of these organisms were hyperthermophiles and thermophiles, which survived in very high temperatures. The genus Acetothermus is believed to be highly related to these ancient organisms.

KEY TAKEAWAYS •

Prokaryotes can live in just about any habitat on earth.

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Prokaryotes are all unicellular but can form different arrangements.

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Gram-negative bacteria are divided into proteobacteria and non-proteobacteria.

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Gram-positive bacteria are divided into high GC bacteria and low GC bacteria.

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Archaea are unicellular. Many prefer very extreme environments.

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Deeply branching bacteria are considered a common ancestor to all other living things throughout evolution.

QUIZ 1. From what source do carbon-fixing bacteria get their organic carbon from? a. Carbohydrates b. Protein c. Carbon dioxide d. Hydrocarbons Answer: c. Carbon-fixing bacteria can make use of the carbon found in carbon dioxide in order to make organic carbon-containing molecules. 2. What does the rhizobium species of bacteria make out of the nitrogen in the air? a. Proteins b. Ammonia c. Amino acids d. Small peptides Answer: b. Rhizobium can fix the nitrogen from the air so that they can make ammonia that is further turned into higher-order nitrogenous compounds by plant species the bacteria live near. 3. The phylum of protobacteria are actually subtypes of what kind of bacterium? a. Gram positive bacteria b. Gram negative bacteria c. Deeply branching bacteria d. Atypical bacteria Answer: b. Protobacteria are a phylum that represents some types of gram-negative bacteria.

4. Which organism is an obligate intracellular pathogen that leads to Rocky Mountain spotted fever? a. Chlamydia b. Clostridium c. Bordetella d. Rickettsia Answer: d. Rickettsia is the genus of an organism that is an obligate intracellular pathogen because it cannot make its own ATP energy. One species of Rickettsia leads to Rocky Mountain spotted fever. 5. Which type of proteobacteria include myxobacteria that can make metabolically inactive myxospores as part of their life cycle? a. Alpha-proteobacteria b. Beta-proteobacteria c. Gamma-proteobacteria d. Delta-proteobacteria Answer: d. Delta-proteobacteria are a small group of proteobacteria that include myxobacteria, which can make metabolically inactive myxospores when necessary. 6. Which type of proteobacteria include campylobacter and helicobacter, both of which cause gastrointestinal diseases in humans? a. Beta-proteobacteria b. Gamma-proteobacteria c. Delta-proteobacteria d. Epsilon-proteobacteria Answer: d. The classification of epsilon-proteobacteria is small but includes both campylobacter and helicobacter species, which lead to certain types of gastrointestinal disorders.

7. Which gram-negative organism reproduces via budding rather than through binary fission? a. Planctomycetes b. Borrelia c. Spirochetes d. Fusobacterium Answer: a. The organisms of the Planctomycetes classification are unique because they reproduce through budding rather than through binary fission like other bacteria. 8. What is a characteristic feature of phototrophic bacteria? a. They contain chlorophyll b. They live in aquatic environments c. They make oxygen d. They get their energy from the sun Answer: d. The defining feature of phototrophic bacteria is that they all participate in some form of photosynthesis and get their energy from the sun. 9. What disease is least likely to be caused by a species in the genus Clostridium? a. Botulism b. Pseudomembranous colitis c. Gas gangrene d. Pneumonia Answer: d. Each of these diseases is specifically caused by a member of the genus Clostridium, except that there aren’t any of these that cause pneumonia.

10. What is the most similar aspect that define both archaea and bacterial organisms? a. They have similar cell walls b. They are both unicellular c. They have similar cell membranes d. They have similar genomes Answer: b. Each of these is a big difference between bacteria and archaea except that both types of organisms are considered unicellular.

CHAPTER FIVE: TYPES OF EUKARYOTIC CELLS While there are many different kinds of eukaryotes, this chapter focuses mainly on eukaryotic cells that qualify as microorganisms. These include unicellular pathogens that are also eukaryotic like protists, helminths, and fungi. Algae and lichens are not pathogenic but are still important microbiological organisms covered in this chapter.

UNICELLULAR EUKARYOTIC PARASITES Because eukaryotes are by nature diverse, the same can be said about unicellular eukaryotic parasites. Each of them is different morphologically, has different life cycles, and involve unique nutritional needs. Some of these are pathogenic, such as the organisms that cause giardiasis, malaria, and other tropical diseases. We’ve already talked a bit about protists, which is not a formal taxonomic group. There are those that are plant-like, those that are, fungus-like, and those similar to animals. Protozoans are a large group that is non-photosynthetic, involves strictly unicellular organisms, and has motile organisms. Planktons include phytoplankton, which are considered photosynthetic, and zooplankton, which are not photosynthetic but are motile. The term planktons, like protists, is a loose definition not used in taxonomic classification systems. Because protozoans are diverse, they can be seen on land and in water. There are those that live freely and those that are parasitic in at least one part of their life cycle. Trophozoites are what protozoans are called when in the growth and feeding part of their life cycle. Some stay this way, while others form cysts in what’s called encystment. The opposite of encystment is called excystment. This is when the cyst becomes a trophozoite. An example of a protozoan life cycle is one of the Eimeria protozoan. There is a trophozoite stage that undergoes asexual reproduction. This produces merozoites, which can be male or female. These merozoites undergo what’s called sexual syngamy that makes an oocyst in the feces of the host. It grows into an unsporulated oocyst that 73

undergoes sporulation. This is infectious and enters the host. The oocyst releases sporocysts that undergoes excystment to become sporozoites that grow to become trophozoites, starting the life cycle all over again. As you can see, the reproductive aspects of the protozoan can be complicated. Some will only reproduce sexually, while others will only reproduce asexually. Others, like Eimeria, will be both asexual and sexual in their reproductive capabilities. Asexual reproduction occurs one of three ways. These are budding, binary fission, and schizogony. With schizogony, the nucleus will divide many times before the smaller cells are split off. The end products are called merozoites, which are stored in schizonts all together. Sexual reproduction is called syngamy. Conjugation, which is the transfer of DNA from one cell to another, can also occur. Ciliates are a group of protozoans that engage in conjugation, which is actually sexual in nature. The plasma membrane or plasmalemma of the protozoan can have a pellicle, which is a structure of the organism called by protein bands inside the plasma membrane. The cytoplasm can be compartmentalized into ectoplasm, which is an outer gel layer, and an endoplasm, which is an inner fluid layer. There can be specialized feeding structures associated with certain protozoans. The cytostome is a feeding structure that can phagocytize nutrients. The cytoproct is another structure than specializes in the exocytosis of waste products. There are holozoic forms that involve those protozoans that ingest whole food particles and saprozoic forms that only digest small or soluble nutrients. Motility in protozoans can be accomplished with flagella, which are long and whip-like, or cilia, which are large in number, short, and hair-like. We’ve already talked a bit about those that use pseudopodia to both attach to surfaces and for forward propulsion. Some organisms will have specialized contractile vacuoles in their cytoplasm that regulate salt and water balance. Others will have kinetoplastids or hydrogenosomes instead of mitochondria. Figure 21 shows the inner structure of a paramecium, in which the contractile vacuoles can be seen:

Figure 21.

Protists are not a taxonomic group because they contain organisms that are not related to one another evolutionarily speaking. The subgroups that are related to one another include the Amoebozoa, the Chromalveolata, and the Excavata. These may have clinical significance when it comes to human diseases. Clinically-relevant groups of eukaryotic microbes include Giardia lamblia, which causes giardiasis, Trichomonas, which causes trichomonas, Leishmania, which causes Leishmaniasis, and Trypanosoma, which causes both Chagas disease and African sleeping sickness. These are all from the Excavata supergroup. Others that are clinically relevant include the dinoflagellates, which cause red types or paralytic shellfish poisoning and others in the Chromalveolata group, such as Plasmodium, which causes malaria, Toxoplasma, which causes toxoplasmosis, Theileria, which causes babesiosis, and Cryptosporidium, which causes cryptosporidiosis. Among the Amoebozoa is Entamoeba, which leads to Amoebiasis. Fungal organisms are also unicellular eukaryotic organisms in some cases. One of these is Ascomycetes, which causes candidiasis. Basidiomycetes are fungal organisms that cause cryptococcosis and Microsporidia cause microsporidiosis. Among animals species

that are parasitic and pathogenic are Cestoda, which causes tapeworm infestations, Nematoda, which includes organisms that cause trichinosis, hookworm infections, and pinworm infections, and Trematoda, which leads to schistosomiasis. The organisms in the Amoebozoa category use amoeboid locomotion. There are actin monofilaments within the pseudopodia, which determine movement. The clinically relevant organisms are the Entamoeba, which includes Entamoeba histolytica, which makes cysts that are transmitted into the feces, leading to amoebic dysentery. There is a brain-eating variety that can cause a severe form of encephalitis. Acanthamoeba is a group that can cause blindness from keratitis. Slime molds are in the Amoebozoa supergroup. There are cellular varieties and plasmodial varieties. The cellular varieties only sometimes aggregate and form fruiting bodies, while the plasmodial slime molds exist in large multinucleated groups. They also produce reproductive organs that make spores. Figure 22 shows the fruiting bodies of a cellular slime mold:

Figure 22.

The Chromalveolata supergroup involves diatoms, ciliates, apicomplexans, and dinoflagellates, among others. The apicomplexans are parasitic and have a specific apical complex on one end of the cell. This is where organelles, microtubules, and vacuoles aggregate and allow the parasite to infect the host cells. Their life cycle is complex, involving things like schizogony and merozoites. They can infect livestock, humans, and insects, among other animals. Plasmodium is one of these. Figure 23 shows the apical tip of the plasmodium organism:

Figure 23.

Plasmodium organisms cause malaria but there are others of this supergroup that cause disease. Cryptosporidium parvum contaminates drinking water as cysts and causes epidemic diarrhea. The Theileria organism is also referred to as Babesia and causes babesiosis after a tick vector bites a victim. Toxoplasma is found in cat feces and in unwashed fruit or uncooked meat, leading to toxoplasmosis, which is dangerous to fetuses in the womb. The ciliates are known for their cilia and are part of the Chromalveolata supergroup. Cilia can be used for either motility or for feeding. Balantidium coli is the only pathogenic organism; it causes gastroenteritis. Paramecium has been extensively studied. It has a small micronucleus that is used for sexual conjugation and a macronucleus that can be polyploid having multiple sets of chromosomes. They can reproduce through conjugation, in which two cells connect and undergo meiosis to produce haploid nuclei, most of which disintegrate, leading to one haploid nucleus that divides and is exchanged between the nuclei. The oomycetes are similar to fungi but are actually protozoans. They have cellulose as part of their cell walls and include the organism called Phytophthora, which is a plant pathogen that led once to the Irish potato famine. The Excavata supergroup has many pathogens. They include Giardia lamblia that causes gastroenteritis from fecal contamination of water. Trichomonas vaginalis leads to vaginal trichomoniasis. It can lead to pregnancy complications. There are also Euglenozoa that include Euglena, which are not pathogenic. Euglena have two flagella and an eyespot called a stigma that senses light. It undergoes photosynthesis and has a pellicle, that supports the cell membrane. Those Euglenozoa that are pathogenic include Trypanosoma, which leads to African trypanosomiasis and American trypanosomiasis, which is also called Chagas disease. African trypanosomiasis is very severe and can lead to mental confusion, insomnia, and death if not treated. Leishmania is in this group as well and can cause leishmaniasis.

FUNGI Fungi are generally saprophytic, meaning they thrive on decay. They are included in the discussion of microbiology because there are many that are microscopic in nature. They lead to mycoses or fungal diseases in humans. Some are strictly opportunistic because they infect only the immunosuppressed human host. Remember that a major feature is a chitin-containing cell wall. There are molds that are multicellular and made from hyphae, which are filaments. Multiple entangled hyphae are called a mycelium. There are those that have walls separating them, called septate hyphae and those that do not have septa, called coenocytic. Pseudohyphae are short chains of daughter cells that have budded off of a budding yeast. Figure 24 depicts the different types of hyphae:

Figure 24.

Dimorphic fungi have more than one morphological appearance, depending on their life cycle. They can be either yeasts or molds and change morphology based on the environment. Histoplasma capsulatum and Candida albicans are two fungal organisms considered to be dimorphic. Histoplasma causes the lung disease called histoplasmosis, while Candida species result in yeast infections. Histoplasma makes hyphae in the environment but are unicellular yeasts in the human body. Fungi reproduce sexually or asexually, through self-fertilization or cross-fertilization. Most of their life cycle is haploid instead of diploid. Spores can be formed that can germinate to begin the haploid mycelial stage. Some groups form zygospores; others will produce basidiospores; and others will form ascospores, depending on their taxonomy. There are seven major groupings of fungi but not all of these are pathogenic. Some will only be pathogenic in plants, causing smuts and plant spores. These can affect the food supply. Others are symbiotic with plant roots, including those that can only survive this way. The Chytrids are small types of fungi that live in water and have motile gametes. They infect amphibians and have threatened their survival. There are four groups that are important in human illnesses, including Ascomycota, Zygomycota, Microsporidia, and Basidiomycota. Zygomycota will make sporangiospores for asexual reproduction and zygospores for sexual reproduction. There is one genus called Rhizopus, which leads to bread mold and Mucor, which is a genus that can cause severe necrotizing human infections. Ascomycota include the morel mushrooms, truffles, and other edible mushrooms. Some will spoil food. They make cup-shaped fruiting bodies called ascocarps. There are sexual spores called ascospores and asexual spores called conidia. Besides bread molds, some will cause human disease, particularly Aspergillus, which makes an aflatoxin when it contaminates grains or nuts. Aflatoxin can cause cancer. Penicillium is what makes penicillin. The ascomycetes an also cause the various skin infections, such as ringworm, jock itch, and athlete’s foot. Blastomyces can cause blastomycosis, which is a respiratory infection. Histoplasma is in this classification. Coccidioides leads to Valley fever and

Candida albicans causes common yeast infections. Saccharomyces are Ascomycota yeast forms that help to brew beer. The basidiomycetes or Basidiomycota have club-shaped structures called basidia that make basidiospores through the budding process and have basidiocarps, which are fruiting bodies. These participate in decomposition. The main clinical organism is Cryptococcus neoformans, which causes lung diseases in the immunosuppressed human. Some poisonous mushrooms are in this category as well. The Microsporidia are strictly unicellular and must replicate inside a host cell. They do not have peroxisomes, centrioles, or mitochondria so they are obligate intracellular pathogens. They do have spores that have a polar tubule, which can pierce the host cell membrane so the fungus can get into the cell. There is one species that will infect the gallbladder, GI tract, and lungs.

HELMINTHS Helminths do not have to be parasitic and are generally not microscopic but their larvae and eggs will be microscopic. The two main classifications are Nematodes, which are roundworms, and Platyhelminthes, which are flatworms. Most have very complex life cycles with the ability to infect more than one host. Those that are monoecious have both male and female reproductive organs, while others are considered dioecious because they are either male or female but not both in the same organism. Nematodes are the roundworms, containing about 15,000 different species. They do not have segments. The largest nematode is Ascaris lumbricoides, which can be a meter in total length. It is more common in developing countries. The most common roundworm infection is Enterobius vermicularis or pinworms. Toxocariasis is a rarer infection that can be gotten from cats or dogs. Hookworm infections are also rare. Each of these primarily infects the GI tract. Trichinellosis or trichinosis is a nematode infection from Trichinella spiralis. It is notable because, when gotten into the system through undercooked meat, the infectious organisms in larval form can form cysts in the muscles as well as the GI tract.

Heartworm in dogs is related to this but is caused by another nematode that is transmitted via a mosquito vector. The Platyhelminths are flatworms. The flukes and tapeworms are pathogenic and parasitic, while the turbellarians are not pathogenic to humans. The flukes or trematodes have an oral sucker feature and are not segmented. They attach to the inner lining of the GI tract, liver, lungs, and blood vessels. They are notable for having multiple hosts. Figure 25 shows the liver fluke with its oral sucker:

Figure 25.

Schistosomiasis is one of the more severe flatworm or fluke diseases in the world. They are found mainly in freshwater snails and can burrow under the skin, where they migrate to several different organs. If untreated, there can be anemia, abdominal pain, fever, malnutrition, and later death. Tapeworms are segmented flatworms that can have suckers or hooks at their head region, which is also called a scolex. They attach to the small intestinal lining. Small proglottids are the segments that can detach, releasing eggs into the feces. There is a host that will ingest the eggs that become larvae, which then form cysticerci that are eaten by the definitive host, where they mature into the adult tapeworm. There are pork tapeworms and tapeworms spread with cattle as the intermediate host.

ALGAE Algae can be unicellular or multicellular. There are those that are not pathogenic but are very important to the environment, such as diatoms, brown algae, red algae, dinoflagellates, and green algae because they make oxygen to a large extent and make up a great amount of the earth’s organic matter. Algae are responsible for making agar and carrageenan, used to solidify and thicken foods. Some are only harmful because they produce toxins when they come from algal blooms, which can cause nervous system and liver damage in humans and water-dwelling animals. Algae will store starches in pyrenoids within their chloroplasts as part of their metabolism. A few will have chloroplasts that have triple or quadruple membranes, depending on their evolutionary origin. Algae can be differently pigmented, depending on what pigments are in them. Seaweed is a form of algae and are not plants, even though they are macroscopic and look like plants. Algae are divided into two supergroups: the Chromalveolata and the Archaeplastida. The dinoflagellates are Chromalveolata and make up plankton. Some are phototrophic, while others are heterotrophic or mixed in how they get their energy. Dinoflagellates whirl because of their two flagella. Some can have an outer plate called a theca made from cellulose. Some are neurotoxic to fish or humans. Red tides come from overgrowth of dinoflagellates. There are stramenopiles that are what make up golden algae, brown algae, and diatoms. These are photosynthetic. The diatoms have an outer skeleton made from crystallized silica, which goes on to make diatomaceous earth. Some diatoms will contribute to toxic algal blooms. Giant kelp organisms are considered brown algae; they have holdfasts that attach them to substrates. Green and red algae are similar to land plants but are not plants. Chlorella are small unicellular algae.

LICHENS Lichens are never pathogenic but are important to the ecosystem. They are not a single organism but are a combination of two organisms—a relationship between green algae and cyanobacteria. They break down rock to make soil and some will fix nitrogen and stabilize soil. There are antibacterial properties to some lichens that make them medically interesting. The relationship between fungi and lichens is partly mutualistic and partly parasitic. It is partly parasitic because the photosynthetic component does not grow as well with the fungus but it can still grow. Lichens themselves grow slowly and may survive for centuries. They can be used as a food source or to make dyes. They are environmental indicators because they die off if there is too much pollution. The thallus or body of the lichen has an outer cortex made of tightly packed fungi and a medulla made of loosely packed fungi. Rhizines are the hyphal bundles that attach the organisms to the substrate. Figure 26 shows what a lichen looks like:

Figure 26.

Together, lichens are considered fungi. There are crustose lichens that are crusty in appearance, foliose lichens, which are leaf-like in appearance, and fruticose lichens, which have a more rounded appearance and can look branched.

KEY TAKEAWAYS •

Protozoans are a classification of eukaryotes that are very diverse.

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Many microbial protozoans have both asexual and sexual reproductive capabilities.

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Protozoans can be pathogenic or nonpathogenic.

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Fungi can also be pathogenic or nonpathogenic. They can form yeasts or hyphae and have chitin as part of their cell walls.

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Helminths are divided into roundworms or flatworms. Flatworms can be segmented or nonsegmented.

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Many helminths will have more than one type of host.

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Algae can be dangerous to humans because they create toxic algal blooms in bodies of water.

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Lichens are made from two different organisms that mostly work mutualistically. They are not pathogenic.

QUIZ 1. What is not a characteristic of protozoans? a. They are motile b. They do not participate in photosynthesis c. They are unicellular d. They product toxins Answer: d. Each of these is a typical characteristic of protozoans except that they do not generally produce any toxins. 2. When talking about protozoans, what is a trophozoite? a. A protozoan that is metabolically inactive. b. A protozoan in the growth and feeding phase. c. The larval form of a larger protozoan. d. The protozoan that is encased in a cyst. Answer: b. A protozoan that is in its growth and feeding phase is referred to as a trophozoite. When it undergoes encystment, it becomes a metabolically inactive cyst. 3. What is the main purpose of the cytoproct in certain types of protozoans? a. Immunity b. Motility c. Cell structure d. Waste removal Answer: d. The cytoproct is a specialized structure with regard to certain protozoans that is involved in the exocytosis of waste products for the organism.

4. What is the contractile vacuole used for in protozoans such as paramecium? a. Salt and water balance b. Cell structure c. Compartmentalization of the cell d. Buoyancy Answer: a. The contractile vacuole is a structure inside certain protozoal cells that are effective in water and salt balance. 5. What is the major characteristic of plasmodial slime molds? a. They cause toxic blooms in the ocean b. They only have asexual reproduction. c. They do not aggregate d. They form multinucleated aggregates Answer; d. A major characteristic of plasmodial slime molds is that they form multinucleated aggregates. The organisms undergo sexual reproduction. 6. Which of the Chromalveolata supergroup is known to cause malaria? a. Theileria b. Babesia c. Plasmodium d. Toxoplasma Answer: c. Plasmodium falciparum causes malaria after going through a complex life cycle that includes an insect vector. The others are of the same supergroup but cause different diseases in humans.

7. Which classification of fungal organisms leads to things like yeast infections and various skin-related fungal infections? a. Zygomycota b. Basidiomycota c. Ascomycota d. Microsporidia Answer: c. Ascomycota is the classification of fungal organisms that cause many different human diseases, including common yeast infections and dermatomycoses or skin infections. 8. What part of the human body is most affected by cryptococcosis, histoplasmosis, and Coccidiomycosis? a. Brain b. Lungs c. GI tract d. Skin Answer: b. These organisms are notable for causing lung infections and many will cause these types of infections only in the immunosuppressed patient. 9. Which type of helminth parasite has proglottids that release eggs into the feces of the definitive host? a. Tapeworms b. Pinworms c. Liver flukes d. Trichinella Answer: a. Tapeworms are segmented, with segments that are called proglottids. The proglottids release eggs into the environment that are then eaten by an intermediate host.

10. What aspect of algae can be pathogenic or dangerous to humans? a. They can infect the Gi tract, leading to gastroenteritis. b. They can make toxins in algal blooms that can be toxic. c. They can cause dermatoses or skin infections. d. They can get into the lungs, leading to lung infections. Answer: b. The most pathogenic part of algae is their ability to make toxins in algal blooms that are toxic to humans and aquatic animals.

CHAPTER SIX: THE BIOCHEMISTRY OF MICROBIOLOGY This chapter teaches you the biochemistry you need to know in order to study microbiology. All living things are basically made structurally of organic molecules and the interactions between the molecules is strictly biochemical in nature. For this reason, you need to understand what the different organic molecules are in living things. Nucleic acids are studied in another chapter but carbohydrates, lipids, and proteins are part of this chapter. The way biochemistry helps in understanding microbiology is also covered in the chapter.

ORGANIC MOLECULES Organic molecules are those that define and create live; however, not all organic molecules do this. An example of this is most hydrocarbons, such as pentane and octane, which are organic but not biochemical. A better definition of organic chemistry is that it is carbon-based. All organic molecules have some type of carbon molecule associate with it. The principles of biochemistry and organic chemistry are the same, however. Atom for atom, the most prevalent element in cells is hydrogen, followed by carbon, oxygen, nitrogen, phosphorus, and sulfur. These are the macronutrients, containing 99 percent of the cell’s dry weight. Other elements are considered micronutrients or trace elements, such as sodium, magnesium, potassium, zinc, calcium, iron, copper, manganese, and molybdenum. These are crucial to cellular function but are found in smaller amounts in the cell. The elements of carbon, nitrogen, oxygen, and hydrogen have a small atomic weight and form covalent bonds with one another. There are different types of bonds in chemistry, with covalent bonds being the tightest and most cohesive. Electrons between two atoms in a covalent bond are roughly shared between the atoms. Other bonds that can happen in chemistry are ionic bonds, van der Waals bonding, hydrogen bonding, and metallic 90

bonding. These bonds are easier to break than covalent bonding. Figure 27 shows some of these types of bonds:

Figure 27.

There are inorganic molecules in living things as well. These include various kinds of salts, oxygen, and water. The biggest difference is that inorganic molecules do not contain carbon. There are some carbon-based molecules that are not organic. This include things like carbonates and CO2. They contain carbon but do not have hydrogen in them. Most organic molecules have chains of carbon atoms linked together. In general, organic molecules are larger than inorganic molecules. Molecules that make up life are called biomolecules. Carbon is a good molecule for organic molecules because it has four valence electrons—each of which is capable of making a covalent bond with another atom. This adds to the diversity of organic molecules. Look for some 91

type of carbon skeleton or backbone in all biochemical and organic molecules. The simplest organic molecule would be methane, which is just one carbon atom and four hydrogen atoms. Figure 28 shows the structure of methane:

Figure 28.

Because of the complexity of organic molecules, there can be isomers of different molecules. An isomer is a molecule that has the same number of atoms in it but has different structures. Compounds with completely different chemical structures are referred to as structural isomers. Figure 29 shows structural isomers:

Figure 29.

In biochemistry, simple sugars are considered structural isomers of one another in some cases. An example of this is galactose, glucose, and fructose. Each of these is a sixcarbon sugar that comes in its own unique structural shape. There are some isomers that have the exact structure of another but different special arrangements of their atoms. These are called stereoisomers. Enantiomers are an example of a stereoisomer and have chirality. It means that they are mirror images of one another. There can be left-handed or right-handed enantiomers, which respond differently from a biochemical perspective. Left-handed molecules are referred to as L-enantiomers, while right-handed molecules are referred to as D-enantiomers. These differentiations are important in pharmacology because generally, the different enantiomers have different pharmacological properties. Enantiomers can be called optical isomers because of the way they are able to rotate a plane of light that is polarized. Molecules in nature will often have a specific functional group, which is where chemical activity takes place and is what defines the different molecular properties of the molecule. Some functional groups are simple, while others are complex. You do not need to know these functional groups specifically for microbiology unless you are studying biochemistry as well. Some of these groups include ketones, carboxylic acids, aldehydes, and amide functional groups. Functional groups will combine with a carbon chain in order to make a biomolecule. Most of the biomolecules in nature are macromolecules because of their large size. Those that are linked together are called polymers. Polymers are made from simpler biomolecules called monomers. An example of a monomer is glucose, which can make several different polymers, such as starch and glycogen. The different biomolecules in nature are proteins, carbohydrates or polysaccharides, lipids, and nucleic acids. As mentioned, this chapter will not cover nucleic acids as these are covered later. When a polymer is made from a monomer, much of the time, this is referred to as a dehydration reaction, because one of the end products is water. In a sense, water is taken out of the monomers to make a polymer.

Each type of macromolecule has a different function. In looking at the different macromolecules, some generalizations can be made. These are the main characteristics of macromolecules in nature: •

Nucleic acid—this molecule makes genetic information or participates in its transfer to make proteins.

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Carbohydrate—these will be energy sources but will also form receptors, cell walls, and exoskeletons, and are involved as a food source.

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Peptides or proteins—these can make enzymes and will be involved in cell structures, receptors, and in nutrient transport.

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Lipids—these will store energy and make up membrane structures, pigments, and insulation in the organism.

LIPIDS The two main atoms in lipids are carbon and hydrogen but lipids can also have nitrogen, oxygen, phosphorus, and sulfur in them. They provide an energy source for cells, help to store carbon atoms, and are the main structural component of the cell membrane. There are different types of lipid molecules you might encounter in your study of microbiology. Fatty acids are common lipids. They contain long hydrocarbon chains that are hydrophobic because they do not like water and are not soluble in water. Fatty acids that do not have any double bonds but are considered saturated with hydrogen atoms are saturated fatty acids. Fatty acids with one or more double bonds are called unsaturated. There are monounsaturated fatty acids with one double bond and polyunsaturated fatty acids with more than one double bond. Cis-fatty acids are shaped differently than trans fatty acids. Figure 30 shows the different fatty acids:

Figure 30.

A triglyceride or triacylglycerol is what the main storage form of lipids are found in. Body fat and the fat in sebaceous or oil glands of the skin are triglycerides. They consist of three fatty acid side chains attached to a three-carbon molecule called glycerol. Between the glycerol and fatty acids are aldehyde linkages. Figure 31 shows a triglyceride molecule:

Figure 31.

While triglycerides are simple types of lipids, there are lipids that are more complex. Phospholipids have a polar phosphate group attached to them that make up most of the cell membrane. Another complex lipid is a glycolipid, which has some type of carbohydrate attached to it. Lipids are largely hydrophobic, meaning that they do not dissolve well in water. For things like phospholipids, which have a polar end, the polar phosphate group will interact with water but the hydrocarbon chains will cluster together away from water. This explains why cell membranes are lipid bilayers that have a hydrocarbon core and a polar exposed surface. Those molecules that have hydrophobic and hydrophilic components are called amphipathic. Lipids can form micelles, which are spherical and contain a completely hydrophobic core along with a hydrophilic outer component. Micelles, by nature of their structure, are mainly seen in phospholipids because of their amphipathic nature. Larger than micelles are lipid bilayers, which are hydrophilic on the inside of the sphere and 96

hydrophilic on the outside of the sphere. The hydrophobic part is between the two layers. Figure 32 shows a micelle formed by a phospholipid:

Figure 32.

Isoprenoids are lipids that are branched. Their name comes from the fact that they come from the isoprene molecule. They are used to make pigments and pharmaceuticals like beta carotene and capsaicin. Fragrances are also isoprenoids and longer chain isoprenoids are used to make waxes. Sebum in humans is a wax in the skin; this wax is used as food by certain skin bacteria. Steroids are ringed lipids found to be part of cell membranes. Most steroids in cells are sterols, which are steroids that contain a hydroxyl group. These are largely hydrophobic but the hydroxyl OH groups or alcohol groups are hydrophilic. Cholesterol is the most common sterol in animal tissues; it allows for strengthening of the cell membrane. Prokaryotes don’t make cholesterol but they do make hopanoids, which are similar. Fungi do not make cholesterol either but will make ergosterol.

PROTEINS Proteins are amino acid in a chain, in which small or large amino acids are bonded together to make different sizes of peptides. Short peptides are called oligopeptides, while long peptides are called polypeptides or proteins. Proteins are largely structural aspects of the cells, but some an be hormones, receptor molecules, transport molecules, and enzymes. Amino acids are organic molecules with two specific groups: a carboxyl COOH group and an amino group or NH2 group. These are attached to what’s called the alpha carbon. Added also to this carbon atom is a side chain, which differs according to the amino acid. The simplest side chain is just hydrogen molecules attached to the alpha carbon. Figure 33 shows the different amino acids found in nature:

Figure 33.

Amino acids attach to one another through a peptide bond. Other bonds that help to shape the protein are disulfide bonds, ionic bonds, and hydrogen bonds. There are also polar and nonpolar aspects to each peptide chain that will attract like amino acids to one another. This is why proteins have three-dimensional shapes instead of linear shapes. The formation of a peptide bond is one that is a dehydration synthetic reaction. This means that water is made to form the bond. Dipeptides and tripeptides have two and three amino acids attached to one another, respectively. Oligopeptides have a small number of amino acids—in the range of 20 amino acids. Polypeptides have about fifty amino acids. Proteins are very long and have the ability to be a subunit that combines with other protein subunits. Figure 34 shows what a peptide bond formation looks like:

Figure 34.

Proteins are categorized by specific aspects of their structure. The primary structure of a protein is the arrangement of their amino acids in the sequence. Because of interaction and bonding strategies in different proteins, they will have a secondary structure. This happens because of hydrogen bonds in the amino and carboxyl groups. Some will become helical and some will form sheets. The tertiary structure is what happens between the side chains. Disulfide bridges will occur between certain amino acids in some cases. In other cases, there will be ionic or hydrogen bonding. This gives the 3D shape of the protein. The quaternary structure is the interactions that happen between two different peptides. Conjugated proteins are those that have some type of non-protein component. If, for example, the peptide is attached to a carbohydrate, it is referred to as a glycoprotein. If the peptide is attached to a lipid, it is called a lipoprotein. These are particularly 99

important when it comes to proteins that are affiliated with the cell membrane. Figure 35 indicates the different types of proteins structures:

Figure 35.

CARBOHYDRATES Carbohydrates are the most abundant molecules on the planet. They provide the world with carbon and water. Carbohydrates always contain carbon, hydrogen, and water. There are a few that are attached to nitrogen, sulfur, or phosphorus but this is uncommon. They are an energy source, are used in certain cell membrane receptors, and are a part of what makes a nucleic acid. Polymers of carbohydrates will make things like glycogen for energy storage, chitin for cell wall formation and cellulose, which is also a cell wall component. Carbohydrates are also referred to as saccharides. Monosaccharides are simple sugars and are the monomers that make polysaccharide chains. Carbohydrates are also named with the suffix -ose. A triose has three carbon atoms; a tetrose has four carbon atoms; a pentose has five carbon atoms and a hexose has six carbon atoms. Hexoses such as glucose are extremely abundant in nature. Disaccharides are also common. These two-

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monomer molecules include things like sucrose, which is table sugar, and lactose, which is milk sugar. Figure 36 shows the sucrose molecule:

Figure 36.

Monosaccharides that have at least four carbon atoms are generally more stable as cyclic molecules. There is a chemical reaction between two functional groups at each end of the molecule so that the molecule become cyclic. Glucose, galactose, and other hexoses prefer to be cyclic in general.

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Polysaccharides are referred to as glycans and have hundreds of monosaccharides all linked together. They are not water soluble and do not taste sweet. The main polysaccharides in nature include cellulose, glycogen, and starch. All are made from strings of glucose linked together in different ways. Glycogen is used for energy storage in bacteria and in animals. Starch is also used for energy storage in plants. Cellulose is a structural molecule used to make cell walls.

BIOCHEMICAL PRINCIPLES IN MICROBIOLOGY The biochemistry of different microorganisms will be unique to the organism. For this reason, biochemical testing of bacteria can help to identify the bacterial species. There are some organisms that can be identified through their phenotype, while others must be identified by their genotype or by their different types of metabolism. There are, for example, different granules inside certain organisms that can be specific to the organism. An example is Pseudomonas aeruginosa, which will fluoresce under the microscope when their storage granules are stained with specific stains for their granules. Another technique is to use biochemistry to identify lipid profiles. Their fatty acids can be unique to the organism. Biochemists can use fatty acid methyl ester analysis to detect the type of fatty acids in the cell membrane using gas chromatography. A related technique for identifying a bacterium is phospholipid-derived fatty acid analysis, which will look at the type of fatty acids in the bacterial cell wall. Certain proteins can be unique to a microorganism so this can be used to identify the organism. The technique is called proteomic analysis, which breaks down the bacterial proteins and identifies the specific peptides that come from this using mass spectrometry. Glycoproteins can also be identifying features ot an organism. Antibodies can be made to attach to these glycoproteins in order to identify which glycoprotein is on the cell wall of the microorganisms. There are serological tests that can be done to identify streptococcus species by virtue of their specific carbohydrates on their cell wall.

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KEY TAKEAWAYS •

There are four types of biomolecules, including carbohydrates, proteins, lipids, and nucleic acids.

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Lipids can be simple fatty acids, phospholipids, and sterols, which have somewhat different properties.

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Peptides and proteins are made from combinations of amino acids. They have different structural features depending on what amino acids are in the peptide chain.

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The major polysaccharides in nature are glycogen, starch, and cellulose.

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Polysaccharides and disaccharides are made from glycosidic linkages between certain carbohydrate monomers.

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Biochemical techniques can be used to identify pathogens according to different characteristics inside the cell or on the cell membrane itself.

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QUIZ 1. Which organic molecule is not considered a biochemical molecule? a. Glucose b. Oligopeptide c. Octane d. Lipoic acid Answer: c. Each of these is representative of an organic molecule; however, hydrocarbons like octane are organic but not biochemical in nature because they do not exist in living things. Only methane is a hydrocarbon that exists in life. 2. What is the definition of an organic molecule? a. A molecule that is neither acidic nor basic. b. A molecule that contains carbon. c. A molecule found in living things. d. A molecule that is hydrophobic or water-hating. Answer: b. Organic molecules, by definition, contain carbon so these are carbon-based molecules. 3. Which of the following is considered a polymer rather than a monomer? a. Amino acid b. Glucose c. Glycogen d. Fructose Answer: c. Glycogen is a long chain of glucose molecules so it is a polymer rather than a monomer. The other choices represent single subunits or monomers.

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4. What needs to be an end product of a chemical reaction that is a dehydration synthetic reaction? a. Some type of monomer b. Water c. Carbon dioxide d. Some type of acid or base Answer: b. In a dehydration reaction in biochemistry, a polymer is often created and water is always one of the end products. Water is taken out of the monomers in order to make the polymer. 5. Which type of lipid has a ringed structure? a. Fatty acid b. Triglyceride c. Phospholipid d. Cholesterol Answer: d. The main difference between cholesterol and other lipids is that cholesterol has a ringed structure and the others are straightchained structures. 6. What part of the phospholipid molecule will most likely associate with water? a. Glycerol b. Phosphate c. Saturated fatty acid d. Unsaturated fatty acid Answer: b. The polar and most hydrophilic component of the phospholipid molecule is phosphate, which will be the part that interacts with water.

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7. What defines the amino group on all amino acids? a. NO group b. COOH group c. NHO group d. NH2 group Answer: d. All amino acids have an NH2 amino functional group as well as a COOH carboxyl functional group that together define what an amino acid is. 8. What type of bonding is not seen in peptides and proteins that give its shape? a. Disulfide bonds b. Hydrogen bonds c. Metallic bonds d. Ionic bonds Answer: c. Metallic bonds are not seen on proteins but the others are seen in a protein or peptide to give the molecule its three-dimensional shape. 9. What kind of carbohydrate is glucose? a. Pentose b. Hexose c. Tetrose d. Triose Answer: b. Glucose is a hexose because it has six carbon atoms in the molecule. Most of the common carbohydrate monomers are hexoses.

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10. What are glycogen and cellulose made of? a. Sucrose b. Galactose c. Glucose d. Fructose Answer: c. All of the major polysaccharides in nature are ultimately made from strings of glucose molecules.

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CHAPTER SEVEN: METABOLIC PROCESSES IN MICROBIOLOGY This chapter talks about cellular metabolism, which is how microbial organisms get their cellular energy. Most of this involves catabolism, which is the breakdown of certain molecules. How cells catabolize carbohydrates, lipids, and proteins is discussed in this chapter. Some organisms derive their energy from the sun. This is called photosynthesis, which is a part of this chapter. Finally, biogeochemical cycles are important to the environment so these are explained in the chapter.

ENZYMES AND CELLULAR ENERGY The chemical processes inside the cell are collectively known as metabolism. Molecules are broken down and synthesized all the time by means of chemical reactions. There are endergonic reactions that require some form of energy in order to happen and exergonic reactions that do not require energy but instead release energy. Anabolism relates best to endergonic reactions because they require energy, while catabolism refers to exergonic reactions, because they release energy. Anabolism is the making of a molecule, while catabolism is the breakdown of a molecule. Anabolism creates bonds, while catabolism breaks bonds. Carbon is what’s used for cellular metabolism. Organisms will differ in their source of carbon. Those organisms that get their carbon from carbon dioxide are called autotrophs, while those that get their carbon from existing organic molecules are called heterotrophs. Most organisms in nature are heterotrophs. Energy sources also differ. Phototrophs get energy from sunshine, while chemotrophs get their energy by breaking chemical bonds. Chemotrophs come in two separate categories. Organotrophs get their energy from organic molecules, while lithotrophs get their energy from inorganic compounds, like hydrogen sulfide and reduced iron. This leads to a specific classification system for microorganisms. Chemoautotrophs are those that get their energy from organic molecules and carbon from inorganic 108

molecules. Chemoheterotrophs are what animals are. They use chemical sources of energy and organic molecules as carbon sources. Photoautotrophs get energy from light and use inorganic molecules as carbon sources. Photoheterotrophs get energy from light and use organic compounds as animal sources. These include green and purple non-sulfur bacteria. Electrons within an atom are considered high energy. For this reason, when electrons are transferred from one molecule to another, this will involve energy. Oxidation reactions will remove electrons from a donor molecule, while reduction reactions will add electrons to a molecule. These reactions do not occur separately from one another. When they are used together, they are called oxidation-reduction reactions or simply “redox” reactions. There are several molecules that provide energy to biochemical reactions in nature. They store energy in the form of a chemical bond. The main energy currency in all cells is ATP, although there are others that can be used. These include NAD+, which is nicotinamide adenine dinucleotide, NADP+, which is nicotinamide adenine dinucleotide phosphate, and FAD, which is flavin adenine dinucleotide. These can be reduced or oxidized. Most of these secondary molecules are used in catabolic processes within the cell. Others are used in anabolism. ATP is interesting. The energy of this cell is provided by its phosphate bonds. Any time that a phosphate bond is broken, energy is released. There is AMP, which has one phosphate molecule, ADP, which has two phosphate molecules, and ATP, which has three phosphate molecules. Figure 37 shows the ATP molecule:

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Figure 37.

The process of breaking off one or more phosphate group from any molecule, including ATP, is called dephosphorylation, which generally releases energy. In living things, exergonic reactions are linked to endergonic reactions. As energy is released from glucose to make ATP, the energy is used to drive the endergonic reaction that actually makes the ATP. Most chemical reactions take place with some type of catalyst, which reduces the amount of energy necessary to help the reaction go. Enzymes are proteins in general that act as catalysts. They do not change the energy level of the reactants or products but simple change the activation energy necessary for the reaction to proceed. In a reaction, there are substrates, which are the beginning substances in a reaction. These will bind to specific actions sites on the enzymes in order to allow a reaction to happen. It used to be believed that the interaction was like a lock and key mechanism. Now it is believed that a conformational change takes place that helps the reactants or substrates become end products. The enzyme is not consumed as part of the reaction. Figure 38 shows how an enzyme works:

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Figure 38.

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Enzymes are highly sensitive to environmental influences. They require certain pH levels, temperatures, and substrate concentrations. Increasing temperature will generally help a reaction up to a point but, with enzymatic reactions, very high heat can denature the enzyme so that it doesn’t occur. The same is true of extremes in pH levels. Substrate concentration increases generally increase the reaction processes but there is a limit to how fast the enzyme will work so high concentrations of substrate do not necessarily increase the reaction rate. There are specific molecules, such as cofactors and coenzymes, that help a reaction to happen. Cofactors tend to be inorganic ions, while coenzymes are organic helper molecules that aid in the speed of the reaction. Some coenzymes are the dietary vitamins a person eats. An apoenzyme is an inactive enzyme that does not have its cofactor or coenzyme. A holoenzyme is an active and complete enzyme. Enzymes can be impacted in other ways. There can be competitive inhibition of an enzyme when a molecule similar to the substrate actually binds to the enzyme in order to block the enzyme’s activity. Some drugs work through competitive inhibition. Noncompetitive inhibition can also occur. This is also called allosteric inhibition. The inhibitor binds to another site besides the active site but change the ability of the enzyme to be active. Feedback inhibition involves the end product concentration to be high enough to block the activity of the enzyme that created it. Allosteric activators do the opposite of allosteric inhibition because they enhance the activity of the enzyme.

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CATABOLISM OF CARBOHYDRATES The catabolism of carbohydrates is very important to the making of ATP energy. These catabolic processes happen sequentially involving a series of reactions and many intermediates that turn simple carbs like glucose into carbon dioxide and water. Glycolysis is the first part of glucose metabolism. It occurs in eukaryotes, bacteria, and most archaea organisms. It provides some energy for the cell. The starting molecule for glycolysis will always be glucose. This process is anaerobic because it does not require oxygen. It can, however, be connected to reactions that do require oxygen at a later step in the process. The end product of glycolysis is called pyruvate, which is a three-carbon molecule. Pyruvate can be broken down further in subsequent reactions. Glycolysis usually involves what’s called the EMP pathway, which is also referred to as the Embden-Meyerhof-Parnas pathway. There are two phases. The first phase is the investment stage, which actually requires energy. The end product is glyceraldehyde-3phosphate. The second phase involves an energy payoff stage, in which more energy is produced than was invested. Four ATP molecules are made, which offsets the two ATP molecules used in the investment phase, for a total of two ATP molecules made using one glucose molecule. The ATP molecules mad are made through what’s called substrate level phosphorylation. This means that phosphate is removed from an organic molecule and is attached to an ADP molecule to make ATP. In the end of glycolysis, two ATO molecules and two NADH molecules are made. Pyruvate, as mentioned, is the end product. Two of these are made per glucose molecule. Figure 39 shows the glycolysis pathway:

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Figure 39.

Most organisms use the EMP pathway. There are a few that use slightly different pathways. The Entner-Doudoroff pathway is used by certain bacteria, such as Pseudomonas aeruginosa and some E. coli organisms, is an alternative pathway. Still another alternative pathway is called the pentose phosphate pathway. All cells will use this pathway as an ancient version of glycolysis. It uses intermediates that help to make nucleic acids and amino acids. All cells can use the pentose phosphate pathway when nucleic acids or amino acids are necessary.

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The pyruvate that is made can be further oxidized after glycolysis to make more energy. There is a transition reaction or bridge reaction that makes NADH from NAD+. A twocarbon molecule called acetyl is connected to a carrier molecule called coenzyme A. This compound then enters the Krebs cycle. The Krebs cycle is also called the citric acid cycle or the tricarboxylic acid cycle. This is a closed loop system. The cycle makes two carbon dioxide molecules, one ATP molecule or one GTP molecule—made through substrate level phosphorylation. Three molecules of NADH and one molecule of FADH2 are made, which become important later on. Oxygen is not required. The coenzyme A molecule is completely recycled. Figure 40 shows the Krebs cycle:

Figure 40.

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FERMENTATION Cells that are unable to undergo respiration may lack a final electron acceptor, may not have the genes to make the complexes necessary to undergo respiration, or may not have the genes to have a Krebs cycle. As you can see, some of these are geneticallyoriented, while others are related to the environment. Many prokaryotes just do not have the genetic makeup to participate in respiration, while others are considered facultative, meaning that they change their biochemistry depending on the environment. The cell’s only way to make ATP energy in some cases is glycolysis. The final electron acceptor is pyruvate, and the organism participates in fermentation. No other sources of ATP energy exist. Fermentation will be made use of in many human food processes that make fermented food, such as yogurt and beer. The fermentation that occurs in yogurt and related products as well as in human muscle tissue that is overextended is called lactic acid fermentation. Pyruvate plus NADH makes lactic acid and NAD+. There are bacteria that can do this. Homolactic fermentation involves only making lactic acid as an end product, while heterolactic fermentation involves the making of lactic acid, plus ethanol or acetic acid plus CO2. These pathways are used in cucumber and cabbage fermentation, which makes pickles or sauerkraut. Lactic acid fermentation is important in medical circles. It is the lactic acid bacteria in the vagina that make the pH of the vaginal milieu more acidic. This prevents things like yeast from proliferating. If these bacteria die off or are eliminated, then yeast forms can overgrow, leading to a yeast infection. This is why antibiotics can lead to yeast infections; they kill off healthy lactic acid-making bacteria. Alcohol fermentation makes ethanol as the end product. Pyruvate is the first substrate and both carbon dioxide gas and ethanol are end products. The yeast form called Saccharomyces cerevisiae is what makes bread rise and what makes alcoholic beverages. Other forms of fermentation include those that make acetone and butanol. Certain

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vitamins and penicillin are made from mixed acid fermentation. Other end products include hydrogen gas, propionic acid, and succinic acid.

CELLULAR RESPIRATION Most of the ATP generated by far in the various pathways of catabolism is made through what’s called oxidative phosphorylation, which is different from substrate level phosphorylation. The NADH and FADH2 made in glycolysis, the transition reaction, and the Krebs cycle will be useful in this process. Oxygen is the final electron acceptor in most cases, except in anaerobic respiration. It takes place on the inner membrane of the mitochondrion or on the inner part of the membrane in prokaryotes. The final component of respiration is the electron transport system. There are membrane complexes that participate in this process. Electrons from FADH2 and NADH get passed from component to component in the system until it reaches the endpoint, which is oxygen, which gets reduced to water. The reaction happens through cytochrome oxidase, which is different in the different organisms. These differences can help to detect the different organisms. Figure 41 shows the electron transport chain:

Figure 41.

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Aerobic respiration cannot take place if there are not the genes to make the different components, if there are not the genes to protect the cell from the oxygen free radicals that are made in oxidative phosphorylation, or if there is not enough oxygen. In anaerobic respiration, an inorganic molecule other than oxygen acts as the final electron receptor in the electron transport chain. This phenomenon is mainly seen in archaea and bacterial organisms. Some of these are those that use nitrate or nitrate, which are reduced to make nitrogen gas. The Krebs cycle is intact in these organisms but the electron transport chain is altered. These organisms are called denitrifiers. The electron in the electron transport chain gradually loses energy with each pass; however, some of this energy is stored by pumping hydrogen ions across a membrane, which can be outside the cell in prokaryotic organisms. When this happens, there is potential energy created, which is also referred to as the proton motive force. It is more acidic on the outside of the membrane than the inside of the membrane. This potential energy can drive certain reactions, such as those involved in flagella rotation and those involved in the intake of nutrients. The flow of hydrogen ions is called chemiosmosis. This force is made use of by an important enzyme called ATP synthase, which makes ATP. The flow of hydrogen ions back across the membrane drives the enzyme to make ATP. The actual number of ATP molecules made in the entire catabolic process varies. The average number is about 30 to 34 ATP molecules per glucose molecule. The maximum is about 38 molecules.

CATABOLISM OF PROTEINS AND LIPIDS Other nutrients can be catabolized besides glucose. What happens when there are lipids or proteins that need to be broken down? It turns out that there are mechanisms in place to metabolize these sorts of molecules. Most of them ultimately feed into the glycolysis, transition reaction, or Krebs cycle in some way so it can progress similarly to glucose at the end of the catabolic process. Triglycerides can be broken down and metabolized. There are lipases and phospholipases that start the process of degradation. Some organisms use these enzymes as part of their virulence because they can use the enzymes to catabolize the 118

cell membranes of the host organism. Glycerol and fatty acids come from triglycerides and get further degraded into two-carbon groups through beta-oxidation. When the two carbon molecules come off the triglyceride chain, FADH2 and NADH are made that go into the electron transport chain. The acetyl groups also made go into the Krebs cycle. Ultimately, CO2 is made from them and more ATP is made via the Krebs cycle. There are protease enzymes that help to break down proteins. Smaller peptides are taken up by the cell for further metabolism. Certain pathogens can be identified by their ability to break down extracellular proteins in differing ways. Peptides are further broken down into amino acids that get deaminated to remove the amino group. The carbon atoms can be metabolized and will break down into compounds that will enter the transition reaction or the Krebs cycle.

PHOTOSYNTHESIS Phototrophic organisms participate in photosynthesis, which takes sun energy and makes chemical energy. This process happens in plants but also happens in a number of microbial organisms. There are two phases to this process: the first is the lightdependent reactions, where energy from sunlight gets absorbed and turned into chemical energy. The second phase is light-independent phase does not depend on light but is responsible for making sugar from CO2. Light-dependent reactions make ATP, NADH, or NADPH in order to temporarily have a means for energy storage. In eukaryotic organisms, photosynthesis takes place in the chloroplast, which contains thylakoids that have pigments that capture light. One stack of thylakoids is called a granum, which is surrounded by stroma. Prokaryotes do not have chloroplasts but have infoldings of their cell membrane that contain photosynthetic pigments. The photosynthetic process involves light-harvesting complexes that get excited when light is absorbed by them. Light energy gets transported to a reaction center, which involves a pigment giving up an electron. Each microorganism has different pigments associated with it. This means that they can be red, purple, green, blue, orange, or yellow, depending on the pigment. The electron given up by the pigment in the reaction

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center goes to an electron transport chain that makes NADH or NADPH. ATP is also made through chemiosmosis, just as is done in oxidative phosphorylation. As we have talked about, there is oxygenic and anoxygenic photosynthesis, depending on the organism. When there is oxygenic photosynthesis, water is split to create the electron in the reaction center. Oxygen is released as a byproduct. In anoxygenic photosynthesis, hydrogen gas, hydrogen sulfide or thiosulfate are electron donors, making elemental sulfur or sulfate as byproducts. Light-independent reactions take the energy made in the light-dependent reactions to make carbohydrates. It mainly involves the Calvin-Benson cycle, which fixes CO2. Figure 42 shows the Calvin-Benson cycle:

Figure 42.

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The enzyme that participates in the Calvin-Benson cycle is called RuBisCO. It is the most abundant enzyme on the earth and is crucial for the fixation of carbon dioxide to make sugar. There are alternative cycles used to fix CO2 in certain green sulfur bacteria that are also photoautotrophic in nature.

BIOGEOCHEMICAL CYCLES Biogeochemical cycles are important in all ecosystems. The most common atoms used in organic compounds are carbon, hydrogen, oxygen, nitrogen, sulfur, and phosphorus. These are found in many forms in nature and will move from one aspect of nature to another with the different organisms contributing to this. Many of these atoms are recycled in what’s called a biogeochemical cycle. There are several of these cycles. The carbon cycle is very important. It connects all the different organisms in the world’s ecosystems. It is completely oxidized as CO2 and is reduced in organic molecules. Heterotrophs will produce CO2 and autotrophs will fix carbon. Autotrophs also participate in respiration and fermentation for their own specific metabolic needs. Certain archaea and bacteria will use methane as a carbon source. These are called methanotrophs. Methanogens are organisms that make methane as part of their fermentation process. This builds up in the ecosystem. Cattle are a big source of methane because the bacteria in their gut produce it. Methane is a greenhouse gas. The nitrogen cycle involves the different forms of nitrogen in the ecosystems. Proteins and nucleic acids have nitrogen in them. Most plants and phytoplankton are unable to fix nitrogen so it takes symbiotic bacteria and some free-living bacteria to help fix them through nitrogen fixation. Cyanobacteria can fix nitrogen from nitrogen gas to make ammonia, which can be made into biological molecules. Nitrogen goes back into nitrogen gas by certain microbes that undergo ammonification, followed by nitrification and denitrification. The ammonia and organic waste get oxidized to make nitrite and nitrate. Then some bacteria, such as Clostridium and Pseudomonas, make nitrogen gas during anaerobic respiration, to make gas that can get into the atmosphere again. Artificial fertilizers contribute to nitrogen gas in the

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environment by adding nitrogenous compounds to the water supply through runoff into the water supply. Sulfur also gets cycled in the environment. The amino acids methionine and cysteine have sulfur in them and there are other biological molecules that have sulfur in them. Anoxygenic photosynthetic bacteria and some archaea use hydrogen sulfide and oxidize it to elemental sulfur and to sulfate. Sulfate can be used as a sulfur source by certain bacteria and plants.

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KEY TAKEAWAYS •

Metabolism basically involves the catabolism of certain organic molecules.

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Glycolysis is the first step in breaking down glucose into pyruvate, yielding some ATP and molecules that can later be used to make more ATP.

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There is a transition reaction between glycolysis and the Krebs cycle.

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Some organisms proceed from glycolysis to fermentation, which has different end products, depending on the organism. These can be things like lactic acid, acetic acid, and CO2.

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The Krebs cycle is cyclic and generates ATP and other molecules that can later be used to make energy.

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Most of the ATP is gotten through oxidative phosphorylation and the electron transport chain.

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Energy is stored by transferring hydrogen ions across a membrane in a cell, which goes on to drive the synthesis of ATP.

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Photosynthesis involves two phases of reactions that capture light to make organic molecules.

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The biogeochemical cycles will involve the movement of molecules into different forms and in different aspects of the environment.

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QUIZ 1. What statement is true? a. Anabolism is molecular breakdown, which is endergonic. b. Catabolism is molecular breakdown, which is exergonic. c. Anabolism is molecular buildup, which is exergonic. d. Catabolism is molecular buildup, which is endergonic. Answer: b. Catabolism is the breakdown of molecules, which releases heat and energy so these are exergonic reactions. Anabolism is the making or buildup of molecules, which requires energy so these are endergonic reactions. 2. Organisms that get their energy from inorganic molecules are called what? a. Phototrophs b. Heterotrophs c. Autotrophs d. Lithotrophs Answer: d. Lithotrophs are organisms that get their energy from inorganic molecules, such as reduced iron and hydrogen sulfide. 3. What is the main energy currency used in most energy-requiring reactions in the cell? a. ATP b. NAD+ c. NADP+ d. FAD Answer: a. Each of these reactions are helpful in energy needs of the cell, except that ATP is the main energy source in most metabolic processes in the cell.

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4. What is not something that an enzyme does? a. It lowers the activation energy of a reaction. b. It binds to the substrate of a reaction. c. It causes a conformational change to occur in a substrate. d. It is consumed as part of the reaction. Answer: d. The reaction with an enzyme occurs because of each of these things. The exception that does not occur is that the enzyme is consumed as part of the reaction. The enzyme is not consumed in an enzymatic reaction. 5. When is the pentose phosphate pathway used instead of regular glycolysis? a. When oxygen is not available b. When there are pentoses and not glucose available c. When nucleic acids or amino acids are needed d. When more ATP energy is required by the cell Answer: c. The pentose phosphate pathway is used in all cells and is the preferred pathway when nucleic acids or amino acids are necessary because these can be made from this pathway’s intermediates. 6. What are the starting product and some end products of the Krebs cycle? a. Pyruvate is the starting substrate and some end products are oxygen and ATP. b. Pyruvate is the starting substrate and some end products are CO2 and GTP. c. Acetyl CoA is the starting substrate and some end products are oxygen and CO2. d. Acetyl CoA is the starting substrate and some end products are CO2 and ATP. Answer: d. The Krebs cycle starts with acetyl CoA and some end products are CO2, ATP, GTP, and NADH.

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7. Which organism makes beer and bread because it participates in alcoholic fermentation? a. Escherichia coli b. Staphylococcus aureus c. Lactobacillus species d. Saccharomyces cerevisiae Answer: d. Saccharomyces cerevisiae is a yeast organism that makes CO2 in the making of bread and fermented alcoholic beverages. 8. Where does oxidative phosphorylation take place in prokaryotes? a. Inner cell membrane b. Outer cell membrane c. Outer mitochondrial membrane d. Inner mitochondrial membrane Answer: a. Oxidative phosphorylation takes place on the inner cell membrane of prokaryotes. They do not have mitochondria. In eukaryotes, the oxidative phosphorylation takes place on the inner mitochondrial membrane. 9. What gets made as part of beta-oxidation of lipids? a. Acetyl groups b. Pyruvate c. Glucose d. Glucose-6-phosphate Answer: a. Acetyl groups are 2-carbon molecules that participate in metabolism by entering the Krebs cycle to make CO2 and ATP energy.

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10. What is the main end product of the light-independent processes in photosynthesis? a. Amino acids b. Glucose c. CO2 d. Pyruvate Answer: b. The end product of this type of metabolism is the making of sugar or glucose from CO2 as a beginning substrate.

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CHAPTER EIGHT: THE GENOME IN MICROBIOLOGY The focus of this chapter is the genome of the cell. Cellular organisms generally have DNA making up their genome. Both DNA and RNA are nucleic acids, which are important in the genetic functioning of the cell. The structure and function of DNA and RNA are covered as part of the chapter. The totality of the DNA in a cell is referred to as the genome. The different characteristics of a cell’s genome are also discussed in this chapter.

DNA STRUCTURE AND FUNCTION We have already talked about proteins, lipids, and carbohydrates. The class of biochemical macromolecules that we are talking about in this chapter is nucleic acids. The monomers of nucleic acids are referred to as nucleotides, which form large strands in a certain order, called a base sequence. The specific base sequence of nucleotides in the DNA of cells determines the heredity and genetic makeup of the organism. DNA stands for deoxyribonucleic acid. It has a certain structure and function, which is what we will also cover in this chapter. The nucleotides in DNA are referred to as deoxyribonucleotides. There are three major components to these molecules. These are deoxyribose, which is a five-carbon sugar, a nitrogenous base, and a phosphate group. A nucleoside is just the nitrogenous base and the five-carbon sugar but not the phosphate group. Figure 43 shows what a nucleotide is:

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Figure 43.

The structure of the nitrogenous base is different throughout the DNA strand. With regard to DNA, there are four different nitrogenous bases. There are the purines, which are adenine and guanine, as well as the pyrimidines, which are cytosine and thymine. When looking at a strand of DNA, it is the first letter of each of the nitrogenous bases that is used to identify the nucleotide. Nucleoside triphosphates on the same DNA chain are linked by what are called fiveprime to three-prime phosphodiester bonds. When referring to the numbering system, deoxyribose carbons are labeled one through five. The phosphate group binds to the five-prime carbon atom and attaches to the three-prime end of the next deoxyribose carbon atom just adjacent to it. This is called a phosphodiester bond. Figure 44 shows a phosphodiester bond:

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Figure 44.

At each end of the linkage, there will be a five-prime phosphate that is free on one end and a three-prime hydroxyl group at the opposite end. These chains are made from a nucleoside triphosphate, with pyrophosphate or two phosphate groups dropped off and used as energy to combine the molecules into a DNA strand. It was in the early 1950s that it was discovered that DNA was the basic genetic molecule of the cell but It wasn’t clear what its structure was. It was then discovered that A always bound to T and G always bound to C. This is referred to as Chargaff’s rule. In was the work of Rosalind Franklin that uncovered the x-ray diffraction technique that would identify the double helix ultimately found in the structure of DNA. James Watson and Francis Crick together used x-ray diffraction to define the double helical structure of DNA and were able to put together than purines always connected to pyrimidines. They published their structure of DNA and were ultimately awarded the Nobel Prize for their work. 130

There are two antiparallel strands of DNA so that the five-prime end of one strand will face the three-prime end of another strand. Remember, the three-prime end has the free hydroxyl group and the five-prime end has the free phosphate group. The outside of the double helix has the deoxyribose molecule and the phosphodiester bond, while the inside of the double helix has the nitrogenous bases that connect to one another through hydrogen bonding. There are ten bases per turn of the helix. There are major grooves and minor grooves that are made from the asymmetry between the base pairs. The fact that thymine goes together with adenine and that guanine goes together with cytosine means that these are considered complementary base pairs and matches completely with Chargaff’s rule. Some complementary base pairs, namely A and T, have two hydrogen bonds, while C and G have three hydrogen bonds between the two molecules. The strands will separate at higher temperatures and in the presence of certain chemicals. This is called denaturing the DNA. The single strands of DNA can be put together again if the circumstances are right. Because of the differences in the number of hydrogen bonds, the DNA with high GC content is harder to break than those that are high in AT. DNA will store the genetic material of the cell. With vertical gene transfer, the mother cell transfers the genetic material to the daughter cells. DNA is replicated anytime the cell divides to make daughter cells. This is basically the only function that DNA has with regard to the cell. There are no structural elements to the DNA molecule.

RNA STRUCTURE AND FUNCTION Ribonucleic acid or RNA is very similar to DNA but it is generally single-stranded, has much shorter strands, and contains ribose instead of deoxyribose. RNA is made from ribonucleotides and is linked together via phosphodiester bonds, just as is seen in DNA. The nitrogenous bases are different with RNA. There is adenine, guanine, and cytosine like in DNA but the uracil replaces thymine that is seen in DNA. DNA is somewhat more stable than RNA so it is better than RNA in keeping genetic information. Adenine matches with uracil in the RNA molecule.

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The main function of RNA is to help turn the DNA message into the different proteins. There are several kinds of RNA, including messenger RNA, ribosomal RNA, transfer RNA, and small nuclear RNA. Messenger RNA takes the DNA code and sends it to the ribosomes, where it is read. Transfer RNA contains the amino acids that combine to make proteins. Ribosomal RNA is part of the structure of the ribosome. Small nuclear RNA is only seen in eukaryotes. There is a transcription process, in which DNA information gets transferred to messenger RNA through the activity of RNA polymerase. Then, the messenger RNA exits the nucleus or travels away from the DNA in order to go to the ribosomes. At the ribosomes, transfer RNA contains a single amino acid per molecule. The message is read in the ribosomes and the protein strand is added one amino acid at a time by transfer RNA. Messenger RNA is an unstable molecule that is easily degraded if protein is not made relatively quickly. Ribosomal RNA and transfer RNA are much more stable than messenger RNA. These are made by DNA and then cut to smaller segments. The nucleolus is where ribosomal RNA processing takes place in eukaryotes but this happens in the cytoplasm of prokaryotes. Ribosomal RNA makes up about sixty percent of the ribosome and this is where the messenger RNA ultimately binds. Ribosomal RNA makes sure that the transfer RNA and messenger RNA are aligned correctly. Ribosomal RNA is the only RNA type that has enzymatic activity, called peptidyl transferase, which helps to make the peptide bond when proteins are made. Transfer RNA is very short and has only 70 to 90 nucleotides per molecule. Each one carries an amino acid that can be inserted into the polypeptide molecule. While it is small, it is essential to the making of proteins so mutations in the making of this RNA type can affect protein synthesis to a great degree. Remember that DNA is the hereditary material in cells but RNA can serve as the genetic information source in the virus particle. In viruses, RNA can be single-stranded or double-stranded. As mentioned, certain RNA types in the virus can be directly

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translated into proteins, while other types need manipulation in order to be replicated into new viral particles.

CELLULAR GENOMES The entirety of the DNA message in the cell is called its genome. The genome is made from genes that are together stored in one or more chromosomes. Organisms vary from one to another in the size and arrangement of their genome. The DNA will code for all cellular activities with many segments that each encode for just one protein. This segment is called a gene. The genotype is all the genes in the genome. Not all genes are expressed at the same time within the cell and the genes are expressed differently, depending on the cell, in multicellular organisms. The gene will determine the phenotype, which is what is actually observed with a specific genotype. Constitutive genes are always expressed so, for this reason, these are known as housekeeping genes. A facultative gene, on the other hand, is only turned on when it is needed. The genotype is always constant but the phenotype is variable, depending on the environment. An example is Streptococcus mutans, which will make a slime layer only when it is exposed to sucrose or table sugar. Temperature also affects the phenotype of a specific organism. The genome is associated into chromosomes, which are considered discrete DNA segments. Prokaryotes generally have a single circular chromosome, while eukaryotes have multiple linear chromosomes. Each chromosome can have thousands of genes. Eukaryotic cells are often diploid, which means they have two copies of each chromosome. The chromosomes are tightly packed so they fit within the cell. This is due to supercoiling of the DNA, which shrinks it size. There are topoisomerases, which are enzymes that maintain the supercoiling of the genome. Histone proteins are DNAbinding proteins that help to organize and coil the DNA. Chromatin is what DNA plus the histone proteins are called.

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Prokaryotes, with their circular DNA, are generally haploid. This means there is one copy of the genome in each cell. Supercoiling also happens to help the DNA shrink to a more acceptable size. Topoisomerases are also involved, one of which is DNA gyrase, which keeps the DNA from winding too much. There are no histone proteins in prokaryotes but there are similar proteins involved in the packaging of DNA. Some regions of DNA are not supercoiled and can be expressed directly. There can be a great many regions of DNA called noncoding DNA, which does not encode for protein or stable RNA segments. These are located between genes. An intron does not code for a protein, while an exon does encode for a protein. There are start and stop sequences at the beginning and end of a gene that are not coding segments. There are regulator segments that are also not coded but help regulate the expression of the gene. Prokaryotes have fewer noncoding segments compared to eukaryotes. Extrachromosomal DNA is located outside the main chromosome. These are a part of the genome. These extrachromosomal DNA segments can be found in chloroplasts, mitochondria, and plastids. The DNA of some latent viruses can also represent extrachromosomal DNA. This is seen in humans when human papillomaviruses infect the cell. Plasmids are found in prokaryotes mainly and can confer certain benefits to the host, such as antibiotic resistance. Each organism has a different size of genome. Humans have 46 chromosomes with three billion base pairs. Plants have larger genomes that are generally polyploid, meaning they have multiple copies of the chromosome. The smallest genomes are found in viruses, although this is not universally true, with small, obligate intracellular organisms having the next smallest genome. Other bacteria will have larger genomes. As mentioned, plants have the largest genomes.

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KEY TAKEAWAYS •

DNA is the genetic material in all cells but may not be the genetic material in viruses.

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A nucleotide has a nitrogenous base, a deoxyribose sugar, and a phosphate group.

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DNA is a double-stranded helix that has the nitrogenous bases making up the rungs of the ladder shape of the molecule.

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RNA contains a ribose sugar, is often single-stranded, contains uracil instead of thymine, and is generally less stable than DNA.

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The genome is the totality of the genes in a cell. A cell can be haploid, diploid, or polyploid, depending on the number of copies of DNA within the cell.

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QUIZ 1. What is not a part of a DNA-based nucleotide? a. Ribose molecule b. Phosphate group c. Deoxyribose molecule d. Nitrogenous base Answer: a. There is just one type of sugar molecule in a DNA nucleotide and this is deoxyribose and not ribose, which is another type of fivecarbon sugar. 2. What would not be seen in a DNA nucleoside? a. Adenine b. Thymine c. Deoxyribose d. Phosphate Answer: d. A DNA nucleoside is just a nitrogenous base like adenine and thymine plus deoxyribose but not the phosphate group. If a phosphate group is associated with this, it is referred instead as a nucleotide. 3. What is the carbon number on the deoxyribose sugar that has the free phosphate end associated with the end of a DNA strand? a. One-prime b. Three-prime c. Four-prime d. Five-prime Answer: d. It is the five-prime end that has the free phosphate group associated with it at the end of the DNA strand. The three-prime end has a free hydroxyl group.

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4. What kind of bonding occurs between nitrogenous bases on a DNA double helix? a. Ionic bonding b. Hydrogen bonding c. Covalent bonding d. Metallic bonding Answer: b. Hydrogen bonding connects two of the nitrogenous bases in a DNA double helix. The purines match with the pyrimidines, and vice versa. 5. What is the type of RNA seen in eukaryotes only and participates in RNA processing? a. Transfer RNA b. Ribosomal RNA c. Messenger RNA d. Small nuclear RNA Answer: d. Small nuclear RNA is found only in eukaryotes and participates in the processing of RNA. 6. Which type of RNA takes the DNA message and sends it outside the nucleus or into the cytoplasm for the making of proteins? a. Transfer RNA b. Ribosomal RNA c. Messenger RNA d. Small nuclear RNA Answer: c. Messenger RNA is the carrier of the DNA message. It takes this message to the ribosomes where it is translated into a protein.

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7. What is it called when proteins are made in the ribosome through the activity of messenger RNA and transfer RNA? a. Translation b. Replication c. Modification d. Transcription Answer: a. Translation is the making of proteins from messenger RNA and transfer RNA. 8. The totality of the genetic material of the cell or organism is called what? a. Chromosome b. Genome c. Gene d. Phenotype Answer: b. The totality of the genetic material of a cell or organism is referred to as its genome. This will be different from organism to organism. 9. What is a segment of a chromosome that directly codes for a protein? a. Exon b. Intron c. Regulator segment d. Stop segment Answer: a. An exon is a segment of a chromosome that directly codes for a protein. The rest of the choices are not coding segments that make a protein.

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10. Which genome type would be considered the smallest? a. Diploid genome b. Polyploid genome c. Haploid genome d. Triploid genome Answer: c. A haploid genome will be the smallest because it has just one copy of the cell’s genome, while the other choices represent the presence of more than one copy of the chromosomes.

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CHAPTER NINE: MICROBIAL GENETICS This chapter expands on the study of DNA by looking into microbial genetics. The ways in which DNA is replicated, the transcription process, and the processes involved in protein synthesis are covered in the chapter. Other things discussed are genetic mutations and the different ways genes are regulated. How each of these things leads to genetic diversity in prokaryotes is also discussed in the chapter.

DNA REPLICATION The process by which DNA makes copies of itself is called DNA replication. One model of DNA replication is semiconservative replication, in which whole strands of DNA are separated and copies made of each strand. Each double strand then has an old strand and a new strand. In conservative replication, the old double strand is conserved and new strands are made from that. In dispersive replication, there are regions of old and new strands dispersed throughout the daughter strands. Research has indicated that the semiconservative approach is the most accurate way to describe DNA replication. DNA replication has best been studied in bacteria. The entire replication process takes about 42 minutes in the E. coli bacterium. DNA polymerase is the main enzyme responsible for this process. There are three types of DNA polymerase in bacteria, with DNA polymerase III responsible for synthesis of DNA and both DNA polymerase I and II involved in DNA repair. DNA polymerase III adds nucleotides onto the three-prime hydroxyl group one nucleotide at a time using the energy stored in the triphosphate bond of the nucleotide. This energy creates the phosphodiester bond. Replication starts with initiation, which occurs at a specific site, known as the origin of replication. E. coli has just one origin of replication, which is true of most prokaryotes. This origin is about 245 base pairs in length. There are other proteins necessary for DNA replication. Remember that DNA is supercoiled with histone proteins or histone-like proteins in bacteria. Topoisomerase II helps to relax this supercoiling. It is also referred to as DNA gyrase. Helicase will separate the different DNA strands.

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Y-shaped structures are created by the replication processes called replication forks. There are two forks started at the origin of replication that open up to make a replication bubble so that replication can occur in both directions along the chromosome. There are single-stranded binding proteins that help prevent the single-stranded DNA from rewinding into a double helix. Remember that DNA polymerase III can only add new nucleotides from the five-prime to three-prime direction. This eliminates the easy possibility of adding DNA to both strands, which are antiparallel. The problem is managed by having an RNA sequence with a three-prime end. This is called a primer sequence. It can be synthesized by an RNA polymerase called RNA primase. They do not need to add to a three-prime end so it creates a free three-prime hydroxyl group that can be extended with DNA polymerase III. About 1000 nucleotides are added per second. There is a leading strand that directly gets copied according to the rules of DNA replication. The other strand is not so simple. The DNA is made in segments called Okazaki fragments that later get spliced together. This is known as the lagging strand, which has discontinuous replication. The direction of the lagging strand synthesis is three-prime to five-prime. The RNA primers are later replaced by DNA. There are exonucleases as part of DNA polymerase I that remove the primers and fill in the gaps with the right DNA fragments. There are nicks in the new DNA segments that get sealed through DNA ligase that catalyzes the making of a phosphodiester bond between adjacent parts of the DNA strand. Figure 45 shows the process of DNA replication:

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Figure 45.

Once the entire replication happens, there is termination of DNA replication. There is the action of bacterial topoisomerase IV, which helps to separate the circular chromosome to keep it from getting tangled. This process only happens in bacteria. Because eukaryotes do not have DNA gyrase or topoisomerase IV, quinolone antibiotics can tackle this aspect of bacterial growth and replication. DNA replication is more complicated than that seen in prokaryotes. The chromosomes are, of course, linear, and there are many more base pairs to work with. There are multiple origins of replication—about 30,000 to 50,000 per genome in humans. The actual rate of replication is much slower, at about ten times slower than that seen in prokaryotes. The steps are basically the same except that there are histone proteins. The DNA polymerases are structurally different with a sliding clamp that holds the DNA polymerase in place. There is an enzyme in eukaryotes called Ribonuclease H, which removes the RNA primer. DNA synthesis in eukaryotes also proceeds in a five-prime to three-prime direction. When the replication fork gets to the end of the chromosome, the last few DNA base

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pairs do not get paired, which shortens the genome over time. These ends are called telomeres and ae not coded. The base-pair sequence of TTAGGG is repetitively copied up to 1000 times to make the telomere. Telomerase is an enzyme that attaches to the end of the chromosome to add telomeres in cells like germ cells and adult stem cells, which remain “youthful” for extended periods of time. It isn’t active in adult somatic cells and may be responsible for the aging of cells because the telomeres shorten. Plasmids also undergo DNA replication. While some replicate like bacterial DNA, others use the rolling circle replication technique. One piece of the double strand of DNA is nicked and then the DNA polymerase will help to make a copy of the un-nicked strand. The nicked strand may then circularize again and can get replicated. This leads to two copies of the plasmid.

RNA TRANSCRIPTION Transcription happens when DNA is used as a copy to make an RNA transcript, which is single-stranded. It requires partial unwinding of the DNA segment and the formation of a transcription bubble. The antisense strand is the one that gets transcribed. This makes the RNA segment made a near-exact copy of the sense strand. Transcription involves RNA polymerase, which works from the five-prime to threeprime end. RNA polymerase does not involve a primer but DNA polymerase does. Complementary RNA base pairs are added to match the DNA segment being transcribed. There are six subunits to the bacterial RNA polymerase. The sigma subunit is responsible for binding of the enzyme to a promotor site at the beginning of transcription. Transcription always starts at a promoter site. The first DNA piece to be transcribed is called the initiation site. Nucleotides are added downstream from the initiation site. There are similarities in the promotor regions of all bacteria, which are the sites of RNA polymerase attachment. This usually involves what’s called a TATA box, which is a TATAAT sequence of DNA bases. Elongation happens at about 40 nucleotides per segment, getting made from the five-prime to three-prime end.

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At some point, the RNA has been completely transcribed and the RNA polymerase dissociates from the template. This is called termination. There will be specific DNA sequences that act as signals for termination so that the RNA polymerase drops off and the RNA segment is freed. Transcription also happens in eukaryotes with a few differences compared to prokaryotes. There are three different RNA polymerases in eukaryotes that transcribe different genes. One of the biggest differences is that one piece of eukaryotic RNA encodes for a single protein, while prokaryotic RNA encodes for multiple proteins at once. This is referred to as polycistronic RNA. RNA is made in the nucleus but must exit the nucleus for protein translation. There are several modifications that need to happen before the RNA will be able to leave the eukaryotic nucleus in order to protect the RNA from being degraded. This allows eukaryotic messenger RNA to last several hours as opposed to prokaryotic messenger RNA, which lasts just a few seconds. There will be a five-prime cap added to the RNA transcript in order to prevent degradation. In addition, a poly-A tail of a couple hundred adenine nucleotides to the three-prime end is added, also to prevent degradation and to signal the need for transport outside the nucleus. It is only eukaryotic organisms that have exons that get expressed and introns that do not get expressed. The introns get spliced out of the pre-messenger RNA as part of the processing of the RNA. This RNA spicing process is done by a spliceosome, that contains small nuclear RNA. After each of these things happens, the RNA is transported outside the nucleus.

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TRANSLATION AND PROTEIN SYNTHESIS The process of protein synthesis is called translation. There are 20 amino acids that together make all the proteins of life. Each is associated with a triplet of base pairs, called a codon. There are sixty-four possible combinations of base pairs so there are 64 codon types. A total of 61 of them code for amino acids, while the rest are either stop codons or start codons. The codon AUG is a start codon. Because there are 20 amino acids, there is more than one codon for the same amino acid. Essentially all forms of life have the same codons matching for the same amino acids. Ribosomes are made from ribosomal RNA and protein material. The different organisms have structurally different ribosomes. Prokaryotes are said to have a 70S ribosome, while eukaryotes are said to have an 80S ribosome. There are two subunits. The small subunit binds messenger RNA, while the large subunit binds transfer RNA. The messenger RNA is read from the five-prime end to the three-prime end, making a protein that starts with an amino group and ends with a carboxyl group. A polyribosome is a ribosome plus a messenger RNA segment. In prokaryotes, translation and transcription happen at the same time. This means that prokaryotes can respond quickly to environmental signals. Transfer RNA is a special type of RNA that specifically helps to extend and elongate the polypeptide chain. It has a unique three-dimensional shape. Figure 46 shows what transfer RNA looks like:

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Figure 46.

The shape is made by different base pairs connecting to one another in the same strand. It allows for positioning of the amino acid binding site. The anticodon is what binds to the messenger RNA codon in order to align these segments. Transfer RNA is either charged with an amino acid or it is not charged. There are enzymes that connect the amino acid to the transfer RNA called aminoacyl transfer RNA synthetases.

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There are similarities between the translation process of prokaryotes and eukaryotes. Initiation starts with the making of an initiation complex. This requires GTP as an energy source. Initiation starts with the start codon, which is AUG. There is a specialized amino acid added to every protein at the time of initiation, called methionine. Figure 47 shows what translation looks like:

Figure 47.

Elongation is the same in all organisms. Peptide bonds are formed to a growing peptide chain. The charged transfer RNAs enter the A site in the ribosome, get moved over to the P site, and leave at the E site or exit site. Peptidyl transferase is the enzyme that makes the peptide bond. Termination happens when a stop codon or nonsense codon is reached. This releases the peptide chain and the process starts all over again.

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There are post-translational modifications that can occur. The signal sequences are removed and the protein is properly folded. There are other chemical modifications to the amino acids that can occur, such as phosphorylation, glycosylation, and methylation.

MUTATIONS At any point in time, a mutation can occur, which will change the protein made or will change the phenotype of the organism. Most mutations happen at the level of transcription, which may or may not affect the protein end product. There are several types of mutations. A point mutation affects just one base pair. An insertion involves the addition of one or more base pairs and a deletion involves the removal of a base pair. These will have differing effects on the protein. Some point mutations won’t affect the amino acid at all, leading to what’s called a silent mutation. A missense mutation happens when a different amino acid gets into the protein. Some are more severe than others, depending on the amino acid’s qualities. Conditional mutations are missense mutations that only sometimes affect the protein. A nonsense mutation turns an aminoacid codon into a stop codon. Frameshift mutations can be very severe because they cause each of the subsequent amino acids to be wrong. If the mutation affects a deletion that isn’t a multiple of three bases in a row, this leads to a frameshift mutation. Mutations can be spontaneous or can be caused by certain mutagens. Mutagens can be chemical or secondary to radiation. Most mutations are also considered carcinogens because they can cause cancer. Chemical mutagens include things called nucleoside analogs that are so similar to nucleosides that they get into the DNA molecule but don’t pair with other bases in the proper way. Intercalating agents get between base pairs, throwing off the sequence, leading to deletions or insertions. Radiation can be ionizing radiation or nonionizing radiation. Ionizing radiation includes gamma rays and x-rays. These can break bonds and can modify bases in the DNA molecule. Nonionizing radiation like ultraviolet light can cause dimer formation between two pyrimidine molecules. This will affect both transcription and replication. In most cases, DNA can repair itself. Most mistakes are fixed through a proofreading function. This happens because of DNA polymerase, which reads the base pair and 148

corrects it if it is not the right one. There are enzymes that can replace the wrong nucleotide with the right one. Dimers of thymine can be excised and repaired as well. The DNA is sealed up again with DNA ligase.

OPERONS AND GENE REGULATION Because all cells contain every possible gene in an organism, there needs to be a mechanism for some genes to be expressed and others to be repressed, depending on conditions in the environment or on the cell with multicellular organisms. There are regulatory genes that specifically turn on or turn off the different structural genes. For microorganisms, this regulation allows the organism to survive under different circumstances. There are differences and similarities in how prokaryotes and eukaryotes undergo gene regulation. Prokaryotes but not eukaryotes have a block of genes with related functions called operons. They are usually transcribed together. This allows the entire operon to be regulated at the same time. The best studied operon is the lac operon, which has a single lac promotor region. Operons are not seen in eukaryotes but some of the same principles exist in eukaryotic genes. There is a regulatory region that includes a promotor region, which binds to certain transcription factors. These transcription factors can influence the ability of RNA polymerase to bind to the promotor region. Transcription factors can be activators, repressors, or inducers. Activator and repressors will increase or decrease the transcription of a gene. An inducer can interact with an activator or repressor. It is a small molecule that can increase or decrease transcription along with the activator or repressor. The repressor will bind to the operator, blocking attachment of RNA polymerase. Activators bind to the promotor site to increase transcription. Figure 48 describes an operon and how it works:

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Figure 48.

Repressors prevent transcription. If an operon is under the control of a repressor, it is called a repressible operon, while others are inducible operon. The tryptophan operon is one of the repressible operons, in which tryptophan itself is a repressor that turns off its own production. The lac operon is an inducible operon, win which lactose induces the activity of the operon. In the trp operon, tryptophan is an inducer molecule that binds to a repressor in order to turn off the trp operon. The lac operon is inducible that can be activated in the absence of glucose. It codes for genes that can consume lactose in the environment, breaking it down into glucose. Lactose needs to be present in the environment for this process to happen. There is a lac repressor active all the time that, when bound to lactose, an inducer molecule, no longer represses the operon.

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The E. coli bacterium prefers to thrive on glucose but it is not always available. When glucose runs out, there is an increase in cyclic AMP, which binds to catabolite activator protein or CAP. This binds to the promotor region of the lac operon, which increases RNA polymerase activity and causes lac operon transcription. This is an example of an activator protein acting on the lac operon. Lactose needs to be present and glucose need to be diminished. Only when both conditions are present will transcription levels of the lac operon be high. Prokaryotes have the ability to sense oncoming stress by releasing alarmones, which are small nucleotide derivatives. They can stimulate the expression of stress-related genes. There are certain virulence genes that can be upregulated in response to alarmone production. There are transcription factors in eukaryotes as well. Proteins called enhancers can bind a distance away from the gene they regulate, with DNA looping putting the enhancer and promotor together. There are bending proteins that allow for DNA bending, which can put the enhancer in proximity to the promotor. There is also what’s called epigenetic regulation, which involves methylation of certain base pairs in order to affect transcription. Usually, the methylation of cytosine will often slow transcription. Histone proteins can also affect transcription.

GENETIC DIVERSITY IN PROKARYOTES Vertical gene transfer involves the passing on of genetic information from a mother cell to daughter cells. This works differently when comparing sexual organisms and asexual organisms. Mutations will add to gene diversity in prokaryotes and eukaryotes. Asexual reproduction usually involves passing on direct copies of the parent genome. Prokaryotes can engage in what’s called horizontal gene transfer, which involves the passing on of genetic material in the same generation. It can occur between species that are not directly related to one another. There are three ways that this can occur. In transformation, naked DNA is taken up from the outside environment. In transduction, there is a viral vector that transmits DNA. In conjugation, pili are used to transfer DNA from one cell directly to another cell. Figure 49 shows these processes in a bacterial cell:

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Figure 49.

In transformation, only single-strand DNA is taken up because it cannot easily be degraded, which is not the case for double-stranded DNA. Transduction involves bacteriophages that have accidentally taken up host genomic material when it separates from the host DNA. This can change the phenotype of the cell, offering it some evolutionary advantages over the original cell. In conjugation, it is usually plasmids that get transmitted from one cell to another. The F pilus or fertility pilus will pass from a donor cell to a recipient cell. There are R plasmids that can be transferred, which encode for things that bring antibacterial resistance to the new cell.

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Transposons are also called “jumping genes” or transposable elements. These must have a gene that encodes for the enzyme called transposase, which allows large sequences of the genome to move from one area to the next. This phenomenon is seen in both eukaryotes and prokaryotes. They basically cut and paste themselves in a different location. They can alter the phenotype of the cell in some cases and can confer antimicrobial resistance.

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KEY TAKEAWAYS •

DNA replication involves making a copy of DNA that will be passed to daughter cells. There are a number of enzymes required.

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Transcription involves the making of messenger RNA from a DNA template, involving RNA polymerase and other enzymes.

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Transcription takes place in the ribosomes and involves the taking of the genetic code, turning the code into a specific polypeptide chain.

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In prokaryotes, transcription and translation happens at the same time and in roughly the same place.

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Gene regulation is different between eukaryotes and prokaryotes. The best studied operons in microbiology are the lac operon and the trp operon.

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Genetic diversity can happen with transduction, transposition, conjugation, and transformation. Transposition can happen in both prokaryotes and eukaryotes.

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QUIZ 1. What kind of replication occurs in the replication of DNA? a. Conservative b. Semi-conservative c. Dispersive d. A combination of semi-conservative and dispersive Answer: b. Research has proven that DNA replication is semiconservative, which involves splitting of the DNA strand and the formation of a new strand half on the opposite side of both parent strands. 2. What enzyme is most responsible for DNA replication? a. DNA polymerase b. Helicase c. DNA gyrase d. Topoisomerase Answer: a. The main enzyme that is responsible for DNA replication is DNA polymerase, which is responsible for creating the daughter strand on the DNA molecule. 3. What is not a difference between eukaryotic DNA replication and prokaryotic DNA replication? a. The eukaryotes synthesize DNA in the three-prime to five-prime direction as well as the five-prime to three-prime direction. b. The prokaryotes have DNA polymerase while the eukaryotes do not. c. Eukaryotes do not have DNA ligase. d. Eukaryotes have multiple origins of replication and prokaryotes have just one of these.

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Answer: d. The main difference between eukaryotic and prokaryotic DNA replication is that eukaryotes have multiple origins of replication, while prokaryotes have just one. 4. Which DNA replication enzyme is unique to eukaryotes? a. DNA polymerase b. Ribonuclease H c. Helicase d. RNA primase Answer: b. Eukaryotes have ribonuclease H, which is not present in prokaryotes. It is responsible for removing the RNA primer. 5. The making of proteins in the ribosomes is called what? a. Replication b. Transcription c. Translation d. Post-translational modification Answer: c. The making of proteins in the ribosomes is specifically referred to as translation. It directly follows the transcription of RNA. 6. What is the function of the poly-A tail added to messenger RNA in the nucleus? a. To prevent degradation b. To identify the exons and introns c. To prevent degradation and to signal the need for exit of the RNA outside the nucleus d. To separate the different protein-coding segments Answer: c. The function of the poly-A tail added to messenger RNA is to prevent degradation of the messenger RNA and to signal the need for exit of the RNA outside the nucleus.

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7. What is the same thing that occurs in both prokaryotes and eukaryotes in the process of translation? a. In both, the same start and stop codons are used. b. In both, the beginning or initiating amino acid is the same. c. In both, translation occurs simultaneously with transcription. d. In both, the same ribosomes are used. Answer: a. The only similarity between the two is that the same start and stop codons are used. The other things are different between the two types of organisms. 8. What type of mutation in a codon leads to the same amino acid getting put into the protein? a. Nonsense mutation b. Conditional mutation c. Missense mutation d. Silent mutation Answer: d. A silent mutation happens when there is a mutation but it doesn’t affect the amino acid that gets put into the protein. 9. In the trp operon, what is the role of tryptophan? a. Tryptophan itself plays no role in gene regulation. b. Tryptophan is an activator amino acid. c. Tryptophan is a repressor molecule. d. Tryptophan is an inducer molecule. Answer: d. In this case, tryptophan is an inducer molecule that binds to a repressor molecule so that it can turn off its own synthesis in the cell when enough of it exists. The repressor molecule binds along with tryptophan to the operator region.

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10. What does lactose bind to in a cell in order to trigger lactose-consuming genes in the lac operon of a eukaryotic cell? a. Operator region b. Promotor region c. A repressor molecule d. An activator molecule Answer: c. Lactose binds to a repressor molecule, causing it to change and fall off the operator so that the lac operon can be transcribed.

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CHAPTER TEN: MICROBIAL GROWTH This chapter touches on aspects of laboratory microbiology by looking into microbial growth. The different patterns of microbial binary fission and bacterial growth in cultures is important to understand as a laboratory microbiologist. There are certain factors that increase or decrease microbial growth, which are covered in the chapter, along with the different physical and chemical methods of controlling microbial growth both in culture and in the environment.

MICROBIAL GROWTH Prokaryotes engage only in asexual reproduction but exhibit genetic diversity through horizontal gene transfer. Almost all bacteria have a single circular chromosome. These cells replicate through binary fission. The cell first grows in size and then the DNA replicates. The origin of replication is located attached to the inner cell membrane. The cell membrane constricts in the middle so that two daughter cells are made. There is a protein structure called the Z ring that constricts the center of the cell. Then the peptidoglycan cell wall makes a septum that ultimately separates the cell. Figure 50 shows bacterial binary fission:

Figure 50.

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The eukaryotic cell will have a generation time, which is the time between the same points in the life cycle of two generations. In humans, this will be about 25 years. This does not work for bacteria, which can divide rapidly or not at all. Instead, the term doubling time is used. This can be as little as 20 minutes or as much as several days. Some, like the organism of tuberculosis, takes 14 to 20 hours, and the organism that causes leprosy takes up to 14 days. In a closed batch culture of bacteria, no nutrients are added during the growth and no wastes are removed. They follow a specific growth pattern. This does not completely mimic the growth inside a host organism. The growth curve measures the culture density over a period of time. There four phases to the growth curve in a batch culture. The first is the lag phase, which does not involve an increase in the bacterial cell count. Then comes the log or logarithmic phase, which involves rapid utilization of nutrients and a large increase in cell counts. In the stationary phase, the rate of cell division and the rate of cell death are equalized so the cell count is high and stationary. In the death phase or decline phase, nutrients are used up and the cell count decreases. In the lag phase, you provide an inoculum to fresh medium that will support growth. The cells get larger and are metabolically activated but do not divide. This can represent somewhat of a shock to the culture cells. The log phase involves exponential cell growth with rapid binary fission. There is a genetic basis behind how well the cells grow in a specific environment. Growth is exponential and not linear. The stationary phase is when the growth rate slows down and is roughly equal to the death rate. This concentration is called the maximum culture density. The death phase involves a marked slowing of the cells’ metabolic rates. All of this can be avoided by continuing to add nutrients and removing some waste by removing an effluent. Bacterial growth can be measured through a direct cell count, which counts cells in a liquid medium or on a culture plate. A specialized Petroff-Hausser chamber can be used, which is a calibrated slide that holds a specific volume of the culture. Individual cells are counted with a microscope, using the chamber to calibrate the count. It works

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well with relatively concentrated solutions and does not tell the difference between living and dead cells unless a fluorescence stain is used. A Coulter counter is an electronic cell counting device that counts cells in an electrolyte solution by detecting electrical resistance changes brought on by the bacteria. This may not be as accurate in a concentrated solution compared to a dilute solution and cannot tell the difference between living and dead cells. Living cells can be counted with a viable plate count. A certain inoculum is spread onto a plate and allowed to grow. The number of colony-forming units is measured. It measures living cells and is best done when the number of colony-forming units is between 30 and 300 CFUs. It is difficult to assess bacteria that grow in chains or cultures. Serial dilution can be used in the viable plate method. It allows at least one of the plates to be in the desirable range. Dilutions are done in concentrations of one-tenth, onehundredth, or more of the original concentration. The dilutions are poured or spread upon the culture plate and incubated until one can see colonies growing. Some of the dilutions will be in the acceptable range for measurement. In dilute solutions, the original sample might need to be concentrated. This is done through a technique called membrane filtration, in which a porous membrane traps the microorganisms. Then the colonies are allowed to grow. The most probably number technique is a way of detecting the number of organisms in dilute samples. Samples of water that might be contaminated are inoculated into a broth. There are chemical methods that induce color changes or changes in turbidity that can detect the most probable number of organisms in the sample. Probability and statistics are used to determine how many organisms are in the different dilutions of the sample. Indirect measures will use things like turbidity to estimate the cell count in a liquid medium. This is detected using a spectrophotometer, which measures the amount of light getting through the sample. High concentrations of organisms will increase turbidity, which is measurable. It can be verified through direct cell count detection

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methods. Another technique is to get a dry weight after filtering, washing, and thoroughly drying the sample. Binary fission isn’t the only way cells divide. Some will have spore formation, others will fragment pieces off, such as is seen in Actinomycetes in the soil. Budding has been known to happen mainly in yeast cells but a few bacteria will do this as well. Many organisms in their natural environment will develop biofilms, which is a specific ecosystem involving microorganisms. There will be specialized channels for nutrient, water, and waste management. Some will attach to a solid surface, while others will form spongy mats in a liquid environment. These represent communities of organisms that combine for overall species survival. There are extracellular polymeric substances that are secreted into the biofilm, accounting for a great deal of the biofilm’s mass. It helps to maintain the biofilm integrity, while allowing for channels to form. Within a biofilm, there is metabolic collaboration so that there is sharing of nutrients and waste products. They are involved in quorum sensing, which allows organisms to detect the density of the biofilm. There can be an increase in pathogenicity and virulence when a certain quorum is detected. Gram-negative organisms communicate with one another through the use of N-acylated homoserine lactones. Gram-positive organisms use small peptides to communicate. These will be at a certain level when a quorum has been sensed. Biofilms can be either beneficial or harmful to humans. Certain respiratory and gastrointestinal biofilms are protective against pathogens, while plaque in the teeth is a harmful type of biofilm. Wound infections can form biofilms and there can be biofilms on artificial devices used in and on the body. Most pathogens are more resistant to antimicrobial agents when they form biofilms. Biofilms involve organisms in close proximity so they can share DNA that results in drug resistance.

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EFFECTS OF THE ENVIRONMENT ON MICROBIAL GROWTH Organisms survive and grow under optimal conditions but exactly what constitutes optimal conditions can vary with the type or organism. Some anaerobic organisms are not equipped to survive the reactive oxygen species that come out of living in a high oxygen environment. Organisms that survive in extreme locations are actually anaerobic and do not live well when oxygen is present. This can include places like sewers, the intestines, deep in the earth’s crust, bogs, and marshes do not survive well with oxygen present. Bacteria can be grown in thioglycolate tube cultures that is low in oxygen. The organism is stabbed into the tube and can become motile within it. It gradually picks up oxygen from the top of the tube on down. The maximum growth occurs at the level where the oxygen concentration is optimal. Organisms at the top are obligate aerobes, while organisms at the bottom are obligate anaerobes. Organisms that grow throughout are called facultative anaerobes but they will concentrate at the top. Aerotolerant anaerobes will growth throughout. Microaerophiles grow somewhere in between, where oxygen content is lower than atmospheric oxygen levels. Obligate anaerobes are difficult to grow. They need to be grown in an anaerobic jar, which chemically removes the oxygen, or in anaerobic chambers, which are also anaerobic. Mixed bacterial infections are common in humans, which usually involve anaerobes and aerobes. Some organisms will operate somewhere between a minimum permissive oxygen concentration and a maximum permissive oxygen concentration. The process of aerobic respiration involves the making of reactive oxygen species as byproducts. These need to be detoxified from the cell. There are three important enzymes that do this: catalase, peroxidase, and superoxide dismutase. Each does a slightly different thing to detoxify reactive oxygen species. Obligate anaerobes will not have any of these enzymes, while other organisms will have just one or two of these. Some organs are called capnophiles because they like high carbon dioxide levels and low oxygen levels. They can grow in candle jars in which a candle has been lit to get rid of oxygen and release carbon dioxide as a byproduct of the burning process.

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Organisms vary also in their desire for growth in an acidic or alkaline environment. Levels of pH less than 7 are acidic environments, while pH levels higher than 7 are alkaline environments. Extremes of pH can denature certain macromolecules. The proton motive force so important in making ATP will fall apart in an alkaline environment. Proteins are especially sensitive to pH levels. And yet, certain bacteria prefer more extremes in pH. These include the organisms that make pickles, yogurt, and other fermented food. Acidic foods can be healthy for digestion because they inhibit the growth of certain bacteria. There will be a minimum growth pH and a maximum growth pH with an optimum pH level for each organism. Most bacteria will be neutrophiles that like a pH near 7. Acidophiles like low pH levels. There are those that live in hot springs or sulfur fields that are considered extreme acidophiles. Lactobacillus in the human vagina like the pH 4 levels of that area. Many of these bacteria will pump hydrogen ions out of the cell actively in order to adapt to low pH levels. Alkaliphiles like higher pH levels. This is true of Vibrio cholerae, which is inactivated by stomach acidity. There are certain lakes that are very alkaline and contain these types of alkaliphile organisms. They have mechanisms to maintain the proton motive force despite the circumstances. Organisms can also tolerate ranges in temperature. There are organisms that thrive in the Antarctic and organisms at the bottom of hot ocean vents. There are optimal temperature levels for most of the organisms. Most organisms are called mesophiles and adapt to moderate environments. Psychrotrophs like cooler environments but those above freezing. Psychrophiles like temperatures below freezing. Thermophiles prefer heat and are seen in hot springs and compost piles. Hyperthermophiles like extremely hot environments so they cannot be killed with an autoclave. Many psychrophiles have characteristics, including antifreeze proteins, that protect it from extremes in temperatures. The lipids are hydrophobic and more flexible at low temperatures. Growth rates in these organisms are, however, quite slow. It should be noted that thermophiles and mesophiles can withstand freezing but of course do not

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grow well in freezing environments. Freeze-drying is a way of storing these organisms for an extended period of time. This is referred to as lyophilization. Thermophiles and hyperthermophiles have differences in their chemical environment. They have more cytosine-guanine base pairs, which are more stable. There are high amounts of saturated lipids to decrease the membrane fluidity. There are proteins that are resistant to denaturation called thermoenzymes. Microbes have rigid cell walls in order to protect them from dilute environments. This prevents cells from bursting under conditions of low osmotic pressure. There are halophiles that live in high-salt environments, usually seen in marine environments or in even higher salt environments like the Great Salt Lake and the Dead Sea. Salt is often pumped actively out of the organism. There are also halotolerant organisms that will survive in high salt environments but do not require such environments. Barophiles are organisms that survive the high-pressure environments of the bottom of the ocean. These are difficult to grow outside of these environments so they are not well studied. As already talked about, organisms differ in their need for light. Some will be photoautotrophs, while others will be photoheterotrophs.

MEDIA AND MICROBIAL GROWTH Organisms are best studied when they can be grown and cultured in a medium. There are several types of media you will come across. Tryptic soy broth or TSB is commonly used. There are media that are particularly enriched with things like vitamins and growth factors for fastidious organisms that do not grow easily in regular media. Chemically defined media have exact concentrations of certain chemicals. Complex media has extracts of meat, plants, or yeasts that are less precisely put together. Selective media will grow certain organisms but not others. Enrichment cultures are selective media that will grow certain organisms that are low in number to the exclusion of other organisms.

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CONTROLLING MICROBIAL GROWTH It is often necessary to control the growth of certain microorganisms, particularly those that grow on fomites, which are inanimate objects that can harbor organisms. Some organisms will be resistant to antimicrobial treatments, such as Clostridium botulinum, which survives canning procedures. There are four biological safety levels as outlined by the CDC. They depend on the infectivity of the organism, the ease at which it is transmitted, the disease severity possible, and the type of work being performed by the organism. A BSL-1 organism does not cause disease in the healthy host and there is little risk in working with them. A BSL-2 organism can cause a moderate disease in workers but are indigenous to the area. Personal protective equipment is required. BSL-3 agents can be lethal and some are considered exotic to the area. These include things like the organism that causes HIV and tuberculosis. BSL-4 organisms are very dangerous and usually fatal with no available treatments, such as smallpox and Ebola. Sterilization will kill cells, viruses, and endospores. Heat, filtration, pressure, or chemicals can kill these microbes. Aseptic technique is required to prevent contamination of surfaces that are considered sterile. Maintaining a sterile field in surgery helps to prevent sepsis in the surgical patient. Foods can be sterilized in commercial sterilization, such as those that get rid of botulism. Disinfection gets rid of most organisms on a fomite surface using chemicals or heat. It does not create sterility but is usually fast and inexpensive to do. Chlorine bleach is an example of a disinfectant. Antiseptics are those antimicrobials that are safe for human tissues. Degerming involves handwashing in order to physically get most of the organisms off the hands. Sanitation will clean fomites in order to prevent disease transmission. It involves things like cleaning bathrooms and using a dishwasher. There are physical and chemical methods of getting rid of microorganisms. Some methods are bactericidal or viricidal because they will kill the organism. Fungicides will kill fungi. Things that are fungistatic or bacteriostatic do not kill the organism but stops the growth of the organism. Bacteriostatic agents in plastics, for example, will inhibit

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growth. Longer exposure times will kill more of the microorganisms. Low population volumes are killed easier than high populations of organisms. Heat is the oldest method of microbial control. Cooking and canning will do this. There is a thermal death point, which is the lowest temperature that will kill the organism in a ten-minute exposure time. Endospore-formers are definitely more heat tolerant. The thermal death time is the time necessary to kill all microorganisms at a specific temperature, which might be boiling temperature or the temperature of an autoclave. Boiling will not kill endospores easily unless high pressures are involved. There is dryheat sterilization and moist-heat sterilization technique. Ovens will use dry heat, while boiling uses moist heat. Autoclaves are moist-heat sterilization techniques. It uses both pressure and steam to kill the organisms. There are indicator tapes in autoclaves that will turn color when a certain temperature has been achieved but it doesn’t indicate how long the heat has been applied. A better indicator is using a suspension of endospores, which will tell if the heat has been applied long enough and high enough. G. stearothermophilus is the bacterium often used because it is very heat-stable. Diack tubes are glass ampules that will contain a melted pellet if the proper sterilization temperature has been reached. Pasteurization is a food-based technique that kills organisms while maintaining normal food quality. The food is heated to reduce organisms but it is not considered sterile. Milk, honey, and apple juice are often treated with pasteurization. There are differences in pasteurization techniques. With HTST pasteurization, normal milk is exposed to a temperature of 72 degrees for 15 seconds. UHT pasteurization or ultra-hightemperature pasteurization is what’s done to store milk unrefrigerated for long periods of time. Freezing and refrigeration will be bacteriostatic for organisms that are not psychrophiles. Growth of the microorganisms is curtailed but not necessarily permanently so. Freezing will kill certain organisms, however. Thawed foods are not protected and should be considered perishable. Dry ice and liquid nitrogen can easily keep bacteria out of the food product.

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High pressure is called pascalization. It is used to kill many pathogens by exposing food to high pressures. It will not always kill the endospores. Hyperbaric oxygen is used in a medical setting to fight the growth of anaerobic organisms using both oxygen and high pressure. Drying or desiccation will preserve many types of foods but the organisms will often grow back if the environmental conditions involve higher temperatures and higher water content. Lyophilization is used in the laboratory, which involves freeze-drying the organism. These can later be stored in dry conditions, even at room temperature. Some foods are dried with salt or sugar to decrease the ability of organisms to grow. Ionizing radiation can kill organisms. It can pass through cells and damage cellular components. There will be mutations to the food products that have been irradiated. Many processed foods in plastic and paper can be treated with ionizing radiation and certain laboratory equipment can be sterilized this way. UV light or nonionizing radiation can kill organisms. UV light-related purifiers will clean water for campers. It does not penetrate solid objects, however, so it has limited capabilities. Other techniques for reducing the organisms in a substance includes sonication, which involves high-frequency ultrasound waves. HEPA filters are used to sterilize the air in certain clinical settings. They are also found in airplanes, automobiles, air purifiers, and air conditioning systems. Membrane filters are used to filter antibiotic and vitamin solutions as well as culture media in order to sterilize the liquid without the damaging effects of heat.

ANTISEPTICS Antiseptics involve chemical control of microorganisms. The earliest antiseptic used was phenol. This is what Listerine is made from. It is too irritating to be used on skin surfaces in surgery. There are related compounds called phenolics. These include products called Lysol and Phisohex. Triclosan is used in antibacterial hand soaps and toothpaste. Triclosan is bactericidal. Other antiseptics contain heavy metals but these are also damaging to humans by denaturing proteins. These include mercury, which was used once to treat syphilis. 168

Tincture of mercury was once used as an antiseptic. Silver is an antiseptic, such as is seen in Silvadene cream. Silver nitrate was once used as an eyedrop in newborns. Others include copper sulfate, nickel-containing products, and zinc. Zinc oxide is used in diaper creams to fight infection. The halogens, such as iodine, fluorine, and chlorine are antimicrobial. Povidone-iodine is used to scrub before surgery and in surgical surfaces. Water is chlorinated in order to remove bacteria, such as is seen in swimming pools. Chloramine tablets are used to disinfect water after a natural disaster or in the military. These substances will not kill all microorganisms. Fluoride is used in toothpastes and in certain water supplies in order to prevent tooth decay. Alcohols will kill bacteria, fungi, and some viruses. They do not kill spores but will inhibit the germination of spores. They can be irritating to skin. Both ethanol and isopropyl alcohol will kill bacteria and will be good anti-infectives. Surfactants involve most soaps or detergents. They are not antiseptics but are degerming agents by removing bacteria specifically from a surface. Some soaps, however, will contain an antiseptic or bacteriostatic agent. Bisbiguanides are newer agents that include things like chlorhexidine. These will kill most organisms except for Pseudomonas aeruginosa. It will also kill enveloped bacteria but is ineffective in killing the organism causing tuberculosis, spores, or nonenveloped viruses. It is currently commonly used for surgical scrubbing and to treat gingivitis. Alkylating agents will inactivate certain enzymes as well as nucleic acids. This is what’s seen in formaldehyde. It will decrease the temperature necessary to sterilize a substance. It is used to store tissues and organs after autopsies and is used in embalming. Glutaraldehyde is similar to formaldehyde and is used to disinfect surgical or other medical equipment but is too irritating to the skin to be used as an antiseptic. Ethylene oxide is a form of gaseous sterilization. All alkylating agents are carcinogenic so they must be used carefully. Peroxygens are things like hydrogen peroxide. They can be used as disinfectants but can be irritating to the skin, delaying skin healing and causing scarring of tissue. It is what’s frequently used in contact lens cleaners. Bacteria that contain peroxidase or catalase 169

will have resistance to hydrogen peroxide because they will be protective against hydrogen peroxide. Gaseous hydrogen peroxide will be used sometimes to sterilize equipment or entire rooms. High-pressure carbon dioxide is a supercritical fluid that can be used in a chamber to penetrate cells and kill endospores. It works better if cycled between pressurization and depressurization or if heat is applied as well. It can treat foods and be used in certain medical equipment sterilization because it doesn’t destroy things or break down things. There are natural and artificial food preservatives, such as nitrites, nitrates, and propionic acid. These will not change the taste or properties of food. Others are benzoic acid or sodium benzoate, used in sweet food preservation by inhibiting the citric acid cycle. Natural food preservatives, such as nisin, which is made by Lactococcus lactis, can be used. It is used to preserve certain beverages, meats, and certain cheeses. Natamycin is a natural antifungal product used in cottage cheese and other types of cheese.

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KEY TAKEAWAYS •

Bacteria usually use binary fission and go through many different phases in a fixed culture.

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There are many things that affect the growth of organisms, including oxygen, temperature, pH levels, salinity, and pressure levels.

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There are several techniques that can estimate the concentration of organisms in a culture.

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There are certain types of media that can be used to grow certain microorganisms.

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Bacterial growth can be inhibited by a wide variety of physical techniques, such as heat, freezing, and freeze-drying.

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There are many different chemical antiseptics and disinfectants used in many different medical, laboratory, and food-related settings.

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QUIZ 1. What is the last thing that separates the cell completely when binary fission takes place in prokaryotic cells? a. A cell wall forms between the daughter cells. b. A Z-ring is formed c. The cell membrane is built between the cell halves. d. The cell membrane constricts like purse strings. Answer: a. The separation of the cell occurs when there is a cell wall built between the two daughter cells. The other choices do not effectively separate the cell. 2. In which phase of cell growth in medium is there shock to the organisms so they do not divide? a. Log phase b. Stationary phase c. Lag phase d. Decline phase Answer: c. In the lag phase, there is the presence of an inoculum. Because of shock to the organisms and the beginnings of cellular growth without cell division, the cells do not immediately begin to divide.

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3. What technique will be able to directly measure the bacterial cell count in a dilute solution containing bacteria? a. Pour method b. Most probable number method c. Membrane filtration d. Spread method Answer: c. With membrane filtration, a known quantity of the suspension is filtered and bacteria are trapped and allowed to grow into colony-forming units. The number of bacterial colonies detected is divided by the quantity of liquid filtered. It directly measures the CFUs in a dilute solution. 4. Which technique is an indirect method for estimating the cell count in a liquid medium? a. Pour method b. Spectrophotometer c. Membrane filtration d. Spread method Answer: b. The spectrophotometer measures turbidity, which will increase as the cell count increases. It indirectly measures the cell count. 5. Which organisms tolerate a specific oxygen concentration that is less than atmospheric oxygen levels? a. Microaerophilic b. Aerotolerant anaerobes c. Facultative anaerobes d. Strict anaerobes Answer: a. The microaerophilic organisms tolerate a specific oxygen concentration but it will be less than the concentration of oxygen in the atmosphere.

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6. What is the purpose of superoxide dismutase, catalase, and peroxidase in the cell? a. They are important in oxygen metabolism. b. They get rid of reactive oxygen species. c. They participate in aerobic respiration. d. They get rid of waste products in the cell. Answer: b. These are enzymes that act to get rid of reactive oxygen species that are generated as part of the respiratory processes in the cell. 7. What is not a way that thermophiles cope with their high-temperature environment? a. They have high guanine-cytosine base pair ratios. b. They have specialized thermoenzymes. c. They have antifreeze proteins in their cytoplasm. d. They have high ratios of saturated to unsaturated fatty acids. Answer: c. Each of these is characteristic of thermophiles except that they do not need antifreeze proteins. 8. Which culture medium is used to grow fastidious organisms? a. Selective media b. Enriched media c. Differential media d. Complex media Answer: b. There are many types of media, including enriched media that contains special vitamins and nutrients to grow particularly fastidious organisms.

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9. Which is an example of moist-heat sterilization in a laboratory? a. Oven-heating b. Flame-heating c. Autoclave-heating d. Incineration Answer: c. Autoclaves use a combination of temperature and pressure in order to kill microorganisms in a laboratory. 10. Which organism is least likely to be killed using boiling techniques? a. Staphylococcus aureus b. Escherichia coli c. Streptococcus pyogenes d. Clostridium botulinum Answer: d. Clostridium botulinum involves the production of heatstable spores that don’t get killed unless there is high pressure and temperature applied for a prolonged period of time.

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CHAPTER ELEVEN: ANTIMICROBIAL AGENTS This chapter focuses on the different antimicrobial agents used to treat infectious diseases. There are different classifications of antimicrobial agents, some being bacteriostatic and some being bactericidal. There are antibiotics, antifungals, and antivirals, which are covered in this chapter. The public and medical professionals face serious challenges with regard to antibiotic and drug resistances. Some of these challenges are discussed as part of this chapter.

ANTIMICROBIAL THERAPY When a doctor prescribes antimicrobial therapy, there are several things to take into account. One of these is whether the drug is bacteriostatic or bactericidal. Others include the spectrum of activity of the drug, the dose to be given, the route to be recommended, and the potential for drug-to-drug interactions. An antibacterial drug can be bacteriostatic to the organisms or bactericidal. In patients who are immunosuppressed, a bactericidal drug is recommended because they do not have the immune system to adequately kill off the organism. In addition, lifethreatening situations, a bactericidal drug is also definitely recommended because of the seriousness of the infection. Certain organisms are by nature severe so bactericidal activity is also recommended. The spectrum of activity is also important. If the organism is known, a narrowspectrum antimicrobial drug is recommended because it is least likely to interact with the body’s normal flora and it targets just ma specific group of pathogens. If the organism isn’t known or if there are multiple pathogens, a broad-spectrum antimicrobial drug is chosen. These are often used empirically when the organisms are not known. Broad-spectrum antibiotics are also used in polymicrobial infections or when preventing infections prior to surgery. These are also used when there are drug resistances involved.

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The biggest problem with using a broad-spectrum antibiotic is that there is often a mass kill-off of the normal microbiota of the individual. This increases the chance of having a superinfection, which happens in the person on antibiotics who has a preexisting infection. One superinfection particularly dangerous is Clostridium difficile, which leads to pseudomembranous colitis. Candida yeast infections can also become superinfections. The dosage must also be considered. An optimal therapeutic level must be achieved at the site of the particular infection and the time between doses must keep the drug levels in the therapeutic range. All of this needs to happen without causing toxic side effects in the patient. An allergy is not generally related to dosage but can also be a side effect. The presence of kidney and liver disease might change the dosage as well as the mass of the patient. The half-life of the drug is important in spacing the doses. The half-life is the time it takes to metabolize or eliminate half of the drug. This will be different from drug to drug and can sometimes relate to the ability of the patient to eliminate the drug. The goal is not to have too many peaks and troughs in the concentration of the drug over time, although some drugs are more effective when given in a single large dose. The route of administration can affect how the drug works. Some drugs can be given orally as long as they are absorbed easily by the GI tract. Intestinal infections are particularly well-suited to giving an oral drug—even those that do not absorb easily into the bloodstream. Parental routes, such as intravenous or intramuscular routes, are better when the drug is not easily absorbed or when the patient is vomiting or otherwise cannot take in oral intake. Levels reached intravenously are generally higher than those levels of the drug given orally. Sometimes, more than one drug is given at a time. These can be synergistic with one another and can better treat the infection. Some bacteriostatic drugs given together become a bactericidal combination. There can be antagonism between two antimicrobial drugs or between an antibiotic and a non-antibiotic drug. An example of this is the giving of an antacid and an antibiotic best absorbed under acidic conditions.

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This is an antagonistic situation because the antibiotic will less efficiently be absorbed in the presence of the antacid.

ANTIBACTERIAL THERAPY The goal of antibacterial therapy is to be selectively toxic. This means that the drug is toxic to the organism but not to the host. It generally also means that the drug will harm prokaryotes but not eukaryotes. Every antibacterial drug has a specific mode of action against the organism. It can attack the cell wall, the plasma membrane, the ribosomes, DNA synthesis, or the metabolic pathways of the organism. Examples of drugs that attack the cell wall synthesis include penicillins and bacitracin. Protein synthesis in the ribosomes is affected by aminoglycosides, macrolides, and lincosamides. Membranes are disrupted by polymyxin B and daptomycin. Nucleic acid synthesis is affected by fluoroquinolones and rifamycin. Folic acid synthesis is affected by trimethoprim and sulfonamides. There are also drugs that affect the synthesis of ATP and mycolic acid. The first antibiotic known in modern times was penicillin. It belongs to the beta-lactam class of antibiotics, which also includes monobactams, carbapenems, and cephalosporins. Beta-lactams affect the synthesis of the bacterial cell wall because it prevents cross-linking of the peptidoglycan layer. These tend to work better on grampositive organisms. Penicillin is a natural antibiotic. Amoxicillin and ampicillin are aminopenicillins synthesized from natural penicillin. Methicillin is also semi-synthetic. Cephalosporins are also beta-lactams with slightly different chemical properties. It is less resistant to the beta-lactamase enzymes that some organisms secrete. There are many generations of cephalosporins that differ in their spectrum of activity. First generation cephalosporins are the most broadly acting of these drugs, while a new fifth generation cephalosporin is only active against MRSA or methicillin-resistant Staphylococcus aureus. Aztreonam is the only monobactam and it is only active against gram-negative organisms.

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Vancomycin is a glycopeptide that also inhibits bacterial cell wall biosynthesis. It is a bactericidal drug often used to treat MRSA. It binds to precursors that make the cell wall, preventing cell wall synthesis and killing the organism. It cannot kill gramnegative bacteria because it cannot get through the outer membrane of these organisms. Bacitracin was once harvested from the Bacillus subtilis organism. It affects the cell membrane and prevents peptidoglycan precursor molecules from getting to the outside of the cell membrane. This is how it blocks cell wall synthesis. It is used mainly topically to kill Staphylococcus and Streptococcus but is toxic to the kidneys if given orally. There are drugs that rely on the fact that the ribosomes in prokaryotes are different from those seen in eukaryotes. Aminoglycosides are antibiotics that do this by blocking the ability of ribosomes to proofread the proteins being made. Abnormal proteins get made quite easily, which kills the cells. These are broad-spectrum antibiotics such as streptomycin, gentamicin, and neomycin. The problem with these drugs is that they are ototoxic and cause deafness, neurotoxic, and nephrotoxic, causing kidney damage. There is a narrow range in the bloodstream where the drug is effective without being toxic. Tetracycline binds to the small subunit of the ribosomes and is bacteriostatic by blocking the activity of transfer RNA. There are natural tetracyclines and semisynthetic tetracyclines. The danger to these drugs is discoloration of the teeth, liver toxicity, and phototoxicity so that sunburn can easily occur while using the drug. There are other drugs that bind to the large subunit of the ribosomes. This is true of the macrolides, which are bacteriostatic and broad-spectrum antibiotics. The first of these drugs was erythromycin. A semisynthetic related drug is azithromycin. They block the elongation of proteins by blocking some peptide bonds, keeping them from forming. Azithromycin is the better drug because it has a considerably longer half-life and the ability to be taken in very short courses. The lincosamides like clindamycin and lincomycin are related to macrolides and also bind to the larger ribosomal subunit. They also block some peptide linkages. Chloramphenicol is distinct but also binds to the larger ribosomal subunit, blocking peptide bonding. It is both a natural and synthesized antibiotic, being the first to be

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mass-produced. It has several adverse effects, mainly that it blocks the activity of the bone marrow to make blood cells. For this reason, it is not often used. Aplastic anemia from this drug is irreversible and very dangerous. Linezolid is a newer antibiotic that binds to the larger subunit of the ribosomes. It acts uniquely by preventing the 50S and 30S ribosomal subunits to bind together, preventing protein synthesis. It keeps the polypeptide chain from moving from the A site to the P site in the ribosomal complex. There are drugs that specifically target the bacterial cell membrane. These include polymyxins, such as polymyxin B. These act like detergents to disrupt the gram-negative outer membrane, killing the bacteria. These can be nephrotoxic and neurotoxic so they are used topically in antibiotic ointment. Oral colistin is used to decontaminate the bowel; it is used intravenously as a last resort when there is a serious infection. Daptomycin is a lipopeptide that disrupts the cell membrane, killing gram-positive organisms. The main side effect of daptomycin is muscle aches. Some antibacterial drugs will block nucleic acid synthesis. Metronidazole can do this for bacterial and protozoal infections. DNA replication is affected. Rifampin targets RNA polymerase which, as you remember, is different between bacteria and eukaryotes. Rifampin is used with other antibiotics to kill tuberculosis-causing organisms. The biggest risk is hepatotoxicity. Fluoroquinolones were originally made from nalidixic acid. Both levofloxacin and ciprofloxacin inhibit DNA gyrase in the microorganism. There are many side effects, including those on the heart, glucose metabolism, nerves, skin, and tendons. These drugs will treat both gram-positive and gram-negative organisms. Drugs can also inhibit bacterial cell metabolism. This is true of sulfonamides, which block folic acid synthesis, which in turn affects nucleic acid synthesis. Sulfonamides are bacteriostatic and are not harmful to humans, who get folic acid from the diet. Allergic reactions to these drugs are common. Sulfones are related drugs used to help people with leprosy or Hansen’s disease. Trimethoprim inhibits another enzyme in this synthetic pathway. This is why trimethoprim and sulfonamides are considered synergistic and bactericidal when used together.

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Isoniazid is an antimetabolite drug used to treat tuberculosis, along with rifampin and streptomycin. It prevents the synthesis of mycolic acid used to make the cell wall of these organisms. Like other drugs against tuberculosis, it can be hepatotoxic but also causes nerve damage and anemia. A new class of drugs is the diarylquinolines, which inhibits ATP synthesis in cases of tuberculosis. It is a risky class of drugs to use because it is hepatotoxic and causes heart arrhythmias. For this reason, it is not used unless there is severe tuberculosis that cannot otherwise be treated effectively.

OTHER ANTIMICROBIAL THERAPIES Other antimicrobial drugs are more difficult to use because fungi, helminths, and protozoans are eukaryotic so selective toxicity is more difficult to manage. Antiviral drugs often damage host human cells because the viruses reproduce intracellularly. Antifungal drugs most often target the cell membrane of fungi because fungi have ergosterol in the cell membrane, which isn’t the case in humans, who use cholesterol. Imidazoles impair ergosterol synthesis, including ketoconazole, miconazole, and clotrimazole, which treat many human fungal infections like yeast infections, athlete’s foot, and ringworm. Related drugs include fluconazole, which can be given intravenously, terbinafine, which targets a different step in ergosterol biosynthesis, and amphotericin B, which creates pores in the fungal cell membrane by binding to ergosterol. Much less commonly used are the echinocandins, which affect fungal cell walls, and nikkomycin Z, which affects chitin synthesis. Griseofulvin interferes with fungal cell division by blocking the mitotic spindle; it is only used topically. Atovaquone is an antimetabolite that mimics coenzyme Q. Antiprotozoal drugs target the differences between protozoans and humans. Atovaquone targets the electron transport chain in protozoal organisms. Proguanil is another antiprotozoal drug that blocks folic acid synthesis. It is used along with atovaquone to treat malaria. Artemisinin is also effective against malaria and tends to build up reactive oxygen species in these organisms. 181

There are other antimetabolite drugs that will treat things like toxoplasmosis. Sulfadiazine blocks folic acid synthesis and can also treat malaria. Pyrimethamine is a related drug that targets a different enzyme in folic acid synthesis. Nitroimidazoles and quinolones block nucleic acid synthesis in protozoal organisms. Metronidazole is used to treat giardia, trichomonas, and Entamoeba infections. The drug breaks DNA strands, blocking DNA replication. The main adverse effect is an increase in cancer risk in humans. Pentamidine is also a drug that blocks DNA replication, used to treat African sleeping sickness and leishmaniasis. In addition to its effects on DNA replication, it also affects the loading of amino acids onto transfer RNA. Quinolones are used to treat malaria and are related to quinine. They block the ability of the protozoan to take up amino acids from hemoglobin. It cannot be used long-term because of serious adverse effects. It will also treat amebiasis. Helminths are hard to treat because these are multicellular eukaryotes. Mebendazole and albendazole can treat these infections by preventing the formation of microtubules. It affects the intestines of the helminth so that glucose cannot be taken up. Ivermectin blocks specific glutamate-gated chloride channels only seen in invertebrates so they die from starvation and paralysis. Several helminthic infections are treated with ivermectin as are infections with bed bugs, lice, and mites. Other drugs against helminths include Niclosamide, used to treat tapeworm infestations because it is not absorbed by the GI tract. Praziquantel will also treat liver flukes, schistosomiasis, and tapeworm infections. Thioxanthenones are related to quinine and treat schistosomiasis. They block RNA synthesis. Antiviral drugs are used against viruses. It is hard for scientists to develop specific drugs that are not also toxic to humans. Most of these drugs are nucleoside analogs that block nucleic acid synthesis. Acyclovir mimics guanosine and is used for herpes viral infections. This includes genital herpes, shingles, chickenpox, and Epstein-Barr viral infections. The biggest risk of using this drug is nephrotoxicity. Ribavirin is another guanosine analog, used to treat hepatitis C along with interferon. Amantadine and rimantadine bind to certain transmembrane proteins so that the influenza virus cannot escape the cell. The biggest problem with this drug is the 182

increased frequency of drug resistances. Neuraminidase inhibitors like those in Tamiflu and Relenza specifically attack influenza viruses by preventing the release of the virus from the host cell, shortening the course of the disease. HIV disease is treated on the basis of the fact that the virus is a retrovirus, turning its RNA into DNA in the host cell. Drugs to treat these infections include reverse transcriptase inhibitors. There are other antiviral drugs that are called protease inhibitors and integrase inhibitors. Protease inhibitors are used to treat hepatitis C. Integrase inhibitors and fusion inhibitors are also used to treat HIV viral infections. There are other drugs for HIV disease that prevent attachment of the virus to the host cell.

DRUG RESISTANCE Microbial organisms are always evolving in order to overcome their environment. This leads to drug resistance, made worse by overprescribing of antimicrobials, using the wrong antimicrobial, patient noncompliance, and subtherapeutic dosing. There will be chromosomal mutations that will cause resistance, with the resistance being transferred vertically to the next generations. Plasmids and transposons can also cause drug resistance. There are four main areas that contribute to drug resistance. The organism can create an efflux pump that pumps the drug out of the cell. Other infectious organisms will have blocked penetration of the drug into the cell. The organism can change its target morphology to be less sensitive to the antimicrobial agent. Still others will inactivate the drug by making a specific enzyme against it. Beta-lactam resistance and aminoglycoside resistance can occur by the organism making a drug that inactivates the drug. Some organisms will break the beta-lactam ring, inactivating the beta-lactam drug. Rifampin can be inactivated through related mechanisms. Gram-negative organisms will make channels to efflux the drug out of the bacterial cell. Drug resistance to tetracyclines, beta-lactams, and fluoroquinolones can also happen through the formation of efflux pumps. There are many ways to also confer resistance by changing the target of the antibiotic.

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Multi-drug resistance is particularly damaging. This will create superbugs that are resistant to many different antimicrobials. A common method of doing this is to have a broad-spectrum efflux pump that can pump out many antimicrobial drugs. Nosocomial infections that occur in a medical setting are much more likely to be related to multidrug resistance. MRSA or methicillin-resistant Staphylococcus aureus is a particularly problematic organism to treat. It is resistant to methicillin and all the beta-lactamase drugs. While it was first just an opportunistic and nosocomial infection, it is now a communityassociated infection, causing about six percent of staphylococcal carrier statuses. There are now vancomycin-resistant Staphylococcus aureus infections and vancomycinresistant enterococcal infections, usually spread in the hospital setting. Resistances also exist with gram-negative organisms that make extended-spectrum beta-lactamases and carbapenem resistances seen with Enterobacter organisms. Many strains of Mycobacterium tuberculosis are resistant to multiple drugs.

IDENTIFYING NEW ANTIMICROBIALS AND DRUG SENSITIVITIES There are tests that can be done to test whether or not an antimicrobial drug is effective against a particular organism. The most common test is the Kirby-Bauer disk diffusion test. Organisms are grown on an agar plate and then disks with antimicrobial drugs impregnated into them are placed on the disk. Antibacterial activity is identified as a clear spot called the zone of inhibition around the disk. The diameter of the disk determines the level of antibacterial activity of the drug. It cannot tell the difference between a bacteriostatic and bactericidal drug. It cannot compare one drug with another. Dilution testing can provide the doctor with the drug’s MIC or minimal inhibitory concentration, which is the lowest concentration of a drug that will inhibit visible growth of the bacterium and minimal bactericidal concentration or MBC, which is the lowest concentration of the drug that kills more than 99.9 percent of the starting organism inoculum. The MIC will be seen by looking for cloudiness of the broth, while

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the MBC is detected by checking to see how many organisms grow. The drug’s level in the serum must be at least three times the MIC to treat an infection. The E-test will combine the Kirby-Bauer test and the dilution testing. Instead of a disk placed on the agar disk, there is a strip that has a gradient of antibacterial concentrations. There will be an elliptical zone around the strip that will determine the MIC. It will compare one drug to another but cannot say what the MBC of the drug will be. The strip is labeled with different dilutions of drug on it. New drugs can be made against pathogens by chemically altering existing drugs until something is found to be both effective and safe. Soil, vegetation, and microbial products are tested to see if there is antimicrobial activity found in any of these sources. There is a special technique called the iChip, which grows organisms right in the actual soil to see if anything in the dirt has antimicrobial effects. Soils from around the world have been tested, but there are marine sources that have not yet been tested.

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KEY TAKEAWAYS •

Antimicrobial drugs can be against bacteria, fungi, protozoans, helminths, and viruses.

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Prokaryotic organisms are easier to act against because of differences between prokaryotes and eukaryotes.

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Antibacterial agents can act on the cell wall, cell membrane, nucleic acid synthesis, protein synthesis, and cell metabolism.

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Antiprotozoal drugs are mainly those against malaria, although there are other protozoal infections that can be treated.

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Antifungal drugs are often topical, although there are oral drugs that affect the fungal cell wall or fungal metabolism.

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Antiviral drugs will include nucleoside analogs as well as drugs preventing viral release and specific drugs for retroviruses that contain RNA instead of DNA.

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There are ways to determine if an antimicrobial drug is inhibitory or bactericidal to a specific organism. This is called looking for drug sensitivity.

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QUIZ 1. What is least likely to be a reason to use a bactericidal drug? a. In an immunocompromised host b. With a particularly virulent organism c. When the organism isn’t known d. When the infection is life-threatening Answer: c. Each of these is a situation in which a bactericidal drug is recommended; however, it is not necessarily recommended when the infection is not known. 2. Which type of antibiotic is generally chosen empirically when the causative organism isn’t known? a. Broad-spectrum b. Narrow-spectrum c. Bactericidal d. Bacteriostatic Answer: a. A broad-spectrum antibiotic is often chosen when the causative agent isn’t known so that there is a greater chance of covering for the most probably organism in the infection. 3. What do the beta-lactam drugs have the most affect on with regard to antibacterial activity? a. DNA synthesis b. Cell membrane c. Cell wall d. Metabolism Answer: c. The beta-lactam antibiotic classification is one that acts directly on cell wall synthesis, which is something relatively unique to prokaryotes.

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4. What part of the bacterial organism is most affected by vancomycin? a. DNA synthesis b. Ribosomes c. ATP synthesis d. Cell wall Answer: d. Vancomycin binds to the precursors of cell wall synthesis so it cannot be made. This is a bactericidal drug that is effective only in killing gram-positive organisms. 5. What adverse effect of chloramphenicol prevents its common usage in humans today? a. Bone marrow suppression b. Kidney failure c. Neurotoxicity d. Cancer-causing effects Answer: a. Chloramphenicol can cause aplastic anemia and other forms of bone marrow suppression so that it is not frequently used today as an antibiotic of choice. 6. What is the site of activity of drugs that block protein synthesis, such as aminoglycosides and tetracyclines? a. Amino acid synthesis is blocked. b. Uptake of amino acids are blocked. c. Post-translational modification is blocked. d. There is prevention of the peptide bond formation. Answer: d. These drugs will prevent the formation of the peptide bonds by binding to the different subunits of the ribosomes. Each drug has a slightly different effect on polypeptide synthesis.

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7. What do most protozoal drugs like artemisinin and proguanil treat? a. Ringworm b. Giardia c. Rickettsia d. Malaria Answer: d. These drugs are particularly effective in the treatment of malaria, often used in combination in order to prevent resistances to these drugs. 8. What do drugs like mebendazole and albendazole treat? a. Protozoans b. Helminths c. Viruses d. Fungi Answer: b. These drugs treat helminthic infections by blocking microtubule formation in the intestinal lining cells of the helminth organism. This blocks glucose uptake by the organism. 9. What type of infection is most likely to be related to multidrug resistance? a. Infections in the elderly b. Nosocomial infections c. Gram-positive infections d. Community-acquired infections Answer: b. Nosocomial infections that occur in medical settings have the greatest risk of being caused by multidrug resistance because of the number of antimicrobial drugs used in these types of settings.

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10. What will the Kirby-Bauer test determine? a. The presence of a bactericidal versus a bacteriostatic drug b. The degree of virulence of an organism c. The presence of a possible effective antimicrobial organism d. The nutrients an organism needs to grow Answer: c. The Kirby-Bauer test will be able to predict a possible presence of an effective antimicrobial agent.

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CHAPTER TWELVE: PATHOGENICITY AND DISEASE The focuses of this chapter are pathogenicity, infectious diseases, and epidemiology of infections. The basic definition of an infectious disease is explained as well as what defines a pathogen. There are specific virulence factors that identify viruses, prokaryotes, and some eukaryotes as being pathogenic in nature, which are discussed. The study of epidemiology as it applies to tracking infectious diseases is also covered in this chapter.

WHAT IS AN INFECTIOUS DISEASE? A disease is defined as any condition that causes damage or impairment to the structure or function of the body or its organs. Not all diseases are infectious in origin. Those diseases that are caused by an infection are said to be caused by a pathogen. In most cases, there will be signs and symptoms of a disease. The signs of a disease involve anything that can be measured or observed. These can include changes in vital signs, laboratory findings, and things that the practitioner can observe directly. Symptoms, on the other hand, will be subjective and will be felt by the patient himself. This can be a certain level of pain or other symptoms like nausea or upset stomach. A syndrome is a collection of signs and symptoms that are related to a certain disease. A particular syndrome can be related to several different microorganisms so it can be difficult to find a one-to-one correlation between an organism and the syndrome it causes. An example is a diarrheal disease, which can be viral, bacterial, or parasitic in nature. Fever could be related to just about any pathogen. Other diseases are either subclinical or asymptomatic. The person could be infected yet they would not know it. Diseases worldwide are defined and monitored by the World Health Organization. According to WHO, an infectious disease will be any disease caused by a pathogen directly. Some of these diseases will be communicable, meaning they can be passed from one person to another directly or indirectly. Some communicable diseases are 191

considered contagious, which means it can easily be spread from one person to another. Some diseases are more contagious than others. It depends on the pathogen and on how it is passed from one to another. There are characteristics of the hosts that also affect contagiousness. Hospital-acquired or medically-acquired diseases are called nosocomial diseases. The patients tend to be sick from the beginning, have weaker immune systems, with more virulent and drug-resistant organisms. Other diseases are considered zoonotic or zoonoses. These are those transmitted from an animal to humans. Bites and stings can cause these types of infections. There are noncommunicable infectious diseases that are not passed from person to person. Examples of these include those that cause tetanus, which comes from soil spores, and those that cause Legionnaire’s disease, which lives in water-cooling towers. These cannot be passed from person to person. There are also many noninfectious diseases that can have many different origins but that are not caused by a pathogen. Some causes are inherited diseases, congenital diseases (which are present at birth), degenerative diseases, nutritional deficiency diseases, endocrine diseases, idiopathic diseases, and neoplastic or cancerous diseases. Infectious diseases have five different periods or stages. The first is the incubation stage, after entry of the pathogen but before there are enough organisms to cause signs or symptoms. Incubation periods can be very short—a couple of days or many years. The incubation period depends on the size of the inoculum, the type of organism, the site of infection, the virulence of the organism, and host defense mechanisms. Next comes the prodromal period. There is an increase in the number of pathogens and nonspecific signs of illness and/or inflammation. Ultimately, the illness period is entered, with the highest number of living pathogens in the body and more typical symptoms suggestive of disease. This is followed by the period of decline. The pathogen number decreases and the symptoms diminish. Secondary infections are always possible because of a weakened immune system. Lastly, there is a period of convalescence, with recovery to normal function.

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Many diseases are most contagious in the prodromal period because of increased viral numbers and the lack of a clear disease state. This confers an advantage to the organism, because it maximizes the spread of disease before a quarantine can be effective. In reality, contagion of the disease can happen at any point in the illness process. Different diseases will be contagious at different times during the infection. There are several types of infectious diseases. In an acute disease, the changes are relatively sudden, with active disease symptoms and short incubation periods. Influenza is generally an acute disease. Chronic diseases can last months or years and may never clear out completely. This involves a prolonged period of infectivity and communicability. Hepatitis C and Helicobacter pylori cause chronic infectious diseases. In latent diseases, the pathogen becomes dormant and is not transmissible. This is what’s seen with mononucleosis, caused by Epstein-Barr virus, herpes simplex viruses, and chickenpox viruses. The reactivation can happen many years later.

PATHOGENS The understanding of pathogens was advanced by the work of Robert Koch, who developed Koch’s postulates. It should be noted that Koch’s postulates do not work in all cases of infectious diseases but are good general rules to follow. According to these postulates, an infectious disease must follow these rules: 1. The pathogen must be present in every person who has the disease but not in healthy persons. 2. The pathogen should be able to be isolated and grown in a pure culture. 3. A healthy person who becomes infected should develop the same symptoms as the sick person. 4. The pathogen should be isolated from the new host and must be the same as in the original host. There are limitations to the postulates. Examples include Helicobacter pylori, which can be present in the healthy person and in the person with disease. In addition, not all healthy subjects can develop the disease when exposed. There are host factors that prevent getting the disease in certain resistant hosts. In addition, two different people

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with an infection by the same pathogen might have different symptoms. Finally, not all organisms can be grown in pure cultures. This includes Rickettsia and Chlamydia, which must be grown with a host, and viruses, which are not easily cultured. Finally, because of ethical reasons, it is not possible to infect a person with HIV or other serious diseases. A revised set of Koch’s postulates was developed during the 1980s. There are called molecular Koch’s postulates and uses the ability to detect a specific gene that is causative of a particular pathogenic disease. It relies on the ability to determine which gene is responsible for pathogenicity. It helps to explain why some organisms of the same species can be pathogenic, while others are not. It also helps to identify intracellular pathogens. The revised postulates include the fact that the disease is only associated with the pathogenic form of a potential pathogen. In addition, inactivation of the offending gene diminishes pathogenicity and reactivation of the gene brings pathogenicity back. An agent that can cause disease is referred as being pathogenic, while the degree of pathogenicity is called virulence. There are those organisms that have little virulence and those that are highly virulent. The symptoms of a low virulence organism will be less than those of a high virulence organism. An example of an organism that his highly virulent is Bacillus anthracis, which causes anthrax. This is a potentially lethal infection. The virulence of an organism is quantified by detecting the median infectious dose or the median lethal dose, which are determined in animal models of the disease. The median infectious dose will be less than the median lethal dose. There is, however, a range of virulence that depends on the host’s age, immune status, environmental factors, host’s health, and pathogen-specific factors. Some organisms are pathogenic after just one organism has been passed to the new host but, if very few people die, it is not particularly virulent. There are primary pathogens and opportunistic pathogens. The primary pathogen will cause disease in all persons, while the opportunistic pathogen will cause disease in hosts that are immunocompromised. These include cancer patients, young patient, old patients, and those with known immunodeficiency states. These types of pathogens can

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be of any type or any virulence and depend mainly on host factors. Certain host environmental factors can lead to opportunistic infections. An example is Candida, which will cause disease depending on where it overgrows. There are four stages of pathogenesis. There must be exposure, then adhesion or colonization, then invasion, and finally infection. Exposure relies on contact with the pathogen. There will be a specific portal of entry that includes the skin, parenteral routes, and mucous membranes. Mucous membranes include the anus, mouth, nose, eyes, or vagina. Parenteral portals of entry include needle injection. Skin can be a portal of entry if the skin is broken. The most important portals of entry are the mucous membranes, which include the urinary tract, genital membranes, GI tract, and respiratory tract. Some mucous membranes are inside the body, while others are near the skin surface. Most organisms have a preferred portal of entry. Breaks in the skin and needle injection are both considered parenteral, while exposure to intact skin can also lead to some diseases. The placenta can be an important portal of entry in the uterus during pregnancy. There are certain TORCH infections particularly known for this type of transmission. These include toxoplasmosis, listeriosis, syphilis, hepatitis B, chickenpox, HIV, Fifth disease, Rubella or German measles, cytomegalovirus, and herpes. Adhesion depends on certain virulence factors called adhesion factors. Some pathogens have protein or glycoprotein adhesins, which bind to the host. Cilia, fimbriae, glycocalyces, capsules, membrane factors, hooks, barbs, spike proteins, and capsids can be adhesion factors. Protozoans will have barbs or hooks, while viruses have capsids and spike proteins that aid in adhesion. Biofilms have an extra-polymeric substance that enhances the attachment of the bacterial community. This is seen in Pseudomonas infections in the immunocompromised host. Invasion proceeds after adhesion happens. The organism must be disseminated or may produce certain toxins or enzymes. Some virulence factors are specifically protective of the organism. There are certain virulence factors that determine what kind of damage is done to the host. This is true of H. pylori, which damages the mucin layer of the stomach lining. Intracellular pathogens will need to enter host cells to become invasive. Some

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organisms enter through endocytosis, while others simply enter the cell by producing an invasive protein that binds to the host cell. Infection involves multiplication of the organism. Local infections involve a small body area, usually near the portal of entry. Some infections are focal but can spread to a secondary infection elsewhere in the body. Systemic infections involve a widely disseminated organism. Primary infections involve just one pathogen, while a secondary infection involves another pathogen. An example is a bacterial pneumonia after getting influenza. The disease needs to be transmitted in order to pass the disease onto a secondary host. This usually means there must be a portal of exit, which can be the same as the portal of entry or not. It can involve sneezing, direct contact, semen, feces, sweat, or tears—each of which can be a good vehicle to pass on the infection. Vectors can also be part of the transmissibility of the organism.

VIRULENCE FACTORS FOR VIRUSES AND PROKARYOTES There are specific virulence factors that confer a certain level of virulence of a pathogen. These are generally genetic in origin. The absence of certain genes that later diminish virulence is in keeping with the molecular Koch’s postulates. An adhesin is either a protein or glycoprotein that will attach to receptors on a host cell. Bacteria, fungi, viruses, and protozoans will all have adhesins. E. coli that is enterotoxigenic has fimbriae that contain an adhesin which binds to the host cell. Many other bacteria have proteinaceous or glycoprotein-containing adhesins. Invasion of the bacteria often involves toxins or enzymes. The invasion process often involves the bloodstream and elements of the immune system. The presence of organisms can involve bacteremia or bacteria in the blood, viremia or viruses in the blood, toxemia or toxins in the blood, or septicemia, which is the multiplication of the bacteria in the bloodstream. Septic shock refers specifically to hypotension and organ failure that can happen with a patient who has septicemia. Toxins can cause septic shock as well.

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Exoenzymes are enzymes in an organism that assists in the organism’s invasion process. Some will help the organism fight the immune system. An example is hyaluronidase, which is an enzyme from bacteria that degrades the cement between adjacent cellular structures in the connective tissue. There are nucleases that degrade DNA, phospholipases that degrade cell walls, and proteases that degrade proteins. One protease is called collagenase, which is a major part of connective tissue. It is seen in gas gangrene caused by Clostridium perfringens. Toxins are important to invasion and tissue damage. Certain pathogens will release toxins. There are endotoxins and exotoxins. Endotoxins are found on the bacterium itself. These can be released when the cell divides or dies off. Lipid A on gram-negative bacteria is an endotoxin. It triggers an immune response in the host. Too much of an immune response can lead to sepsis and host death. Exotoxins are mostly made by gram-positive bacteria. These are different from endotoxins, which have specific actions on the host cells rather than just triggering an immune response. Exotoxins are heat labile so they can be inactivated but they are far more lethal than endotoxins. Exotoxins are proteins, while endotoxin is a part of a lipopolysaccharide. There are three types of exotoxins. There are those that target something within the cell, called intracellular targeting toxins. There are membrane disrupting toxins, which damage the host cell membrane, and there are superantigen toxins, which activate the immune system. Examples of intracellular targeting toxins are those leading to cholera, tetanus, botulism, and diphtheria. Membrane-disrupting toxins include those from Pseudomonas, Clostridium perfringens, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae. Superantigens are those that cause staphylococcal toxic shock syndrome and certain streptococcal infections from Streptococcus pyogenes. There are two parts to an intracellular targeting toxin, an A subunit and a B subunit. The B subunit is the one that gives the toxin its host cell specificity, while the A subunit confers the actual toxic response. The toxin is brought into the cell through endocytosis.

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Diphtheria exotoxin is an AB toxin that inhibits protein synthesis. Cholera enterotoxin has an A subunit that increases cyclic AMP, leading to excessive fluid and electrolyte secretion into the gut. Botulinum toxin is a neurotoxin that is the most toxic substance in the world. The A subunit is a protease that blocks the release of the neurotransmitter that helps skeletal muscle contract. The tetanus toxin blocks the release of GABA, another neurotransmitter, in the tissues, leading to permanent muscle contractions. Membrane disrupting toxins may form pores in the cell membrane or may disrupt the lipid bilayer of the cell. Hemolysins and leukocidins cause pores to form in the cell membrane. Streptolysin is made by Streptococcus pyogenes; it causes a pore to form in erythrocytes or red blood cells. Staphylococcus aureus and Streptococcus pneumoniae both produce these types of toxins. Toxins that degrade the cell membrane include phospholipases, made by Rickettsia, Legionella, and Bacillus anthracis. These can lyse the phagosomes inside phagocytes. Clostridium perfringens, Pseudomonas aeruginosa, and certain beta-Staphylococcus aureus species make phospholipases that degrade the cell wall. Superantigens trigger a massive immune response. They cause cells to secrete cytokines, which are chemical messengers in the immune system. There will be hypotension, high fever, and multiple organ failure from toxic shock. Toxic shock syndrome is an example of this but it can also happen with Streptococcus pyogenes. There are virulence factors in prokaryotes that help the organism evade the immune system. Bacterial capsules can help prevent ingestion of the organism by the phagocytes. Streptococcus pneumoniae can do this. Those that are encapsulated tend to be more virulent than those without capsules. Other organisms will produce proteases to prevent phagocytosis. They can attack and destroy antibodies that aid in the phagocytic process. Fimbriae will contain proteins that block complement from binding so that phagocytosis doesn’t occur. Mycobacterium tuberculosis will make a mycolic acid substance that resists killing by the phagolysosome of the phagocyte. Other bacteria make coagulase, which causes blood clotting and the coating of the bacterium with fibrin, which prevents phagocytosis. This is true of Staphylococcus aureus. There are also kinases that digest fibrin clots so that pathogens can escape a

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blood clot and invade the host. Staphylococcus aureus makes both coagulase and staphylokinase. They can produce either, depending on the nutrient supply surrounding the bacteria. Antigenic variation can protect a pathogen. The pathogen can alter its extracellular proteins so that the cell is no longer recognized by the immune system. This is what happens with the agents that cause both gonorrhea and Lyme disease. Viruses also have certain virulence factors. There are adhesins also in viruses but they exist on the viral protein coat. These adhesins will facilitate viral adhesion to the host cell. In addition, enveloped viruses will have a great degree of antigenic variation that will prevent an immune response. Influenza virus has a spike protein that will bind to host intestinal and respiratory membrane cells. HIV has a viral adhesin that binds to CD4 helper cells in the immune system. Herpes simplex has certain glycoproteins that bind to genital or oral mucus membranes. There are two types of antigenic variation in viruses. Antigenic drift involves small point mutations that slightly change the spike proteins. Antigenic shift involves a major change in the spike proteins. This is from gene reassortment. Antigenic variation is a major way that viruses change in order to be infective to the host cell. It is the reason why the influenza vaccine is given every year to fight the disease.

VIRULENCE FACTORS FOR EUKARYOTIC PATHOGENS Eukaryotes can also exhibit certain virulence factors. Fungi have some similar virulence factors when compared to bacteria. Candida albicans produces surface glycoproteins that are adhesins, assisting in attachment. There are also exoenzymes like proteases and phospholipases made by the organism. Cryptococcus makes a large capsule that makes it resistant to phagocytosis. Other fungi will make mycotoxin, such as the organisms that make ergot toxin found in certain grain products contaminated with fungi. The toxin will cause gangrene, mania, and sometimes hallucinations. Aspergillus makes aflatoxin as a virulence factor. Aflatoxin

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can cause genetic mutations and cancer. Aspergillus will also make gliotoxin that causes host cell destruction and it makes proteases, such as elastase and catalase, which will break down elastin and otherwise protect the fungus from the immune system. Protozoans can make adhesins and toxins; they also undergo antigenic variation and some can survive inside the cell’s phagocytic vesicles. Giardia lamblia has an adhesive disk that binds to mucosal cells in the intestines. It causes inflammation of the intestinal lining but doesn’t invade the cells directly. Antigenic variation can happen with organisms that cause malaria. The organism Trypanosoma brucei, which causes African sleeping sickness, makes a capsule and undergoes antigenic variation. Helminths, such as those that cause schistosomiasis, can penetrate intact skin by making elastin and other proteases. Helminths that are very large will evade the immune system and organisms like trichinella and other roundworms will have a cuticle surrounding them that evades the immune system. Still others express glycans on the cell surface that mimic host cells so they evade the immune system. Some will make proteases that degrade antibodies.

TRACKING INFECTIOUS DISEASES Infectious diseases are tracked by epidemiologists. This field of study looks at both disease transmission and disease etiologies. It studies populations of individuals and the specifics of the population that make it susceptible to a given disease. Individuals that have a disease are said to have morbidity with the morbidity rate being a certain number of diseased persons in a standard set of the population or a percentage of the population. Two important topics in epidemiology are incidence and prevalence. Prevalence is the number of people with the disease at a particular point in time, while the incidence is the number of new cases of a disease over a specified period of time. The lifetime prevalence is the number of people who get the disease in their lifetime. The prevalence of a chronic disease will always be higher than the incidence because the disease doesn’t go away. Mortality relates to death from a disease compared to a standard number in the population.

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Some diseases are called sporadic diseases that aren’t seen often and have no geographic concentration. Tetanus would be considered a sporadic disease. Those diseases that are present within a certain geographic area are called endemic diseases. Malaria is an endemic disease in some areas. Those diseases that peak over a short period of time are called epidemic diseases. Influenza is an epidemic disease. Epidemic diseases are often caused by antigenic drift. Pandemic diseases are those that have worldwide impact. The first goal of the epidemiologist is to identify the causative agent or etiological agent. Experimentation needs to happen so as to identify if there is a pathogen leading to the disease. It is particularly hard to do when the symptoms are nonspecific. Much of understanding of the causative agent relates to Koch’s postulates. The CDC or Centers for Disease Control and Prevention is the agency involved in diseases and their causes. There are registers of reportable diseases that must be reported to the CDC by physicians, including things like measles and HIV disease. There is a weekly report made on each of these diseases that is published for doctors and other healthcare providers. As mentioned in a previous chapter, the first epidemiological study was done in London by John Snow, who studied cholera in 1854. The origin of the disease was mapped to certain water sources and public health changes were made to eliminate the causative agent in the water. Some diseases have a common source spread of the disease, such as a specific water source. This can be from a point source spread, with a short time period of contamination, a continuous common source spread, such as an infected kitchen with Norovirus, or intermittent common source spread, which does not cause infection all of the time. Propagated spread involves the infectability of each person who gets the disease with no single source of the infection. This usually involves person-to-person spread. Epidemiological studies come with different study designs. Observational studies are gathered through measuring different findings or doing a questionnaire or survey with no manipulation of the patients. These tend to be ethically easier to do.

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There are different approaches to doing an observational study. Descriptive studies look at the patients with the disease and examine medical records. They are often early studies to see if there is a particular pattern to the disease. The analytical epidemiological study looks at certain populations affected by a disease. This narrows down the possible etiologies of the disease. Retrospective studies look at information from the past regarding current cases of a disease, while prospective studies monitor the disease course after the disease has been established. d Cohort studies look at a particular age of people or a particular group of people and follow them prospectively and retrospectively. Case-control studies look at small groups of people with the disease and study their past. Cross-sectional studies look at all people in a specific point in time. Experimental studies are different from observational studies because they actually do something with the participants. Certain treatments can be given to see what the outcome of the treatment might be. The proofs made in Koch’s postulates requires experimental studies—some of which might not be ethical to accomplish. A good experimental study is double-blinded so that neither the patient nor the researcher knows what treatment is being given.

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KEY TAKEAWAYS •

Diseases vary in their communicability and contagiousness.

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There are certain stages to an infectious disease.

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Bacteria have certain virulence factors that enhance adhesion, increase infectivity, and protect the bacterium from the immune system.

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Viruses exhibit increases in adhesion and antigenic shift that increase their ability to infect the host organism.

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Fungi have many virulence factors, including toxins, which cause symptoms in the host.

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Protozoans and helminths also have virulence factors that increase infectivity.

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Epidemiologists study infectious and noninfectious diseases.

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There are different types of sources of an infection, depending on when and where the source comes from and the infectiousness of the persons who get the disease.

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Observational studies are more ethical than experimental studies but they might not adhere to Koch’s postulates.

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QUIZ 1. What least likely affects the contagiousness of an infectious disease? a. How deadly the disease is b. Characteristics of the host c. Characteristics of the pathogen d. Route of infectivity Answer: a. Each of these will affect the contagiousness of an infectious disease except for the deadliness of the disease itself. Minor or major infections can be equally contagious. 2. What disease is infectious in origin but noncommunicable? a. Ebola virus b. Clostridium tetani or tetanus c. Hepatitis D d. Cholera Answer: b. Clostridium tetani causes tetanus, which comes from soil spores. The disease comes through an open wound but cannot be passed from person to person or from animal to person. The other diseases are directly or indirectly communicable. 3. What is true of Koch’s original postulates? a. It is valid in all cases of an infectious disease. b. It applies only to bacterial diseases. c. There are several exceptions to these postulates. d. The only postulate that is incorrect is the second postulate. Answer: c. There are several exceptions to these postulates that make it difficult for them to be used for all infectious diseases. It can apply to many diseases that are not bacterial. More than one postulate can be invalid.

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4. What are the molecular Koch’s postulates most based on? a. The ability to grow a pathogen in tissue culture rather than pure culture. b. The presence of different metabolic effects in different pathogens. c. The presence of host effects that confer disease resistance. d. The presence of genes that confer pathogenicity. Answer: d. The molecular Koch’s postulates specifically talk about the presence of certain genes that confer pathogenicity in the offending organisms. 5. Which type of organism will have spike proteins that act in pathogen adherence? a. Viruses b. Bacteria c. Protozoans d. Fungi Answer: a. Viruses will have spike proteins that affect their ability to adhere to the host. This is not seen in other pathogens. 6. What is least likely to be an adhesion factor in a bacterial infection? a. Biofilm formation b. Capsules c. Barbs d. Glycocalyces Answer: c. Each of these is an adhesion factor in a bacterial infection except for barbs, which are seen in protozoal infections.

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7. Which of the following exotoxins in bacteria does not directly target an intracellular process in the cell but is a superantigen instead? a. Diphtheria toxin b. Botulinum toxin c. Cholera toxin d. Staphylococcus aureus toxin Answer: d. Certain toxins from strains of Staphylococcus aureus will lead to toxic shock syndrome from specific activation of the immune system because the toxin is a superantigen. The others are intracellular targeting toxins. 8. What is the activity of the exotoxins that are also phospholipases? a. The degrade the host cell membrane b. They create pores in the host cell membrane c. They activate cyclic AMP d. They act as superantigens to the host immune system Answer: a. Phospholipases are made by certain bacteria. They degrade the cell wall, killing the host cell. 9. What is the name of a disease that has impact throughout the world? a. Sporadic disease b. Endemic disease c. Epidemic disease d. Pandemic disease Answer: d. A pandemic disease is one that has a major impact throughout the world.

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10. Which spread of a disease involves no specific source because each infected person becomes infectious themselves? a. Point source spread b. Intermittent source spread c. Propagated spread d. Continuous source spread Answer: c. With propagated spread, there is person to person transmission of a disease in which each infected person becomes a source of the infection.

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CHAPTER THIRTEEN: INNATE IMMUNE SYSTEM This chapter is about the innate immune system. It starts with physical mechanisms in the host used to prevent infection as well as chemical protective mechanisms. The innate immune system involves a nonspecific host response, including inflammation, which is explained in the chapter. The process of phagocytosis is crucial to the innate immune response; how this works is discussed in the chapter.

PHYSICAL DEFENSES There are multiple aspects of the innate immune system, which does not target a specific organism but involves a more generalized response to pathogens. These part all work together to fight infectious diseases. Physical defenses start with having actual physical barriers to infection. Many physical barriers involve cells that are tightly compacted with junctions between them that prevent disease. There are these types of barriers in the mucosal surfaces of the GI tract and respiratory tract, in the skin, and in the cells that line the blood vessels. The cell to cell junctions are made of proteins that make connections with the extracellular matrix or with other cell proteins to prevent the passage of pathogens. The names of these junctions are desmosomes, tight junctions, or gap junctions. Figure 51 shows a tight junction:

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Figure 51.

The skin barrier is the main physical barrier in the body. There are three layers to the skin, with the epidermis being the outer layer, the dermis being the middle layer, and the hypodermis being the inner layer. The epidermis is packed with keratin. The outermost cells are resistant to degradation. The outermost cell layer will slough off with bacteria going along with it. New epithelial cells are replaced all the time. If the skin is broken, infection can get into the body. Mucous membranes are the barriers that line the lungs, mouth, nose, digestive tract, and urinary tract. There are tight junctions between the cells that prevent the passage of pathogens. These membranes contain mucus that trap debris, particulate matter, and microbes. There are antimicrobial peptides in the mucus that protect against certain pathogens. There are mechanical ways of clearing the debris, such as cilia that beat to move mucus and debris from the area. These cilia and mucus together involve what’s called the mucociliary escalator. Things like cystic fibrosis and smoking affect the activity of the mucociliary elevator, which leads to an increase infection and, in particular, an increase in lower respiratory infections. The mucus and cilia lead to very low microbial counts in the lower parts of the respiratory tract. 209

The digestive tract has tight junctions and goblet cells that secrete mucus but it generally does not have cilia like the respiratory tract. Instead, there is peristalsis or strong muscular contractions that propel the mucus out of the body along with feces. The goblet cells are always the cells in the body responsible for making mucus through secretory vesicles near the cell’s surface. The cells that line the urogenital tract, lymph vessels, and blood vessels are called endothelial cells. These are also the cells that make the blood-brain barrier, which prevents disease in the central nervous system. These cell junctions are the tightest in the body because they prevent serious infections from getting into the delicate tissues of the brain and spinal cord. There are other mechanical defenses besides skin sloughing and cilia movement. The urine provides a mechanical defense by flushing bacteria out of the urinary tract. Eyelashes, eyelids, and tears also prevent the invasion of bacteria in the eyes. Peristalsis is a mechanical defense system in the GI tract. The normal microbiome of the body will be a good line of defense against invading microorganisms. They compete for nutrients and occupy the area so pathogens can’t get into the body. Places where the microbiome is important include the vagina, the GI tract, the respiratory tract, and the skin. There are chemical contributions to the microbiome as well. The lack of a good microbiome plays a role in getting an opportunistic infection after taking an antibiotic.

CHEMICAL DEFENSE SYSTEMS The innate immune system has chemical defense mechanisms that also block pathogens. Some are produced in the human body itself, while others are produced by friendly microbes. The sebaceous glands secrete sebum that seals off the hair follicle the gland is attached to. There are bacteria in the microbiome that feed off of sebum to make a waste product called oleic acid, which is acidic and prevents bacterial contamination. The oleic acid is not endogenous to humans but is made by skin bacteria and fungi.

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The digestive tract also produces chemicals that protect the body against infection. There are lactoperoxidase enzymes in saliva and lysozyme in the esophagus. Gastric acid in the stomach is also protective against pathogens. There are antibacterial peptides and bile in the small intestine; lysozyme is produced in the small intestine as well. Acidity is important in the female reproductive system and the urinary tract. The urine contains acids that protect the urinary tract. There is lactate in the vagina that lowers the pH. This is made by lactobacilli that ferment glycogen as part of their metabolism, which results in a lowered pH. The eyes have tears that are made with lysozyme and lactoferrin, which block microbial activity. Lysozyme affects the peptidoglycan layer of the bacteria and lactoferrin sequesters iron so it cannot be used by microbes. Ears make cerumen or earwax that decreases the pH of the ears and makes fatty acids that inhibit bacteria. The respiratory tract makes all of the major enzymes, such as lactoferrin, lysozyme, and lactoperoxidase, which block bacterial growth. Surfactant in the lungs is also antibacterial. Antimicrobial peptides have diffuse antimicrobial properties. Some are naturally produced by the body, while others are made only when there is a bacterial invasion. They can damage the cell membrane, damage RNA or DNA, or block cell wall synthesis. Some act only on bacteria, while others will act against a variety of pathogens. Most of these peptides are found in the skin. Others are called defensins, made by epithelial and other cells. There are actually several types of antimicrobial peptides. The genes that make these are often transferred from cell to cell through plasmids. Bacteriocins are made in the GI tract. Defensins are made by macrophages, epithelial cells, and neutrophils. Dermcidin is made by sweat glands. Cathelicidin is made by cells in the skin. Histatins are made by the salivary glands. Plasma protein mediators are found in blood plasma. These include certain acute-phase proteins, cytokines, and complement proteins that exert a nonspecific response in the innate immune system. There are numerous acute-phase proteins made by the liver and sent to the blood. These include ferritin, transferrin, fibrinogen, serum amyloid A, and C-reactive protein—each of which is antimicrobial.

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The complement system is important to the immune response. There are more than thirty proteins in this system; they get activated when stimulated by microbial invasion. There are several pathways involved, including the classic pathway, the alternative pathway, and the lectin pathway. These are actually cascades activated under specific circumstances. The function of the complement cascade is to cause opsonization. This is when bacteria get coated with a substance that is recognized by phagocytic cells. Opsonin is what the coating is called. There are opsonins made by proteins in the complement cascade, antibodies, and mannose-binding proteins. Certain complement proteins called C3a and C5a strongly affect the inflammatory response, triggering mast cells to release their chemical mediators, including heparin and histamine. C5a will attract neutrophils and other white blood cells. There are certain complement proteins that aggregate to form a membrane attack complex, which participates in making pores in the cell membrane of gram-negative bacteria so that there is bursting of the cell and death of the pathogen. It is only effective against gram-negative bacteria. Cytokines are also important proteins in the innate immune system. These are stimulators of the innate immune system. Cytokines can do several things which can be described as autocrine, paracrine, or endocrine. Autocrine responses affect the same cell that makes it. Paracrine responses affect nearby cells. Endocrine responses affect distant cells. Cytokines can be interleukins, chemokines, or interferons. Interleukins do many things to moderate the immune system and are mainly made by white blood cells. Chemokines attract other white blood cells to the site of an infection. Interferons are signaling molecules in the immune system. They do many things but mainly defend against viruses. Cytokines will stimulate basophils and mast cells, which release histamine and heparin as part of the immune response. Histamine can also cause bronchoconstriction. Mast cells will also release leukotrienes that are potent, proinflammatory molecules. These

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molecules together will cause vomiting, diarrhea, and coughing, which help to get rid of pathogens from the body. Some cytokines will trigger the production and release of prostaglandins. Prostaglandins increase the body temperature, which increases white blood cell activity and blocks the growth of bacteria. Bradykinin can be released as well, which causes tissue edema in an area of infection.

CELLULAR DEFENSE There are many cells that contribute to the immune response. All cells in the bloodstream are made in the bone marrow by stem cells in the differentiation process called hematopoiesis. Most stem cells make red blood cells but there are others that make platelets, lymphocytes, neutrophils, basophils, monocytes, and eosinophils. Monocytes further differentiate into dendritic cells and macrophages. Lymphocytes further differentiate into natural killer cells, T lymphocytes, and B lymphocytes, which later mature to become plasma cells. Figure 52 shows the hematopoietic process:

Figure 52.

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The three types of granulocytes are neutrophils, eosinophils, and basophils. These all have granules that get released as part of the immune response. Neutrophils participate in phagocytosis and are important to the overall immune response. They make defensins and enzymes that destroy bacteria. Pus is a collection of neutrophils at the site of infection along with cell debris and dead bacteria. Eosinophils are important in fighting helminths and protozoa; they also participate in the allergic response. Basophils release histamine as part of the immune response. Mast cells are derived from the same cells that make granulocytes. They also release histamine, heparin, and leukotrienes. The reside in tissues rather than in the bloodstream like basophils do. They are found near mucous membranes and near the skin. Agranulocytes do not have visible granules and can be either monocytes or lymphocytes. Lymphocytes make natural killer cells or NK cells, which are crucial to the innate immune system. Lymphocytes involve B cells or T cells, which are more important to the adaptive immune response, which will soon be discussed. Monocytes make dendritic cells and macrophages, which also participate in phagocytosis like neutrophils. Natural killer cells or NK cells can destroy abnormal cells. Virally infected cells and cancer cells are two types of cells that are killed by natural killer cells. They can recognize normal cells by their receptors and generally leave them alone. Damaged cells are recognized as being abnormal and will be killed. It is the major histocompatibility I complex that needs to be recognized as belong to the self so as to avoid destruction by NK cells. Natural killer cells can do several things. They can cause apoptosis, which is controlled cell death or cellular suicide. They can release perforin, which is a protein that causes pores in the target cell, killing it. They can also release granzymes, which are proteases that get into the pores and trigger cells to be killed from the inside. Monocytes are large while blood cells that are not granulocytes. They can differentiate into macrophages and dendritic cells that reside in the tissues and participate in phagocytosis. Macrophages can last in a specific tissue for a long period of time. They also release cytokines that are involved in the immune response. Tissue-resident

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macrophages include microglial cells in the central nervous system, Kupffer cells in the liver, alveolar macrophages in the lungs, and peritoneal macrophages in the abdomen.

INFLAMMATORY PROCESSES Inflammation is part of the innate immune response. It happens when there are pathogens in the body or damaged tissue. Inflammation is both necessary and helpful in the immune response, although excessive inflammation is not always helpful and can be lethal to the human host. In acute inflammation, the response is nearly instantaneous. There will be vasoconstriction in order to limit blood loss. It does not last long and will later become vasodilation and an increase in vascular permeability because of histamine release in the mast cells. The process will dilute both bacteria and toxins. The overall inflammatory response involves five visible signs: redness or erythema, heat, swelling or edema, pain, and alteration of function. Phagocytes enter the system through an increase in vascular permeability. These will release inflammatory mediators that further the response to infection. The complement system gets activated and complement factor C5a, also called anaphylatoxin, increases the inflammatory response. Bradykinin increases tissue edema, neutrophils rush in, and sometimes there can be pus formation. Chronic inflammation happens when the body cannot clear the pathogen. This leads to a chronic battle between the immune system and the pathogens. One phenomenon of chronic inflammation is granuloma formation. This is a pocket of infection and white blood cells in a cluster. Figure 53 shows a granuloma:

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Figure 53.

Granulomas often occur in the setting of tuberculosis, in which the granuloma is called a tubercle. Viral infections can also lead to this type of inflammation, with granulomas forming when the infection does not clear out. Fever is another part of inflammation that can affect the whole body. There are some pathogens that result in pyrogen formation in the body, which resets the thermostat in the hypothalamus of the brain. Pyrogens can involve the endotoxin lipopolysaccharide of gram-negative bacteria, which causes leukocytes to release some of their own pyrogens. These can include tumor necrosis factor, certain interleukins, and interferongamma. These lead to prostaglandin formation that ultimately causes the fever. Fever makes leukocytes more effective and decreases the growth of many pathogens. There can be a crisis phase, when the fever is said to “break”. Vasodilation and sweating occur, which help to cool the body. Sometimes the fever is too extensive; this can be

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harmful to the human host. This is what can happen in superantigen infections like toxic shock syndrome. In such cases, the fever can be life-threatening.

PATHOGEN RECOGNITION AND PHAGOCYTOSIS Phagocytosis happens in phagocytes. It is the process of engulfing and killing pathogens as a way of nonspecifically killing off the infection. It starts with the extravasation or diapedesis of white blood cells, which involves the cells leaving the blood vessels through gaps in the endothelial lining because of complement factor C5a and the release of cytokines. This process is also called transendothelial migration. Extravasation only happens in the capillaries because they have very thin walls and low levels of turbulence. The white blood cells need to adhere to the endothelium before they can get through the capillary walls. Remember that opsonization occurs because of complement factors, lectins, and antibodies that bind to the pathogen. This leads to pathogen recognition, necessary for the phagocytic process. Not all organisms need to be opsonized before they are recognized as pathogens. There are some parts of pathogens that are inherently seen as pathogenic. These are called pathogen-associated molecular patterns or PAMPs. Some PAMPs, which are automatically recognized as pathogenic, include peptidoglycan cell wall material, lipopolysaccharide from gram-negative bacteria, bacterial lipopeptides, flagellin seen in bacterial flagella, and bacterial or viral nucleic acid fragments. There are specialized structures on phagocytic cells that specifically recognize PAMPs. These are called pattern recognition receptors. One type of these is called a toll-like receptor, which binds to certain PAMPs to cause phagocytosis. Some are on the cell membrane, while others are on internal organelle membrane. The binding of a pattern recognition receptor and a PAMP will activate the phagocyte so that it becomes ready to engulf the pathogen. More cytokines are released to enhance the inflammatory response and bring in more phagocytes. This leads to a larger response than could happen with just one or a few phagocytes. In addition, proliferation of the phagocyte occurs as part of the process. 217

After attachment and activation, the phagocyte engulfs the pathogen, bringing it inside the cell into a vesicle. This vesicle is called a phagosome. It later becomes fused with lysosomes, causing the formation of a phagolysosome. This acidifies the vesicle and activates lysosomal enzymes, hydrogen peroxide, and reactive oxygen species. The lysosomal enzymes include phospholipase, lysozyme, and proteases. Each of these participates in digesting the pathogen. There is an increased oxygen need, which is necessary to make the reactive oxygen species that help to destroy the pathogen. There are also reactive nitrogen compounds that kill the cell similar to reactive oxygen species. After digestion and degradation, there will be leftover waste from the pathogen. These are mostly excreted by the cell as part of exocytosis. Not all of the pathogen does this, however. Both macrophages and dendritic cells are called antigen-presenting cells. They take up antigenic proteins from the pathogen and present them on their cell surface in order to activate the adaptive immune response, which will be discussed next.

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KEY TAKEAWAYS •

The innate immune system is nonspecific. It starts with physical barriers, mechanical defense systems, and chemical defense systems.

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There are many different chemical components to the innate immune system, including cytokines, histamine, bradykinin, and complement proteins.

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The goal of inflammation is to increase the response to the pathogen through increased redness, increased body temperature, localized heat, edema, and loss of function of the affected area.

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There are multiple cells that participate in the innate immune response, including granulocytes and agranulocytes.

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Macrophages, neutrophils, and dendritic cells are involved in phagocytosis.

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Phagocytosis can happen after opsonization or the recognition of PAMPs, which activates the phagocyte to increase the immune response and engulf the pathogen.

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Phagocytosis involves digesting the pathogen and getting rid of the leftover waste products afterward.

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QUIZ 1. What protein toughens the outer layer of the skin so it can be a barrier against infection? a. Elastin b. Keratin c. Collagen d. Actin Answer: b. Keratin is part of the epidermis. It leads to toughening of the skin so that it can better resist the passage of pathogens through the tissues. It helps provide a mechanical barrier for the skin. 2. What component of mucous membranes least likely prevents disease? a. Movement of cilia b. Antibacterial proteins c. Mucus d. Phagocytosis Answer: d. Each of these is important in helping mucous membranes fight disease except for phagocytosis, which is not a function of the mucous membranes. 3. What part of the GI tract contains hydrochloric acid to protect the system against pathogens? a. Esophagus b. Mouth c. Stomach d. Small intestine Answer: c. There are specialized cells in the stomach that make hydrochloric acid that is protective against pathogenic bacteria.

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4. Lysozyme is an important antibacterial substance. What does it do? a. It binds iron so it cannot be used by bacteria. b. It breaks down the bacterial peptidoglycan layer. c. It forms pores in the bacterial cell membrane. d. It blocks DNA synthesis in bacterial organisms. Answer: b. Lysozyme breaks down the peptidoglycan layer of the cell wall, mainly affecting gram-positive bacteria. 5. What is the membrane attack complex made of? a. Antibodies b. Complement proteins c. Cell wall fragments d. Enzymes Answer; b. The membrane attack complex or MAC is made from complement proteins that aggregate to kill bacteria. 6. What do mast cells produce in the innate immune system? a. Heparin and histamine b. Complement proteins c. Antibodies d. Chemoattractants Answer: a. Mast cells make heparin and histamine, which aid in the inflammatory response.

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7. Which cells are most important in the allergic response and in killing off helminths and protozoans? a. Mast cells b. Basophils c. Eosinophils d. Neutrophils Answer: c. The eosinophil count will go up in allergies and when there is an infection with helminths or protozoans because these cells are important in these types of responses. 8. Which cells do not participate in phagocytosis? a. Neutrophils b. Dendritic cells c. Macrophages d. NK cells Answer: d. Each of these cells participate in phagocytosis, except for NK cells or natural killer cells. 9. What do pattern recognition receptors on phagocytes recognize? a. Bacterial antibodies b. Cytokines c. Pyrogens d. Pathogen-associated molecular patterns Answer: d. These pattern recognition receptors are designed to recognize PAMPs or pathogen-associated molecular patterns, which identify a certain organism as being pathogenic.

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10. What happens to the pathogen after it is bound to a phagocyte? a. The pathogen is taken inside and digested. b. A compound is secreted that surrounds and deactivates the pathogen. c. Proteases exit the phagocyte to kill the pathogen. d. Antibodies are made that congeal pathogens together. Answer: a. After binding to the phagocyte, the pathogen is taken inside and digested inside a phagolysosome.

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CHAPTER FOURTEEN: ADAPTIVE IMMUNE SYSTEM The topic of this chapter is the adaptive immune system, which is far more specific against certain pathogens than the innate immune system. There are B cells that are responsible for making antibodies and T cells that participate in killing infected cells that have been marked with specific antibodies. Both the T cell line and the B cell line have memory cells that retain the memory of a past infection. Vaccines are given to provide immunity to individuals before they get an infection.

ADAPTIVE IMMUNITY The adaptive immune system is involved in pathogen specificity and in the development of memory to infections. After any infection, this is the part of the immune system that develops a specific memory of the prior infection in order to prevent recurrent infection. There are two responses associated with the adaptive immune system. The first is the primary response, which is a new response to a pathogen the body has never been exposed to. The secondary response occurs at a later time and is the faster response that happens after subsequent exposure to the same pathogen. The two cell types involved in these responses are B cells and T cells, both of which are lymphocytes. B cells completely mature in the bone marrow. When mature, they are called plasma cells; these are the cells that make antibodies. Antibodies are also called immunoglobulins. They participate in what’s called humoral immunity, which directly involves B cells. Another aspect of the adaptive immune system is cellular immunity or cell-mediated immunity, which mainly involves T cells. Antigens involve anything that triggers an antibody response. Antigens are like PAMPs except they are specific to a particular pathogen and not to pathogens in general. Any molecule that can trigger an antibody response is antigenic in nature. Antigens, however, do not necessarily trigger an antigen. They can also trigger cellular immunity unrelated to antibody production. Antigens can be toxins, enzymes, call wall fragments,

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capsules, flagella, pili, or fimbriae that belong to a specific bacterium. Viral antigens can be spike proteins, envelopes, or capsids. Antigens can be any kind of molecule but are usually on the outside of the cell or virus. They can stimulate the humoral or cell-mediated immune system. The more complex the molecule, the better it is as an antigen. The best antigen is a three-dimensional structure that will stimulate all aspects of the adaptive immune system. Carbohydrates are less effective than proteins as antigens and nucleic acids or lipids are least antigenic in nature. Nucleoproteins, lipoproteins, and glycolipids make better antigens. The part of the antigen recognized as being important to the T cell or antibody recognition is called an epitope. It is just part of the larger whole; more than one epitope can be in one antigen with different antibodies responding to the different epitopes. Some small molecules that are too small to be an epitope by themselves are called haptens. Haptens need to be combined to a larger protein to make a conjugate antigen, which will be antigenic. Haptens are more important to allergic responses than to immune responses. Antibodies or immunoglobulins are made of glycoprotein molecules. There are four chains to an antibody that are connected by disulfide bonds. There are two heavy chains and two light chains, which form a Y shape. Figure 54 shows what an antibody looks like:

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Figure 54.

The arms of the antibody are called the Fab region, which stands for fragment of antigen binding. The tips of the Fab region are the variable regions, which do the actual binding of the antigen to the antibody. When this binding happens, the pathogen can be neutralized, aggregated, or agglutinated. The antigen-antibody complex can also initiate cell-mediated cytotoxicity. The trunk of the antibody is called the Fc region, which is constant and doesn’t depend on the antigen.

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There are five types of antibodies, called isotypes. Each has its own unique features that do different things for the purpose of negating a pathogen. IgG is the most abundant antibody in the bloodstream. It is the only one that crosses the placenta to provide passive immunity to the fetus. IgM is a membrane-bound antibody that is secreted in a large group of five called a pentamer. It is the first antibody to be released by the B cells. IgA is the class of antibodies found mainly in mucous membranes, tears, saliva, and breast milk. It helps to trap pathogens. IgD is found on the B cell membrane and acts to bind antigens. It is not secreted directly. IgE is found when there are parasitic infections. It binds to mast cells and contributes to the allergic response. Antigens and antibodies together can do several things. They provide a link between the innate immune system and the adaptive immune system. Neutralization can happen when some antibodies bind to antigens. This is what happens to toxins so they do not attach to target cells. Viruses can also be neutralized as will pathogens that are bound to IgA in the mucus of the body. The IgG antibodies act as opsonins to aid in phagocytosis. Agglutination can also happen when antibodies bind to pathogens. IgG has two antigen binding sites that can agglutinate two pathogens together. Large antigen-antibody complexes can form if many IgG antibodies are involved. IgM Is pentameric so it easily agglutinates pathogens by having 10 Fab segments. These large aggregates are easier to phagocytize and easier to be filtered by the spleen. Antibodies can also trigger activation of the complement cascade. This can stimulate the innate immune response through the process of opsonization. The MAC can also be activated in order to kill gram-negative bacteria. Antibody-dependent cell-mediated cytotoxicity is another function of antibodies, which can kill those pathogens that cannot be phagocytized because of their size. The Fab region of IgG antibodies binds to the pathogen, while the Fc region binds to natural killer cells or other phagocytic cells. The immune cell will secrete perforins and granzymes that help to destroy the pathogen that is now in close proximity to the immune cell.

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MAJOR HISTOCOMPATIBILITY COMPLEXES AND ANTIGEN PRESENTATION As we’ve discussed briefly, there are major histocompatibility or MHC molecules on the surface of each cell in the body that allows immune cells to recognize the self. The MHC is found on all nucleated cells; in humans, they are called the human leukocyte antigens, made by HLA genes. Only mature RBCs do not express these because they have no nuclei. There are two types of MHC molecules, called MHC I and MHC II. MHC I molecules are found on all cells, while MHC II molecules are found only on dendritic cells, macrophages, and B cells. These MHC II molecules will activate the T cells in the immune system. MHC molecules are glycoproteins that span the cell membrane. There are several subunits to these molecules and a cleft at the outer edge of these molecules that binds to antigens. Figure 55 shows the structure of these MHC molecules:

Figure 55.

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Antigen presenting cells or APCs are B cells, dendritic cells, or macrophages that can activate the T cells by presenting antigens on their surfaces. Remember that macrophages and dendritic cells participate in phagocytosis, while B cells mainly make antibodies. B cells also present antigens to T cells. All APCs will have MHC II molecules on their surfaces. They ingest antigens and spit them back out on their surface to present to other immune cells. Dendritic cells are classic APCs. They recognize a pathogen and attach to it, eventually internalizing it. There are lysosomes that digest most of the pathogen, except for parts that are separated by proteases in order to become epitopes that will cause an immune response to happen. Only selected epitopes are presented by the APCs. They ultimately get attached to the MHC II molecules, which is where the presentation happens. MHC I molecules are found on all nucleated cells, presuming they are healthy cells. NK cells and other immune cells recognize the MHC I molecules, leaving the cells with these molecules on them alone. Infected cells are associated with pathogen-specific antigens attached to their MHC I molecule, which marks them for destruction.

T LYMPHOCYTE FUNCTION Humoral immunity is associated with mainly extracellular pathogens. Cells that are already infected with a pathogen need to be gotten rid of by T cells. T cells are responsible for cell-mediated immunity. T cells are made in the bone marrow but mature in the thymus, where they are referred to as thymocytes before they become mature. T cells need to be properly selected by the thymus before they can be released. This is called thymic selection. The first step involves the making of T-cell receptors necessary for APC activation. Those that are defective and do not make these receptors are killed through apoptosis. This is called negative selection. Next, there is positive selection. Those cells that do not interact with the body’s MHC molecules are selected out. The third step involves removing cells that react too much to the self MHC molecules. This prevents things like autoimmunity, which involves accidentally recognizing the self-cells as being foreign. This is also referred to as central tolerance.

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If central tolerance fails, there is a process called peripheral tolerance. This also prevents autoimmune diseases. It involves blocking the activity of self-reacting T cells by cells called regulatory T cells. They block the activity of these reactive T cells and make anti-inflammatory cytokines. After thymic selection, only 2 percent of thymocytes persist and these become mature naïve T cells until they can be activated. There are actually several types of T cells. Helper T cells or CD4 cells, cytotoxic T cells or CD8 cells, and regulatory T cells. The CD4 cells and CD8 cells are called this because they make certain kinds of cluster of differentiation or CD molecules. Helper and regulatory T cells can only be activated by antigen presenting cells, while cytotoxic T cells will respond to any MHC I cell or basically any nucleated cell. Helper T cells augment the function of cytotoxic T cells and cytotoxic T cells do the actual cell killing. A cell that participates in cell killing is called an effector cell. There are specialized T cell receptors on the different T cells. These are related to antibodies like IgD and IgM and also have a variable and constant region. They are actually smaller than antibodies, however, and are less complex than antibodies, made of two peptide chains. Each T cell receptor binds to a specific epitope. Genetic rearrangement in the T cell allows for millions of different and unique receptors on the T cells. When a helper T cell gets activated, it can form four different types of cells. There are T helper 1 cells and T helper 2 cells as well as memory helper T cells. The first two types of helper cells do not live long and are involved in immediate immune activities. Memory T cells, however, live a long time in order to remember a specific epitope. Helper T 1 cells make cytokines and work in all aspects of the immune system. Helper T 2 cells act in the humoral immune system by activating B cells and by directing B cell maturation and antibody production. There is a third helper cell type called helper T 17 cells. A lack of these cells causes chronic mucocutaneous infections. Cytotoxic or CD8 cells respond to any type of cell that makes an MHC molecule. They differentiate and clone themselves after recognizing a cell as being foreign or infected. These are the cells that act in similar ways to NK cells by killing infected cells. Like NK cells, they make perforin and granzymes that can induce cell apoptosis. The major

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difference is that the reaction of cytotoxic T cells is specific and directed at an infected cell that is recognized by the T cell receptor. Sometimes, in the presence of superantigens, the T cells get too activated. This can happen with certain enterotoxins, exotoxins, and viruses. There is an excessive release of cytokines and overactivity of the T cells. The release of cytokines to such a degree is called a cytokine storm. It can lead to shock and death from multiple organ failure.

B LYMPHOCYTE FUNCTION B cells participate mainly in the humoral immune cell. As mentioned, they are matured in the bone marrow, where positive and negative selection occurs as it does with T cells. Those B cells that survive the selection process are called naïve B cells. B cells have B cell receptors, which are basically IgM and IgG antibodies bound to the B cell membrane. There is genetic rearrangement in order to have the diversity of B cell receptors necessary for immunity. These receptors do not need to have MHC reactivity in order to respond to an antigen. They can react to free antigens or antigens attached to membranes. Some B cells need T cells to react. The antigens they react to are called T-dependent antigens. These are mainly proteins. Nonprotein antigens, like carbohydrates and lipopolysaccharides, are T-independent antigens because they don’t need antigen processing and T cell activity. An activated B cell that can proliferate into daughter cells is called a plasma cell. Plasma cells can make many different antibodies. B cells can differentiate into memory B cells, which will last a long time. The helper 2 T cells have the ability to trigger the B cell to switch classes and make different types of antibodies than the initial IgM antibodies made by these cells. The constant region changes in these cases but not the variable region so the antigen-antibody relationship will be the same. There will be a lag time or latent period of about 10 days, where no antibody to an organism will be detected. This happens during the primary response to a new infecting organism. IgM levels rise first and then other antibody types. After that, the IgG antibody levels rise, peaking at three weeks after the initial infection. Memory cells that 231

are made allow for a much quicker and stronger response to the infection should the infectious organism enter the host again.

VACCINATIONS Vaccinations are important to the prevention of infection. It artificially stimulates the adaptive immune system in a way that resembles a primary immune response. The difference is that no actual infection really occurs with a vaccination. Immunity can be active or passive. Natural active immunity happens after a person gets the infection itself. This confers lifelong immunity to the pathogen and is how many people get immunity to a particular pathogen. Natural passive immunity is when a fetus gets temporary immunity through the transfer of IgG antibodies. This lasts up to six months, although IgA antibodies will come to the infant through breast milk. Artificial passive immunity comes from getting antibodies to an infection by a donor. This is when immunoglobulins are given to a person who has been exposed to a particular antigen and when the person will be greatly harmed by getting the infection. It can be used to protect against toxin-related illnesses. Artificial active immunity is what immunizations or vaccinations involve. The adaptive immune system is activated by receiving epitopes that confer immunity but do not cause infection. Herd immunity refers to the fact that, if most of the people in a community have resistance to an infection, this means that fewer people have the ability to pass on the infection to others and fewer actual infections happen, even if not everyone is immune. This has been a problem with certain infections that some parents object to giving their child. If too few are immune, the risk of infection in the community increases for every nonimmune person. There are different types of vaccines that can protect against disease. Live attenuated vaccines have living organisms injected into the person but the infected organisms are weakened and do not usually have the ability to cause real disease. The risk is that the attenuated organism can mutate to become a pathogenic organism, which can lead to disease, particularly in immunocompromised host.

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Inactivated vaccines have whole pathogens in them that are either killed or inactivated somehow. The inactivation must be mild enough not to destroy the architecture of the epitopes. These are more easily stored and transported but do not produce any active infection so the immune response is not as strong. Multiple booster shots usually need to be given. Other vaccine types are subunit vaccines that have only antigens and not whole organisms. Toxoid vaccines only contain inactivated bacterial toxins, known as toxoids. Toxoid vaccines are used in infections that have toxins associated with them. Conjugate vaccines have a protein that is attached to a polysaccharide capsule to fight infections that involve encapsulated organisms.

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Key Takeaways

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The adaptive immune system involves specificity to an organism and memory for the infection.

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The main cells of the adaptive immune system are T cells and B cells.

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Adaptive immune cells undergo selection in order to have a cell that does not selfreact to self-cells.

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Cytotoxic T cells react similarly to NK cells that specifically kill infected or damaged cells with perforins and granzymes.

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Immunizations provide immunity to an infection, usually without causing an actual infection.

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QUIZ 1. What statement is not true of B lymphocytes or B cells? a. Mature B cells are called plasma cells. b. These cells make antibodies. c. The cells mature in the bone marrow. d. These cells are not directly associated with memory. Answer: d. Each of these is true of the B cells except they are directly associated with memory. There are memory B cells that retain the ability to make antibodies to a subsequent infection. 2. What is the difference between PAMPs and antigens? a. Antigens are not related to pathogens and PAMPs are related to pathogens. b. Antigens are specific to a particular pathogen and PAMPs are not specific. c. Antigens are made from glycoproteins and PAMPs are lipids. d. Antigens are associated with viruses and PAMPs are associated with bacteria. Answer: b. Antigens and PAMPs are very similar except that PAMPs are not specific to a particular pathogen, while antigens are very specific to a pathogen. 3. What structure is least likely to be an antigen? a. Viral spike protein b. Bacterial capsule c. Nucleic acid segment d. Fimbriae components Answer: c. Antigens tend to be something on the outside of a virus, bacterium, or other pathogen but nucleic acids, being internal, are not generally antigenic.

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4. Which type of antibody is the most abundant antibody and is the only one that crosses the placenta? a. IgM b. IgG c. IgE d. IgA Answer: b. IgG makes up 80 percent of antibodies and is the only antibody that directly crosses the placenta in order to provide passive immunity to the fetus. 5. Which type of antibody is found in mucus and mucous membranes so that it can trap pathogens there? a. IgG b. IgE c. IgA d. IgM Answer: c. IgA is predominantly found in the mucous membranes and in mucus, where it traps pathogens before they can get into the body tissues themselves. 6. Which antibody has the best ability to agglutinate pathogens because it has ten Fab regions together involved in its structure for binding to antigens? a. IgG b. IgE c. IgA d. IgM Answer: d. IgM is pentameric, meaning that it has ten Fab regions. This makes it the best antibody for agglutination of pathogens, which can more easily be phagocytized.

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7. What type of molecule is a major histocompatibility complex molecule? a. Nucleic acid b. Glycoprotein c. Lipid d. Carbohydrate Answer: b. The MHC molecules are glycoproteins that span the membrane of the cells they exist on. Their role is to be recognizable as self-antigens. 8. Where does the selection of T cells happen? a. Bone marrow b. Lymph nodes c. Thymus d. Peripheral tissues Answer: c. The selection of T cells to be used for the immune system happens in the thymus, which has a cortex and a medulla for these processes. 9. Which type of cell is involved with peripheral tolerance because they help to deactivate self-reacting T cells? a. Memory T cells b. Cytotoxic T cells c. Immature thymocytes d. Regulatory T cells Answer: d. Regulatory T cells participate in peripheral tolerance by getting rid of self-activating T cells that would otherwise cause an autoimmune reaction to self-cells.

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10. Which cell type of the adaptive immune system is most closely related to NK cells in terms of what they can do? a. CD4 cells b. CD8 cells c. Memory T cells d. Regulatory T cells Answer: b. CD8 cells are called cytotoxic T cells. They are activated by specific infected cells and kill them in similar ways as NK cells do.

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CHAPTER FIFTEEN: ADVANCED LABORATORY METHODS This chapter explains several different advance laboratory techniques used in the laboratory in the making of certain drugs and in the detection of diseases that cannot be identified with cultures. Antibodies are specific to a certain pathogen so there are techniques used to identify infectious diseases, which are generally viral in nature or involve fastidious pathogens for which antibodies have been made in the affected patient. Enzyme-linked immunosorbent assays or ELISA testing and fluorescent antibody testing are two of the techniques discussed in the chapter.

POLYCLONAL AND MONOCLONAL ANTIBODIES Antibodies are helpful in diagnostics and other aspect of laboratory microbiology because they are highly specific and can be studied in vitro. In the laboratory, antibodies can be used to agglutinate cells, neutralize toxins and viruses, precipitate antigens, and kill bacteria. Remember that the variable region of the antibody has selective binding ability so they identify only those things they can directly attach to. It should also be remembered that, while antibodies are specific, there can be cross-reactivity of similar epitopes, particularly with antibodies and antigens that have low affinity to one another. Avidity measures the strength of the antigen and antibody binding sites. Affinity and avidity often go together so that antibodies with high affinity and high avidity are least likely to cross-react. Sometimes it is necessary to make antibodies for diagnostic or research purposes. A laboratory animal is injected with an antigen. After a few weeks, the animal will make antibodies against the antigen, leading to the collection of an antiserum, which is the animal’s serum after the antigen has been given. There will generally be more than one antibody made to the substance, which means that there will be a polyclonal antibody response. Most animals are injected twice in order to get high levels of IgG antibody. 239

A side effect of injecting the animal twice is that memory cells will be activated that have what’s called affinity maturation, which is a higher than normal affinity for the antigens. Those antibodies with a lower affinity will not be produced in numbers high enough compared to the high-affinity antibodies. Often, an adjuvant is given, which is a substance that activates the immune system in a general way. Polyclonal antisera are generally used to see if a patient is making antibodies to a pathogen. This is a good test but it has limitations. A false-positive test is gotten when the antigen is said to be present when it is not, and a false-negative test is gotten when antibody is not detected when in actuality, it is present. In general, a test is considered sensitive when it can easily detect the presence of a disease but there are often false positives. There won’t be many false negatives. A test is considered specific or to have specificity when it detects the correct disease with a low false positive rate. The risk of this is that there will be higher false negatives. False positive antibody tests happen because of cross-reactivity between related epitope. Because of this risk, confirmatory tests are needed on all positive tests. The best screening test is one with a high sensitivity but a lower specificity. In general, any confirmatory test done after a positive antibody screening test will be both time-consuming and expensive. If, for example, a person tested positive for the hepatitis C antigen, they would need a hepatitis C viral RNA test of their blood to confirm the presence of the virus. A false-negative test can happen too if the patient has a weakened immune system and doesn’t make antibodies to much of anything. Antibodies will be present in a patient a long time after the infection is over with. For this reason, antibody testing is a better measure of the past experience with an infection, particularly if it is recent, than it is of detecting a current or active infection. Sometimes, it is not specific enough to use a polyclonal serum and a higher specificity is required. This is why it might be necessary to have serum with monoclonal antibodies in it. Monoclonal antibodies cannot be gotten using an animal. Instead a tissue culture is used. An animal is injected with an antigen several times and the spleen is removed. B cells are not able to proliferate by themselves to they are fused with myeloma cells, which are cancerous cells. The end result is hybridoma cells. Only the hybridomas grow

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in culture; the cells are then screened for the antibody in question. These hybridomas are separately grown, leading to pure antibodies being made. As you can imagine, this is expensive and time-consuming. What is the purpose of making monoclonal antibodies? Part of the purpose is to turn mouse antibodies into human monoclonal antibodies. Mouse antibodies will be recognized as foreign by human so they will be neutralized by the human immune system. Genes can be manipulated that make a hybrid antibody that will be mostly human in origin. These are used to treat certain cancers by making antibodies against the cancer cell. Because of the expense, it is necessary to find an alternative way of making hybrid antibodies. Genetically-engineered plants can be used to make plant-related antibodies that can be humanized. These might be cheap enough to be made for use in treating common infectious diseases. Animal-derived monoclonal antibodies are just too expensive to make otherwise. Making these types of antibodies in plants is called making plantibodies.

DETECTION OF ANTIGEN-ANTIBODY COMPLEXES In vitro assays involve the detection of antibody-antigen complexes in a test tube or other medium outside of the body. When an antigen and antibody can be seen, it is referred to as a precipitin. In this type of reaction, an antibody and antigen are mixed together. If they combine, they precipitate out of solution, leading to a precipitin. Most of these tests are done using polyclonal antibodies, which form a precipitate more easily. The ratio of antibodies and antigens is important in a precipitation reaction. If there are too many antibodies, they will not combine well to make a precipitate. If there are too many antigens, the amount of precipitation will also decrease. There is an equivalence zone that provides an optimal ratio between antigens and antibodies in the reaction. A related test is called a precipitin ring test. It can tell the relative amounts of antigen and antibody in a sample. Test tubes are made that have the same amount of antigen in them. Glycerol is added to the antigen; then there is serial dilution of the antiserum. The glycerol allows antigen-antibody complexes to form just at the interface between the 241

glycerol and water. The highest dilution that produces a visible ring of precipitate is called the titer. The higher the titer, the more antibodies are in the sample. It won’t give an absolute concentration of antibodies, however. Another test is called an Ouchterlony assay, which uses a matrix made of agar gel rather than water and glycerol testing. The gel is clear and holes are punched into the gel to make wells. Antigen and antibodies are added to adjacent wells with the diffusion of proteins between the wells showing a precipitate between them. It can quickly tell if there is an antigen and antibody connection. Another related test is the radial immunodiffusion assay. This will actually quantify the concentration of antigen in the serum. Antiserum is mixed with liquid agar and allowed to cool in a plate. Wells are added and the antigen is added to the well. If they are a match, there will be a zone of precipitation. The zone of precipitation is measured and the concentration of antigen is mathematically determined. There is a similar test called a flocculation test. It is used for antigens that are not already soluble in water, such as lipids. It creates a flocculant instead of a precipitin. Flocculants won’t precipitate in solution but instead forms a foam in the test tube. This is a test used for syphilis testing. The VDRL test is a syphilis test that is essentially a flocculation test. Another test is a neutralization test, usually done to detect viruses. It makes use of antiviral antibodies that will coat the viral particles that block viral binding. Because viruses will lyse and damage cells, if they are neutralized, this can be seen as an area on a plate containing cells that isn’t damaged by the virus particle. The test makes use of a serial dilution of patient serum to see if the plaques of dead cells go away. The end result is some type of titer so that the highest titer involves a greater concentration of antibodies. It will not be able to detect an active infection unless the test is repeated in a couple of weeks. A PAGE assay or a polyacrylamide gel electrophoresis assay is done when a patient has too many or too few proteins in the blood. It can tell which proteins are present in the patient’s serum. A related test is an immunoelectrophoresis or IEP test. This test uses a

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PAGE test to separate the proteins and antisera to detect the exact proteins that are present in the sample. The IEP can detect monoclonal antibodies in excess, such as is seen in multiple myeloma. Patients with multiple myeloma make too many monoclonal antibodies in a disease that is cancerous to the individual. A protein peak in a certain spot will indicate that a given antibody is being made in excess. The Western Blot test is one that is used after protein gel electrophoresis. The PAGE test is done to separate proteins and then the proteins are immobilized on a nitrocellulose membrane. Antibodies and the membrane are mixed together along with a second antibody that is coupled to something that fluoresces, an enzyme, or something that changes color. This identifies particular antigenic proteins by the presence of a color change or fluorescence. Antibodies in general will also activate complement proteins. These complement proteins will opsonize bacteria in order to facilitate phagocytosis, while others will cause gram-negative bacterial cell lysis. The fact that this can happen can be used to detect the presence of certain antibodies in the patient’s serum. Red blood cells can also be used in a related test called the complement fixation test, which can detect antibodies against fungi, intracellular pathogens, and viruses, which cannot easily be cultured. If there is an antibody and antigen mixed together, all of the complement will be fixed and there won’t be enough complement to lyse the RBCs when these are added along with RBC antibodies. If the antibodies are present, the cells won’t lyse but if the antibodies are absent, the cells will lyse. This is how the complement fixation test is performed.

AGGLUTINATION ASSAYS Agglutination tests clump cells together or antigen-coated latex beads together. It can detect antibodies against red blood cells or against bacteria. These are easy to do and are done on a slide or on a microtiter plate, which is an array of wells used to look for agglutination. There can be direct agglutination assays or indirect agglutination assays. Direct assays look at bacterial cells that agglutinate themselves. Indirect assays use 243

antibodies or antigens connected to latex beads that also can agglutinate. IgM antibodies are best detected because they easily agglutinate. The rheumatoid factor test looks for agglutination of beads in rheumatoid arthritis patients. Agglutination tests are done in parts of the world where culturing of bacteria is expensive and difficult. It isn’t as accurate as bacterial culturing but is inexpensive and quick to do. It is also limited by the fact that it sometimes takes weeks for seroconversion or the presence of enough antibodies to occur. Agglutination tests work for antibody and antigen testing. Red blood cell agglutination is called hemagglutination. The direct Coomb’s test is a hemagglutination test. This is also called a direct antihuman globulin test, which looks for antibodies that do not agglutinate. It is used in looking for antibodies to the child from the mother when the child has neonatal jaundice. It is also used to look for transfusion reactions, syphilis, infectious mononucleosis, and Mycoplasma pneumonia. Coomb’s reagent is an antiserum that contains antihuman IgG antibodies. It will agglutinate red blood cells that have antibodies already attached to them. Without the reagent, binding and agglutination does not usually occur. Indirect Coomb’s testing screens for antibodies made against red blood cell antigens. It is used to detect antibodies that aren’t bound in the serum and is done before giving a blood transfusion. Some of this kind of testing can be done to detect viral infections. This can be done to check for the presence of influenza viruses, which can agglutinate red blood cells in a direct hemagglutination assay. It will also detect rubella and mumps virus infections. As in related testing, a titer can be determined to see what the concentration of the virus is in the serum. This only works for agglutinating viruses. The test can be modified in order to measure how many antiviral antibodies are in the patient’s serum. If there are antibodies in the patient’s serum, the virus will be neutralized and won’t agglutinate red blood cells. The titer involved is the highest dilution that will block agglutination of red blood cells. Blood typing involves an agglutination assay. The patient’s blood is mixed with antibodies to the different red blood cell antigens. If the antigen is on the red blood cells, there will be agglutination. This type of test is done when cross-matching blood prior to 244

a transfusion. Blood cross-matching mixes patient’s serum with donor red blood cells to see if there is a reaction.

EIAS AND ELISA TESTING Enzyme immunoassays or EIAs will use antibodies in order to check for antigens in the patient’s serum. They are done on microtiter plates or done in vivo. The antibody interacts with an enzyme that can do a specific reaction, which can be detected. Often the enzyme is a chromogen that is turned into a colored product that can be detected. Others are attached to fluorogens that will be converted into a fluorescent form after a reaction occurs. EIAs can be used in immunostaining. This is used to look at whole tissues that are stained with monoclonal antibodies. Immunocytochemistry is related to immunohistochemistry testing but it strips away the extracellular membrane and makes the cell membranes of tissues permeable to antibodies so antibodies can be detected against organelles and proteins within the cell. This can involve a color-changing enzyme or a fluorescent staining technique. The ELISA tests or the enzyme-linked immunosorbent assays are commonly used EIA tests. There is a direct ELISA that involves antigens immobilized inside a well on a plate. Specific antibodies are added to look for an antigen. If the antigen-antibody complex exists, a color-changing enzyme will be used to look for the existence of the complex. The sandwich ELISA test will quantify the amount of the antigen in the solution. The antigen can be anything, such as a serum protein, hormone, or pathogenic antigen. A primary antibody is added to the wells, with the unbound antibody washed away. A blocking protein is then added to bind nonspecific antibodies in the well. Then a secondary polyclonal antibody is added that is attached to an enzyme. If there is a complex formed, there will be a colored end product. The actual amount of color found is detected with a spectrophotometer so the antigen can be quantified. Indirect ELISA testing quantifies the antibody instead of an antigen. It can detect antibodies to HIV disease and Lyme disease. The test affixes a known antigen to a well 245

on a plate and then serum hopefully containing antibodies is mixed with the antigen. Then the complex is detected through fluorescence or a chromogen reaction. Low concentrations of antibodies or antigens can be detected using immunofiltration techniques. This basically filters a large volume of fluid through a porous pad. The antigen or antibody can be captured. This has been adapted for strip testing that can be used in a clinic. Testing for TORCH infections can be done with this type of testing. Pregnancy tests done at home are strip testing, also called lateral flow testing. In such cases, urine is allowed to adhere to an absorbent pad. This hydrates the dried reagents and, if antigens are present, a stripe will form.

USING FLUORESCENT ANTIBODY METHODS Fluorescent antibody techniques can be used by attaching a fluorescent marker to the constant region of a specific antibody. Cells can also be labeled with fluorescent antibodies in related testing. In direct fluorescent antibody testing a monoclonal antibody is bound to a fluorescent label. This is done to test for Group A streptococcus in a throat swab and to detect Mycoplasma infections and Legionella infections. Fluorescent antibodies bind to the bacteria so they can be detected on a slide. Indirect fluorescent antibody testing looks instead for antibodies in patient’s serum. It is used to check for syphilis. Syphilis organisms are prepared from a laboratory animal that are smeared on a glass slide. If the antibodies are present, they won’t wash off the slide and, when a secondary antibody is added that has a fluorogen added to it, there will be fluorescent bacteria seen on a slide. This is a confirmatory test to the VDRL test for syphilis. A related test is the fluorescent ANA test, which detects antinuclear antibodies. This is a test for autoimmune diseases and will be generally positive in a variety of autoimmune diseases. In order for it to work, the cells must be permeable to the anti-DNA antibody. It checks for the presence of anti-nuclear antibodies in the patient’s serum. The results are determined as a titer. Flow cytometry can be combined with fluorescence in order to count cells that pass through an automated, cell-counting system that will specifically detect fluorescing cells. 246

It can be used to count the number of helper T cells in patients who have HIV disease. Cells are mixed with fluorescing antibodies and then are counted as they pass through a narrow tube. This allows for specific counting of the number of cells present in the sample.

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KEY TAKEAWAYS •

There are ways to make polyclonal and monoclonal antibodies in animals or in plants.

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There are tests that can detect antigens and antibodies that precipitate in solution.

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Flocculation is a way of detecting antigens that are lipid-soluble.

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Agglutination tests will allow for agglutination of organisms or cells.

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Enzymes that change color or fluorescent tagging can be used to detect antigen and antibody complexes.

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QUIZ 1. What feature of an antibody and antigen complex makes for the least chance of cross-reactivity? a. Low affinity and low avidity b. Low affinity and high avidity c. High affinity and low avidity d. High affinity and high avidity Answer: d. Affinity is the match between an antigen and antibody and avidity is the strength of their attachment. A complex that his high affinity and high avidity will have the least chance of cross-reactivity with another antigen. 2. What is the advantage of giving an adjuvant to an animal when an antiserum is desired? a. It increases the affinity maturation for the antigen. b. It generally heightens the immune response. c. It prevents IgM antibodies from being made. d. It leads to a faster production of memory B cells. Answer: b. An adjuvant is given along with an antigen when an antiserum is desired because it generally heightens the immune response in the animal being injected.

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3. What is true of tests that are done as screening tests for the presence of an antibody? a. They have high sensitivity but low specificity. b. They have high sensitivity and high specificity. c. They have low sensitivity but high specificity. d. They have low sensitivity and low specificity. Answer: a. The ideal test has both high sensitivity and high specificity but, in reality antibody test are highly sensitive but have low specificity because of the problem of cross-reactivity to other antigens. 4. In the laboratory setting, what is the purpose of using a hybridoma? a. To get the strongest polyclonal antibody response. b. To have a ready supply of antibodies when needed for diagnostic purposes. c. To proliferate independently, making cells that make just one antibody. d. To inject into the animal for a more specific antibody response. Answer: c. Hybridoma cells are a hybrid of B cells and myeloma cells. They proliferate independently to make cells that, when selected properly, will just make a single antibody. 5. What is the advantage of making plantibodies rather than animal-derived monoclonal antibodies? a. Plantibodies tend to be more specific for pathogens than animal antibodies. b. Plantibodies will treat toxin-related diseases, which can’t be done with animal antibodies. c. Plantibodies can be made in larger numbers than animal antibodies. d. Plantibody making is much cheaper and easier to make than animal antibodies. Answer: d. Plantibodies involve a hybrid of plant and human antibodies. They are easier to grow than tissue cultures and represent a much cheaper process overall.

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6. What two things are combined to make a precipitin? a. A monoclonal antibody and a soluble antigen b. A polyclonal antibody and soluble antigens c. Two related monoclonal antibodies d. Solid antigens and a monoclonal antibody Answer: b. In a precipitin reaction, the best precipitate forms when a soluble antigen is mixed with a polyclonal antibody, which together form a lattice that can be seen with the naked eye. 7. What does the precipitin ring test test for? a. The antibody titer in the patient’s serum b. The concentration of an antigen in a solution c. The concentration of antibodies in a certain volume of serum d. The presence or absence of an antigen-antibody complex in a patient’s serum Answer: a. These tests will detect a certain antibody titer in the patient’s serum but they will not detect the absolute concentration of the antibodies. 8. What type of pathogen or infection is detected in a neutralization assay? a. Intracellular pathogens b. Lipid-related antigens c. Viruses d. Parasites Answer: c. Neutralization reactions will detect the presence of viruses that will be neutralized by the presence of the proper antibody.

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9. What is the main diseases or disease type that is detected with an immunoelectrophoresis assay? a. Syphilis b. Multiple myeloma c. Viral infections d. Cancer Answer: b. Multiple myeloma is a cancerous disease that allows for the making of too many monoclonal antibodies, which can be detected with an IEP assay. 10. What type of test is involved in blood typing for ABO antigens? a. Precipitin b. Flocculation c. Immunoassay d. Agglutination Answer: d. Agglutination testing is done by agglutinating red blood cells to see what cells agglutinate and which cells do not agglutinate.

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SUMMARY The purpose of this course has been to introduce the college-level student to the science of living things on a small scale or the field of microbiology. As you have learned, microbiology touches on many related topics, including the biochemistry of living things, the different features of cells, pathogens, and the immune system. Hopefully, you have come to understand the biochemistry involved in living structures and to know how organic molecules combine to make living things. Molecular genetics is also important and involves the structure and function of DNA and RNA, as well as how they are made and participate in protein synthesis. Pathogens, such as bacteria, viruses, and fungal organisms, cause human diseases and activate the immune system, which was also covered in detail during this course. Chapter one in the course introduced the topic of microbiology by first covering the different types of microorganism you might uncover in your quest to understand the fundamentals of this subject. In the laboratory setting, you may have to learn the different staining techniques involved in the identification of microbes, so this was discussed. There are different types of microscopy used to study pathogens and other microorganisms, including light microscopy, dark field microscopy, and electron microscopy—each of which was covered as part of the chapter. Chapter two started with a discussion of the origins of cell theory as well as the different historical aspects of how cells are viewed today. The two types of cells had been introduced in chapter one and these were further expanded upon in this chapter. Features that make prokaryotic cells unique and things that define what results in a cell being called eukaryotic were also covered in this chapter. Chapter three in the course involved the study of acellular pathogens, which mainly involves viruses. Viruses may or may not be pathogenic and do not have the capability of surviving outside of a cell. There are viruses that can infect all forms of life. The life cycle of viruses was discussed in the chapter along with the ways that viruses are cultured and isolated. There are other acellular pathogens less complex than viruses that were talked about in the chapter, including viroids, virusoids, and prions. The topic of chapter four was prokaryotic cells. There are features of prokaryotic habitats and their microbiomes you need to know about, so this was discussed. In addition, prokaryotes are

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divided into bacteria and archaea. The different type of bacteria, such as proteobacteria, Gramnegative bacteria, Gram-positive bacteria, and photobacteria were discussed in this chapter. The different features that describe the Archaea domain were also covered as part of the chapter. While there are many different kinds of eukaryotes, chapter five in the course focused mainly on eukaryotic cells that qualify as microorganisms. These include unicellular pathogens that are also eukaryotic like protists, helminths, and fungi. Algae and lichens are not pathogenic but are still important microbiological organisms that were covered in this chapter. Chapter six taught you the biochemistry you needed to know in order to study microbiology. All living things are basically made structurally of organic molecules and the interactions between the molecules is strictly biochemical in nature. For this reason, you needed to understand what the different organic molecules are in living things. Nucleic acids are studied in another chapter but carbohydrates, lipids, and proteins were part of this chapter. The way biochemistry helps in understanding microbiology was also covered in the chapter. Chapter seven in the course talked about cellular metabolism, which is how microbial organisms get their cellular energy. Most of this involves catabolism, which is the breakdown of certain molecules. How cells catabolize carbohydrates, lipids, and proteins was discussed in this chapter. Some organisms derive their energy from the sun. This is called photosynthesis, which was a part of this chapter. Finally, biogeochemical cycles are important to the environment so these were explained in the chapter. The focus of chapter eight in was the genome of the cell. Cellular organisms generally have DNA making up their genome. Both DNA and RNA are nucleic acids, which are important in the genetic functioning of the cell. The structure and function of DNA and RNA were covered as part of the chapter. The totality of the DNA in a cell is referred to as the genome. The different characteristics of a cell’s genome were also discussed in this chapter. Chapter nine in the course expanded on the study of DNA by looking into microbial genetics. The ways in which DNA is replicated, the transcription process, and the processes involved in protein synthesis were covered in the chapter. Other things discussed were genetic mutations and the different ways genes are regulated. How each of these things leads to genetic diversity in prokaryotes was also discussed in the chapter. Chapter ten touched on aspects of laboratory microbiology by looking into microbial growth. The different patterns of microbial binary fission and bacterial growth in cultures is important to understand as a laboratory microbiologist. There are certain factors that increase or decrease

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microbial growth, which were covered in the chapter, along with the different physical and chemical methods of controlling microbial growth both in culture and in the environment. Chapter eleven in the course focused on the different antimicrobial agents used to treat infectious diseases. There are different classifications of antimicrobial agents, some being bacteriostatic and some being bactericidal. There are antibiotics, antifungals, and antivirals, which were covered in this chapter. The public and medical professionals face serious challenges with regard to antibiotic and drug resistances. Some of these challenges were discussed as part of this chapter. The focuses of chapter twelve were pathogenicity, infectious diseases, and epidemiology of infections. The basic definition of an infectious disease was explained as well as what defines a pathogen. There are specific virulence factors that identify viruses, prokaryotes, and some eukaryotes as being pathogenic in nature, which were discussed. The study of epidemiology as it applies to tracking infectious diseases was also covered in this chapter. Chapter thirteen in the course was about the innate immune system. It started with a discussion of the physical mechanisms in the host used to prevent infection as well as chemical protective mechanisms. The innate immune system involves a nonspecific host response, including inflammation, which was explained in the chapter. The process of phagocytosis is crucial to the innate immune response; how this works was discussed in the chapter. The topic of chapter fourteen was the adaptive immune system, which is far more specific against certain pathogens than the innate immune system. There are B cells that are responsible for making antibodies and T cells that participate in killing infected cells that have been marked with specific antibodies. Both the T cell line and the B cell line have memory cells that retain the memory of a past infection. Vaccines are given to provide immunity to individuals before they get an infection. Chapter fifteen in the course talked about several different advance laboratory techniques used in the laboratory in the making of certain drugs and in the detection of diseases that cannot be identified with cultures. Antibodies are specific to a certain pathogen so there are techniques used to identify infectious diseases, which are generally viral in nature or involve fastidious pathogens for which antibodies have been made in the affected patient. Enzyme-linked immunosorbent assays or ELISA testing and fluorescent antibody testing are two of the techniques that were discussed in the chapter.

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COURSE QUESTIONS AND ANSWERS 11. What type of bacterial species is rod-shaped? e. Vibrio f. Bacillus g. Coccus h. Spirochete Answer: b. The rod-shaped bacterial species is referred to as a Bacillus bacterium, of which the plural is called bacilli. 12. What type of organism is an amoeba? a. Bacteria b. Algae c. Protozoa d. Fungi Answer: c. Protozoa include amoeba, which are eukaryotic and move because they have pseudopodia or “false feet” that extend out from the cell body in a certain direction of movement in order to propel the organism from one place to another. 13. Which microorganism classically has a cell wall that is made from chitin? a. Fungi b. Algae c. Archaea d. Bacteria Answer: a. While each of these microorganisms has a cell wall, only fungi have a cell wall that is made from chitin specifically.

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14. What is the cell wall of bacteria made from? a. Cellulose b. Chitin c. Pseudopeptidoglycan d. Peptidoglycan Answer: d. A characteristic that defines a bacterium is that it contains a peptidoglycan cell wall. The structure and component of the cell wall is often used as a defining feature to differentiate pathogens from one another. 15. Who was the most likely inventor of the first microscopes? a. Zaccharias Janssen b. Galileo Galilei c. Robert Hooke d. Antonie van Leeuwenhoek Answer: a. While each of these individuals contributed to the field of microscopy, the actual inventers who predated everyone were Hans and Zaccharias Janssen. They are credited with the findings, even though they never actually published their work. 16. If a compound microscope has an ocular lens that is 10 times magnification and an objective lens that has a four times magnification, what is the total magnification of this microscope? a. 400 times b. 14 times c. 40 times d. 4000 times Answer: c. The total magnification of the microscope is going to be the product of the magnifications of each lens, making the total 400 times magnification.

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17. What feature on a light microscope will least likely affect the light reaching the specimen? a. Illuminator b. Diaphragm c. Rheostat d. Coarse focusing knob Answer: d. Each of these will have a role in controlling the level of light that reaches the specimen in different ways; however, the course focusing know primarily deals with putting the item in focus. 18. What is the maximum magnification you can get out of a typical laboratory compound light microscope? a. 40 times b. 100 times c. 1000 times d. 10,000 times Answer: c. The maximum that can be reached is about 1000 times. At this high magnitude, oil immersion of the specimen must take place in order to better focus the light and to maximize the resolution of the image. 19. Which microscopy technique will most detect specific pathogens in clinical microbiology? a. Electron microscopy b. Light microscopy c. Fluorescence microscopy d. Dark field microscopy Answer: c. Fluorescence microscopy makes use of tagged fluorescent antibodies that can specifically find certain pathogens that bind to the pathogen and fluoresce under the microscope.

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20. Which type of microscopy uses low energy infrared light and fluorochromes to visualize objects under the microscope? a. Confocal microscopy b. Darkfield microscopy c. Fluorescence microscopy d. Two-photon microscopy Answer: d. With the two-photon microscope, fluorochromes are used and it uses such low energy light that two photons are necessary to excite the fluorochrome. 21. What is the magnification that can be gotten from an electron microscope? a. 1000 times b. 10,000 times c. 100,000 times d. 2 million times Answer: d. The magnification that can be gotten using an electron microscope is 2 million times that of the actual specimen. 22. What microscopy technique uses gold in order to highlight the specimen? a. Scanning electron microscopy b. Two-photon microscopy c. Fluorescence microscopy d. Confocal microscopy Answer: a. In scanning electron microscopy, the surface of the specimen is coated with gold in order to better highlight the specimen under the microscope.

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23. What type of dye or stain is absorbed specifically by what you want to be able to see under the microscope? a. Acidic dye b. Basic dye c. Negative stain d. Positive stain Answer: d. The positive stain is any stain or dye that preferentially stains the item you are wanting to visualize under the microscope. 24. What is the first thing added to the four-step Gram stain procedure? a. Iodine b. Crystal violet c. Safranin d. Ethanol Answer: b. The first step is the addition of the crystal violet stain, which colorizes cells with a thick peptidoglycan layer first. 25. What color happens when safranin stains the cell wall of a bacterium? a. Pink b. Purple c. Orange d. Green Answer: a. The two main colors you will see with the Gram stain is purple and pink or red. When the thin peptidoglycan layers are decolorized and counterstained with safranin, they appear pink or red in color. 26. Which cellular organelles contain their own DNA? a. Chloroplasts and mitochondria b. Mitochondria and Golgi bodies c. Lysosomes and Endoplasmic reticulum d. Peroxisomes and Golgi bodies 260

Answer: a. The two organelles, chloroplasts and mitochondria, each have their own DNA, which suggests that these structures originated from prokaryotic cells. 27. What is not a supportive fact indicating that chloroplasts and mitochondria descended from prokaryotic organisms? a. These structures have their own DNA. b. These structures divide through binary fission. c. The ribosomes of these structures are similar to prokaryotic cells. d. These structures can exist and replicate outside of the cell. Answer: d. Each of these statements is true and supportive of the endosymbiotic theory except that they do not divide or replicate outside of the eukaryotic cell. 28. The study of what disease caused Semmelweis to propose the germ theory of disease? a. Wound infections b. Puerperal fever c. Gastroenteritis d. Plague Answer: b. The first research indicating the probability of the germ theory of disease came out of a high incidence of puerperal fever among women who were examined by doctors rather than midwives. 29. What was the first epidemiological study about when it was performed in the middle 1800s? a. Plague b. Common cold c. Cholera d. Influenza

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Answer: c. The first epidemiological study was done by John Snow, who studied patterns of cholera in London as they related to certain water sources. 30. What best describes a chain of round bacteria in an arrangement? a.

Streptococcus

b. Staphylococcus c. Streptobacillus d. Diplococcus Answer: a. Streptococcus best describes round bacterial organisms arranged in a chain. 31. What will happen to a cell placed in a hypertonic medium? a. It will burst or swell. b. It will shrink or crenate. c. It will neither shrink nor burst. d. What happens depends on the type of cell. Answer: b. When placed in a hypertonic medium, the water in the cell will flow out of the cell, resulting in shrinkage or crenation of the cell. 32. What is the function of the nucleoid-associated proteins in prokaryotic cells? a. They help in cell division. b. They assist in protein synthesis. c. They organize the genetic material. d. They assist in cellular respiration. Answer: c. Nucleoid-associated proteins in prokaryotes will help to organize the nucleic acids or genetic material in the cell’s chromosome.

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33. What is the most common substance found in a prokaryotic inclusion? a. Iron oxide b. Volutin c. Gas d. Glycogen or starch Answer: d. Most inclusions will store glycogen or starch for metabolism but the other choices can less commonly be seen in inclusions found in prokaryotic cells. 34. What is the main function of the glycocalyx in the immune response to bacteria? a. It causes a decreased antibody response. b. It prevents immune cells from recognizing the cells as being foreign. c. It prevents clumping of the bacteria. d. It decreases the uptake of the bacterial cell by immune cells that might phagocytize them. Answer: d. The main immune function of the glycocalyx is that it helps prevent bacterial uptake by phagocytic immune cells. 35. What prokaryotic cell structure is involved in the exchange of genetic material between two cells? a. Fimbriae b. Flagella c. Pilus d. Cilia Answer: c. There are certain types of pili called sex pili that confer the passage of genetic information between two cells of the same species.

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36. Which cell structure is not seen in a prokaryotic cell at all? a. Fimbriae b. Flagella c. Pilus d. Cilia Answer: d. Prokaryotic cells have each of these appendages but they do not have cilia. Cilia are seen in animal eukaryotic cells. 37. What is the arrangement of flagella called that involves flagella that are all around the bacterium? a. Peritrichous b. Amphitrichous c. Lophotrichous d. Monotrichous Answer: a. Peritrichous flagella are those that surround the bacterial cell. 38. What structure in the eukaryotic cell is often referred to as the post office of the cell? a. Smooth endoplasmic reticulum b. Golgi apparatus c. Mitochondria d. Lysosomes Answer: b. The Golgi apparatus will sort and modify organic molecules in order to place them into vesicles, sometimes for transport out of the cell itself.

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39. Which eukaryotic cell structure contains hydrogen peroxide for the breakdown of small molecules within the cell? a. Peroxisome b. Lysosome c. Mitochondria d. Golgi apparatus Answer: a. The peroxisome of the cell contains hydrogen peroxide, which is highly reactive and can break down multiple types of cellular molecules. 40. What two proteins in a cell are responsible for movement, such as is seen in muscle contraction? a. Tubulin and keratin b. Keratin and Myosin c. Actin and myosin d. Actin and tubulin Answer: c. The action of actin and myosin together help to create cell movement as can be seen in the contraction of muscle cells. 41. What is a protein found in certain intermediate filaments in the cell? a. Actin b. Myosin c. Tubulin d. Desmin Answer: d. Desmin is a protein found in intermediate filaments and makes up desmosomes, which bind two adjacent cells together.

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42. Which process of the eukaryotic cell membrane is also referred to as cell drinking, which involves the intake of solutes and water in the cell? a. Phagocytosis b. Exocytosis c. Pinocytosis d. Endocytosis Answer: c. Pinocytosis is “cell drinking”, in which solutes and water are taken up by the cell from the outside. 43. What motor protein drives the movement of the flagella in eukaryotic organisms? a. Kinesin b. Actin c. Tubulin d. Dynein Answer: d. Dynein is the protein that makes up the motor abilities of the eukaryotic flagella, which themselves are flexible and made from microtubules. 44. What is a major difference between flagella and cilia? a. Flagella are made from microtubules and cilia are not. b. Cilia can participate in eating nutrients and flagella cannot. c. Flagella are flexible while cilia are not. d. Cilia have basal bodies and flagella do not. Answer: b. One of the major differences between cilia and flagella is that cilia can participate in sweeping nutrients into some organisms’ mouthparts, which is not the case with flagella that participate in movement only.

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45. Transmission of a virus through getting bitten by an insect is called what? a. Transmission through a biological vector b. Transmission through a mechanical vector c. Direct transmission d. Transmission through a fomite Answer: a. In a biological vector transmission, one can get bit by an insect, which passes on the virus that was once internally located within the insect. 46. The part of the virus particle that is made from phospholipids is called what? a. Capsid b. Capsomere c. Envelope d. Genome Answer: c. Not all viruses have envelopes but some do. The viral envelope contains phospholipids and originate from somewhere in the host cell at the time the virion is released after being replicated. 47. Which type of virus has a sheath and tail pins extending from a polyhedral head? a. Poxviruses b. Ebola c. Tobacco mosaic virus d. Bacteriophages Answer: d. Bacteriophages have unique shapes that involve a headpiece and extensions that include a sheath and tail pins, which aid in the attachment of the viral particle.

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48. What is not a way that virus particles are separately differentiated? a. Positive or negative strandedness b. Degree of virulence c. Enveloped or naked d. Type of nucleic acid Answer: b. Each of these is a way of differentiating different kinds of viruses, except they are not differentiated by their degree of virulence. 49. What stage of the viral life cycle happens just before biosynthesis? a. Penetration b. Attachment c. Maturation d. Lysis Answer: a. Penetration happens just before biosynthesis. In the viral life cycle, penetration involves injection of the genetic material, after which biosynthesis or the making of viral components occurs. 50. In which stage of the viral life cycle does genetic material get injected out of the bacteriophage? a. Attachment b. Penetration c. Biosynthesis d. Maturation Answer: b. During penetration, the viral genome is injected into the bacterial organism, while the actual viral particle remains on the outside of the bacterium.

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51. What is the bacteriophage genome called when it incorporates into the host genome rather than kill the cell? a. Lysogeny b. Phage conversion c. Prophage d. Lysogen Answer: c. When the bacteriophage enters the host cell and becomes incorporated with the host genome, the phage is known as a prophage. 52. What is it called when the virus infecting a host increases the pathogenicity of the host bacterium? a. Induction b. Lytic phase c. Maturation d. Lysogenic conversion Answer: d. With lysogenic conversion, the characteristics of the host are changed by the presence of the prophage, generally causing an increase in the host cell’s pathogenicity. 53. Which type of transduction is linked to which type of viral life cycle? a. General transduction and lysogenic life cycle b. General transduction and both lysogenic and lytic life cycle c. Specialized transduction and lytic life cycle d. Specialized transduction and lysogenic life cycle Answer: d. The only true association listed involves specialized transduction, which is only associated with the lysogenic life cycle.

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54. What is a provirus? a. A newly released viral particle b. A piece of viral DNA incorporated into the DNA of a host cell c. A piece of viral nucleic acid that has just been taken up by the host cell d. The virus particle that has been excised out of the host genome Answer: b. A provirus is what the viral genome is called when it gets incorporated into the DNA of a host cell. 55. Where in the human body does the latent virus that causes chickenpox remain dormant? a. Brain b. Respiratory tract c. Nerve cell bodies d. Skin cells Answer: c. The chickenpox virus will lie dormant in the nerve cell bodies near the spinal cord before they come out to cause a herpes zoster infection along the route of the nerve in the body. 56. What type of viral genome gets directly translated into proteins? a. Positive single stranded RNA b. Double-stranded DNA c. Negative single-stranded RNA d. Negative single-stranded DNA Answer: a. Only positive single-stranded RNA viral genomes get directly translated into proteins. This is the case with most plant viruses.

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57. What is mixed together in a hemagglutinin inhibition assay? a. Red blood cells and viruses in serum b. Red blood cells, viruses in serum, and virus-specific antibodies c. White blood cells and viruses d. Patient’s whole blood and antibodies against the virus Answer: b. In a hemagglutinin inhibition assay, red blood cells and the patient’s serum are mixed with virus-specific antibodies. The antibodies bind the virus and inhibit the hemagglutination of the red blood cells. 58. What does a viroid consist of? a. Circular RNA segments only b. DNA segments and a simple protein coat c. RNA segments and a lipid coat d. Protein segments only Answer: a. A viroid is a circular RNA segment only that does not have a protein coat. The RNA segment can hijack the host cell’s replication enzymes. 59. What is the primary difference between a virusoid and a viroid? a. A viroid is an RNA particle and a virusoid is a DNA particle. b. A viroid is more complex than a virusoid. c. A virusoid requires a helper virus to cause the infection. d. A viroid will kill a cell, while a virusoid is a latent infection. Answer: c. A virusoid requires a helper virus in order to cause the infection while a viroid can cause an infection without having a helper virus infection.

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60. Which type of symbiosis involves one population benefiting from the relationship and the other population being harmed? a. Mutualism b. Commensalism c. Amensalism d. Parasitism Answer: d. With parasitism, one population in the relationship receives a benefit, while others are harmed by the relationship. 61. Which type symbiosis involves both populations in the relationship benefitting from the relationship between the two? a. Mutualism b. Neutralism c. Commensalism d. Amensalism Answer: a. In mutualism, both populations in the relationship will receive benefit from being in the relationship. 62. Which type of bacterium is not considered atypical? a. Rickettsia b. Mycoplasma c. Haemophilus d. Chlamydia Answer: c. Haemophilus is a gram-negative organism, while the rest of them are atypical because they either do not stain with gram staining procedures or are too small to see.

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63. Which type of proteobacteria is the most diverse? a. Alpha-proteobacteria b. Beta-proteobacteria c. Gamma-proteobacteria d. Delta-proteobacteria Answer: c. There are many different organisms contained within the category of gamma-proteobacteria. These include Vibrio cholerae, Pseudomonas, Legionella, and Pasteurella. 64. Which type of proteobacteria can live on very few nutrients? a. Alpha-proteobacteria b. Beta-proteobacteria c. Gamma-proteobacteria d. Delta-proteobacteria Answer: a. Those who are alpha-proteobacteria can live on very few nutrients. They are often found in deep soil or deep oceanic sedimentary habitats. 65. Which type of proteobacteria are very fastidious and difficult to grow because they need a great many nutrients to survive? a. Alpha-proteobacteria b. Beta-proteobacteria c. Gamma-proteobacteria d. Delta-proteobacteria Answer: b. The beta-proteobacteria live in different environments and cause human diseases but they require a great many nutrients and are difficult to grow outside of the human host.

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66. What is not a characteristic of spirochetes? a. They can be grown in agar or tissue cultures. b. They are highly motile. c. They can cause Lyme disease and syphilis. d. They cannot easily be seen using regular light microscopy. Answer: a. Each of these is a characteristic of spirochetes, except that they are nearly impossible to grow in any type of culture. 67. Which bacterial genus is not a part of the CFB phylum of gram-negative bacteria? a. Fusobacterium b. Bacillus c. Bacteroides d. Cytophaga Answer: b. Each of these organisms belongs to the CFB phylum and are similar to one another because they share certain genetic characteristics. 68. What genus of gram-negative bacteria comprise the majority of the normal human intestinal biome? a. Bacillus b. Enterobacter c. Bacteroides d. Fusobacterium Answer: c. The majority of the human intestinal biome consists of species of the genus Bacteroides, which are largely mutualistic to humans because they prevent the overgrowth of pathogenic bacteria.

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69. What is made mostly in those phototrophic bacteria that do not make oxygen? a. Nitrogen b. Sulfur c. Carbon dioxide d. They all make oxygen Answer: b. Those phototrophs that do not make oxygen are called anoxygenic species. They generally make elemental sulfur as their end product. 70. What species of bacterial organism is considered an acid-fast bacillus? a. Corynebacterium b. Listeria c. Mycobacterium d. Actinomyces Answer: c. Mycobacterium species are particularly well known to be acid-fast in nature because their coat contains mycolic acid that must be specially stained in order to be visualized. 71. What is not a similar feature of organisms like Mycobacteria, Corynebacterium, Bifidobacterium, and Propionibacterium? a. They are all motile bacteria. b. They are all Actinobacteria. c. They are all high-GC bacteria. d. They fall under the category of gram-positive bacteria. Answer: a. Each of these is a feature of these organisms except that they are not all motile. In fact, very few of them are motile.

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72. Which organism type is considered to be evolutionarily the oldest and most ancient? a. Eukarya b. Bacteria c. Deeply branching bacteria d. Archaea Answer: c. The deeply branching bacteria are called what they’re called because they represent the furthest branches and the oldest ancestors of common organisms found today. 73. What is sexual reproduction called in protozoal life cycles? a. Syngamy b. Encapsulation c. Encystment d. Sporulation Answer: a. Syngamy is the reproductive life cycle aspect of protozoans. It involves sexual reproduction with male and female merozoites. 74. Which is not a form of asexual reproduction in protozoans? a. Schizogony b. Binary fission c. Conjugation d. Budding Answer: c. Each of these is a form of asexual reproduction in protozoans except for syngamy and conjugation, which are considered sexual reproductive methods of passing on progeny.

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75. What is the main purpose of the protostome in protozoal organisms? a. Motility b. Feeding c. Waste production d. Immunity Answer: b. The protostome is a specialized feeding structure that engages in the phagocytosis of nutrients by the protozoan. 76. What organelle does the kinetoplastid replace inside some protozoal organisms? a. Microtubules b. Nucleolus c. Mitochondrion d. Golgi apparatus Answer: c. The kinetoplastid is a modified mitochondrion seen in some types of protozoans instead of true mitochondria. 77. Which eukaryotic microbe is not of the Excavata supergroup? a. Leishmania b. Entamoeba c. Trichomonas d. Giardia Answer: b. Entamoeba does not belong to the Excavata supergroup but instead belongs to the Amoebozoa supergroup. The others belong to the Excavata supergroup.

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78. Which is not an animal-based parasitic infection? a. Candidiasis b. Schistosomiasis c. Trichinosis d. Cestode infections Answer: a. Each of these is an animal-based parasitic infection with the exception of candidiasis, which is a fungal infection instead. 79. In the paramecium, what is the micronucleus used for? a. Metabolism b. Conjugation c. Formation of fruiting bodies d. As a backup nucleus Answer: b. The micronucleus is used for conjugation. It contains a diploid set of chromosomes that undergo meiosis and get exchanged between two adjacent paramecium cells. 80.Which Excavata organism is not pathogenic? a. Euglena b. Giardia c. Trypanosoma d. Trichomonas Answer: a. Euglena is a subgroup of Excavata that has been well studied. It is photosynthetic and is generally not considered a pathogenic organism.

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81. What type of hyphae are characterized by having cell walls between the different cells of the hypha? a. Coenocytic b. Thallus c. Pseudohyphae d. Septate Answer: d. Septate hyphae are those that have cell walls between the different organism that together form the hyphae of the fungal organism. 82. Which nematode infection is the most common of these types of infections in the US? a. Pinworms b. Hookworms c. Ascaris d. Toxocariasis Answer: a. Pinworm infestation are the most common nematode infection in the United States, primarily affecting the GI tracts of young children. 83. Which part of the body besides the GI tract is affected by the larval forms of the Trichinella nematode? a. Skin b. Brain c. Muscle d. Kidneys Answer: c. Trichinellosis comes from eating undercooked meat. It can encyst the larval forms within muscle tissue, leading to muscle aches and related muscle symptoms.

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84. Which helminth infection is found in freshwater snails and burrows through the skin, leading to disseminated disease? a. Liver flukes b. Intestinal flukes c. Lung flukes d. Schistosomiasis Answer: d. Schistosomiasis is a common infection worldwide that is found in freshwater snails. It can lead to a disseminated infection and death if not treated. 85. What is the hard shell of diatoms made of? a. Chitin b. Cellulose c. Peptidoglycan d. Silica Answer: d. Diatoms have a hard shell from silica that contributes to diatomaceous earth. 86. What two organisms combine to make a lichen? a. Green algae and cyanobacteria b. Green plants and diatoms c. Diatoms and cyanobacteria d. Protozoans and brown algae Answer: a. Lichens are a combination of green algae and cyanobacteria that work cooperatively to do things like break down rocks, stabilize soil, and fix nitrogen.

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87. What element is among the micronutrients or trace elements inside living things? a. Potassium b. Phosphorus c. Nitrogen d. Hydrogen Answer: a. Each of these represents a macronutrient except for potassium which, along with other metallic substances, are primarily trace elements or micronutrients. 88. What type of chemical bond is most commonly seen with organic molecules? a. Ionic bond b. Hydrogen bond c. Metallic bond d. Covalent bond Answer: d. A covalent bond is one in which electrons are roughly shared with one another; these bonds are relatively difficult to break compared to the other types of bonds. 89. Organic molecules with the same number of atoms that are arranged differently in the molecule are referred to as what specifically? a. Structural isomers b. D-enantiomers c. Chiral d. L-enantiomers Answer: a. Structural isomers have the same basic atoms in them but have different arrangements of the atoms in the molecule.

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90. Which biomolecule type is involved in the storage or transfer of genetic information? a. Lipids b. Proteins c. Carbohydrates d. Nucleic acids Answer: d. Nucleic acids are unique in that they come together to store or participate in the transfer of the cell’s genetic information. 91. What type of fatty acid has no double bonds in it between carbon atoms? a. Saturated fatty acids b. Trans fatty acids c. Monounsaturated fatty acids d. Polyunsaturated fatty acids Answer: a. Saturated fatty acids are saturated with hydrogen atoms and have no double bonds between the different carbon atoms. 92. Which type of lipid makes up the most of the cell membrane of a typical cell? a. Fatty acid b. Triglyceride c. Phospholipid d. Cholesterol Answer: c. The majority of the cell membrane is made from phospholipids, which have a polar end and a nonpolar end.

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93. What type of lipid makes waxes, fragrances, and pigments? a. Phospholipids b. Sterols c. Isoprenoids d. Ergosterols Answer: c. Isoprenoids will make many different things, including pharmaceuticals, waxes, fragrances, and pigments. 94. What molecule in the cell membrane of fungi differentiate it from animal and bacterial cell membranes? a. Ergosterols b. Cholesterol c. Hopene d. Hopanoids Answer: a. Ergosterol is the main component that is not phospholipids in the fungal cell membrane, which differentiates these compounds from other organisms. 95. What is the smallest component of a protein molecule? a. Protein b. Amino acids c. Oligopeptides d. Polypeptides Answer: b. Amino acids are the smallest component of these molecules, which makes them the building blocks of the molecules.

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96. What aspect of protein structure involves the exact arrangement of the amino acids in the chain? a. Primary structure b. Secondary structure c. Tertiary structure d. Quaternary structure Answer: a. the primary structure of a protein is determined specifically by the arrangement of the amino acids in the protein structure. 97. What aspect of a protein gives it a specific shape because of interactions between the side chains, such as the formation of disulfide bonds? a. Primary structure b. Secondary structure c. Tertiary structure d. Quaternary structure Answer: c. The tertiary structure of a protein molecule involves an interaction between the side chains. It includes things like the formation of a disulfide bond, hydrogen bonding, and ionic bonding. 98. Which element is not routinely found in a carbohydrate molecule? a. Carbon b. Hydrogen c. Oxygen d. Nitrogen Answer: d. The elements of carbon, hydrogen, and oxygen are necessary in order to make a carbohydrate. Only in rare circumstances would nitrogen be attached to a carbohydrate molecule.

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99. Which is not one of the major polysaccharides found in nature? a. Maltose b. Glycogen c. Starch d. Cellulose Answer: a. Each of these is a polysaccharide found in nature but maltose is an uncommon disaccharide and is not considered a polysaccharide. 100.

Organisms that get their carbon source from carbon dioxide are called

what? a. Phototrophs b. Heterotrophs c. Autotrophs d. Lithotrophs Answer: c. Autotrophs have the ability to get carbon for their organic molecules from carbon dioxide. This is opposed to heterotrophs, which get their energy from organic molecules. 101.

What kind of organism includes humans and other animals when it comes

to energy and carbon sources? a. Chemoautotrophs b. Chemoheterotrophs c. Photoautotrophs d. Photoheterotrophs Answer: b. All animals as well as fungi and other species are chemoheterotrophs. They get their energy from organic molecules and get their carbon sources also from organic molecules.

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102.

Which kind of organism includes all plants when it comes to energy and

carbon sources? a. Chemoautotrophs b. Chemoheterotrophs c. Photoautotrophs d. Photoheterotrophs Answer: c. Plants are classified as photoautotrophs because they get their energy source from the sun and they get their carbon sources from carbon dioxide, which is an inorganic molecule. 103.

What is an enzyme called that is inactive because it does not have its

helper enzymes associated with it? a. Holoenzyme b. Apoenzyme c. Cofactor d. Coenzyme Answer: b. An enzyme that is inactive because it does not have its cofactors or coenzymes associated with it. When this happens, it is referred to as a holoenzyme. 104.

What is the beginning and ending molecule in glycolysis?

a. Glucose is the starting substrate and pyruvate is the end product. b. Glucose is the starting substrate and carbon dioxide is the end product. c. Amino acids are the starting product and glucose is the end product. d. Lipids are the starting product and carbon dioxide is the end product. Answer: a. In glycolysis, there is glucose as the starting product, and pyruvate is the end product of multiple reactions.

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105.

In the glycolysis pathway, how many net ATP molecules are made per

glucose molecule? a. One b. Two c. Three d. Four Answer: b. There are two ATP molecules used up in the investment phase, while four ATP molecules are made later, making a total of two ATP molecules made net in the reaction. 106.

What is not true of the Krebs cycle?

a. It consumes ATP energy. b. It makes two molecules of CO2. c. It is a closed loop. d. It involves the participation of coenzyme A. Answer: a. Each of these is true of the Krebs cycle, except that it makes a molecule of ATP but does not consume ATP energy. 107.

What is least likely to be an end product of heterolactic fermentation?

a. CO2 b. Oxygen c. Acetic acid d. Ethanol Answer: b. In heterolactic fermentation, things like CO2, acetic acid, and ethanol can be made as part of the fermentation process.

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108.

What is the end product of muscle fermentation in the absence of oxygen?

a. Ethanol b. CO2 c. Lactic acid d. Acetic acid Answer: c. Muscle cells are homolactic, meaning they only make lactic acid as an end product of fermentation. 109.

What is not a reason why a cell will not be able to undergo oxidative

phosphorylation? a. There is insufficient oxygen in the environment. b. There has not been enough pyruvate made. c. The genes to make electron transport chain proteins are missing. d. The genes to protect the cell from oxygen free radicals are missing. Answer: b. Each of these is a reason why oxidative phosphorylation cannot happen, except that there will not be enough pyruvate, which is uninvolved in the electron transport chain. 110.

Those organisms that engage in anaerobic respiration will make what after

the electron chain is complete? a. Sulfur b. Sulfite c. Nitrate d. Nitrogen gas Answer: d. Those organisms that participate in anaerobic respiration are involved in denitrification so they take nitrate or nitrite to make nitrogen gas as an end product.

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111.

What does the hydrogen ion gradient made through chemiosmosis get

used to do? a. Help the electron transport chain make CO2 and water. b. Help drive the sodium-potassium ATPase pump. c. Help drive ATP synthase to make ATP. d. Help in acid-base balance inside the cell. Answer: c. The hydrogen ion gradient can drive several kinds of reactions but is most important in driving ATP synthase to make ATP energy. 112.

Which is not likely to be a temporary storage molecule made in the light-

independent phase of photosynthesis? a. ATP b. NADH c. NADPH d. FADH2 Answer: d. Each of these is made as a temporary storage molecule in the light-independent phase of photosynthesis; however, FADH2 is not made. 113.

What process most directly gets nitrogen gas back into the atmosphere?

a. Nitrogen fixation b. Ammonification c. Anaerobic respiration d. Aerobic respiration Answer: c. Anaerobic respiration uses nitrite or nitrate and make nitrogen gas that get back into the atmosphere. Certain bacteria will participate in this process.

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114.

The phosphodiester bond in DNA links which carbon atoms on the

deoxyribose sugar together? a. Five-prime to five-prime b. Five-prime to three-prime c. Four-prime to two-prime d. Three-prime to one-prime Answer: b. The phosphodiester linkage in DNA links a five-prime carbon atom on one molecule or deoxyribose to the three-prime carbon atom on an adjacent sugar molecule. 115.

What is said with regard to Chargaff’s rule in the structure of DNA?

a. That DNA is made from nucleotides b. That adenine and thymine go together and guanine and cytosine go together c. That the genetic material inside the cell comes from DNA d. That DNA consists of a double helical shape Answer: b. Chargaff’s rule was related to the fact that adenine binds to thymine and cytosine binds to guanine. 116.

Which nitrogenous base is not found in DNA?

a. Adenine b. Guanine c. Cytosine d. Uracil Answer: d. While each of these is a nitrogenous base, only adenine, guanine, cytosine, and thymine are found in DNA. Uracil is a nitrogenous base that is instead found in RNA.

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117.

What is not a difference between DNA and RNA?

a. RNA does not have a phosphodiester bond. b. RNA segments tend to be shorter. c. RNA contains ribose and DNA contains deoxyribose. d. RNA tends to be single stranded. Answer: a. These are the major differences between RNA and DNA except that RNA is the same as DNA when it comes to having phosphodiester bonds. 118.

What nitrogenous base in DNA will be replaced by uracil?

a. Adenine b. Guanine c. Thymine d. Cytosine Answer: c. Thymine is not present in RNA but is replaced completely by uracil instead. In such cases, adenine pairs with uracil. 119.

Which base pair in DNA is hydrogen bonded to Adenine?

a. Cytosine b. Thymine c. Uracil d. Guanine Answer: b. Adenine will form base pairs with thymine in the DNA molecule.

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120.

Where does ribosomal RNA processing happen in eukaryotes?

a. Nucleolus b. Nucleus c. Cytoplasm d. Ribosomes Answer: a. Processing of ribosomal RNA in eukaryotes happens in the nucleolus but in prokaryotes, it happens in the cytoplasm. 121.

Which RNA type has enzymatic activity, helping to make the peptide

bond? a. Messenger RNA b. Transfer RNA c. Ribosomal RNA d. Small nuclear RNA Answer: c. Ribosomal RNA is the only type of RNA known to have enzymatic activity because it helps to form the peptide bond in the ribosome. 122.

What is it called when DNA makes a copy of itself?

a. Translation b. Replication c. Modification d. Transcription Answer: b. Replication is the synthesis of DNA from another DNA strand.

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123.

What is the name given to a region of DNA that encodes for a single

protein? a. Chromosome b. Genome c. Gene d. Constitutive gene Answer: c. A segment of a genome that is a region of DNA that can encode for a single protein is called a gene. There are hundreds or thousands of genes per genome, depending on the organism. 124.

What is the name of a gene that is always turned on?

a. Unexpressed gene b. Genotype c. Facultative gene d. Constitutive gene Answer: d. A constitutive gene or housekeeping gene is a gene that is so important that it is always turned on. On the other hand, a facultative gene is one that is only turned on or expressed when needed. 125.

What is the protein or enzyme that causes supercoiling of the DNA in a

genome? a. DNA polymerase b. Topoisomerase c. Histone d. Chromatin Answer: b. Topoisomerase is the enzyme that supercoils the DNA so that it can fit better inside a given cell. Otherwise, the DNA would be too long to fit inside each cell.

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126.

Which type of organism tends to have the largest genomes?

a. Bacteria b. Fungi c. Viruses d. Plants Answer: d. Plants often have very large genomes and are often polyploid, having many copies of the same chromosomes. 127.

What is the main function of DNA polymerase I and DNA polymerase II in

DNA replication? a. DNA synthesis b. DNA strand separation c. DNA repair d. DNA uncoiling Answer: c. These types of DNA polymerase are most involved in the repair of DNA segments rather than actual DNA synthesis. 128.

What enzyme in DNA replication is responsible for is responsible for

separating the DNA strands in order to make the replication fork? a. Topoisomerase II b. DNA gyrase c. DNA polymerase d. Helicase Answer: d. Helicase is the enzyme in the cell that is involved in the separation of the DNA strands. This helps to create the replication fork.

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129.

What is not true of the lagging strand in DNA replication?

a. It involves a single primer b. It involves the making of Okazaki fragments c. Synthesis is discontinuous d. There needs to be multiple primers Answer: a. The lagging strand involves multiple primers and the making of Okazaki fragments. The synthesis is discontinuous. 130.

Which enzyme in DNA synthesis will connect two different segments of

the DNA strand? a. Helicase b. DNA polymerase I c. DNA ligase d. DNA polymerase III Answer: c. DNA ligase is responsible for creating a phosphodiester bond between two adjacent sections of DNA. 131.

The shortening of what part of the chromosome might be responsible for

cellular aging? a. Telomeres b. Origin of replication c. RNA primers d. Structural genes Answer: a. It is believed that the shortening of the telomeres is responsible for the aging of the cells. Telomeres shorten with each cell division.

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132.

What is involved with plasmid replication that isn’t present in regular

bacterial replication? a. Synthesis happens at the three-prime to five-prime end. b. DNA polymerase is not involved. c. The DNA strand gets nicked instead of making a replication bubble. d. The plasmid does not replicate. Answer: c. In the replication of plasmid DNA, the strand gets nicked and comes off, causing the nicked strand to circularize and both strands to get replicated with DNA polymerase. 133.

RNA polymerase adds nucleotides in which direction?

a. The three-prime to five-prime direction b. The five-prime to three-prime direction c. In both directions d. They get added at the same time and then spliced together Answer: b. The RNA polymerase always makes RNA in the five-prime to three-prime direction. 134.

What does it mean that a segment of RNA is polycistronic?

a. It means that there are segments that do not code for a protein. b. It means that it is transcribed without a promotor region. c. It means that it is turned on by an operator region. d. It means that it codes for several proteins at the same time. Answer: d. The polycistronic RNA segment will code simultaneously for multiple proteins at the same time. This is common in prokaryotes but not in archaea and eukaryotes.

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135.

What does not happen to pre-messenger RNA in order to prepare it for

export out of the nucleus? a. The different polypeptide coding regions get separated. b. The Poly-A tail gets added to the messenger RNA. c. The five-prime cap gets added to the messenger RNA. d. The introns get spliced out of the pre-messenger RNA. Answer: a. Each of these things happens as part of the processing of RNA before it is exported out of the nucleus but, in eukaryotes, there is generally one messenger RNA per protein so there is no need to separate polypeptide coding regions. 136.

How many base pairs constitute a codon?

a. Two b. Three c. Four d. Six Answer: b. One codon is a triplet of base pairs, each of which translates into a different amino acid. 137.

How many amino acids are there in nature that make proteins?

a. 10 b. 20 c. 32 d. 64 Answer: b. There are 20 different amino acids in nature that together make the different proteins.

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138.

Which amino acid is the first amino acid added to the peptide when

translation takes place in the eukaryote? a. Glutamine b. Histidine c. Alanine d. Methionine Answer: d. Methionine is always the first amino acid in eukaryotes to be added to make a protein in translation. A modified form of methionine is the initial amino acid in prokaryotes. 139.

Which type of mutation in a codon only affects the cell in certain

environmental circumstances? a. Nonsense mutation b. Conditional mutation c. Missense mutation d. Silent mutation Answer: b. A conditional mutation is some type of missense mutation that only affects the cell under certain environmental circumstances. 140.

What type of mutagen most likely leads to dimer formation between

adjacent pyrimidines? a. Intercalating agents b. Nucleoside analogs c. Nonionizing radiation d. Ionizing radiation Answer: c. Nonionizing radiation, which is what ultraviolet rays are, will lead to dimer formation between adjacent pyrimidines, which can lead to mutations.

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141.

What does the activator bind to in order to increase transcription of an

operon in a prokaryote? a. RNA polymerase b. Promotor site c. Repressor d. Operator Answer: b. The activator will bind to the promotor site, increasing the ability of the RNA polymerase to bind to the DNA molecule and cause transcription. 142.

Which is a small regulatory molecule in prokaryotes that can interact with

other molecules in order to activate or repress transcription? a. Inducer b. Promotor c. Operator d. Repressor Answer: a. An inducer is a small molecule that interacts with either an activator or repressor in order to increase or decrease transcription of a prokaryotic operon. 143.

Under what physiological conditions does the lac operon get transcribed to

the greatest degree? a. Glucose is low and lactose is low b. Glucose is low and lactose is high c. Glucose is high and lactose is high d. Glucose is high and lactose is low Answer: b. Under conditions of low glucose and high lactose, the lac operon transcription is turned on the most. This is because the cell prefers glucose as a fuel source, so it needs to be low before lactose is used as an energy source.

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144.

What gene regulation type is available to eukaryotes but not to

prokaryotes? a. Inducers b. Activators c. Repressors d. Enhancers Answer: d. Enhancers involve the presence of a specific DNA sequence a distance away from promotor sites that get close to the promotor site through looping of DNA. It will be seen in eukaryotes but not in prokaryotes. 145.

Which method of gene transfer does not occur between members of the

same generation in prokaryotes? a. Vertical transmission b. Transduction c. Conjugation d. Transformation Answer: a. With vertical transmission, the DNA is passed from one cell to the daughter cells in the next generation. 146.

Which method of gene transfer in prokaryotes involves the transfer of

DNA through pili between the cells? a. Vertical transmission b. Transduction c. Conjugation d. Transformation Answer: c. Conjugation in prokaryotes involves the direct transfer of DNA through pili that connect two cells together.

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147.

Which genetic change occurs in both prokaryotes and eukaryotes?

a. Transduction b. Transposition c. Conjugation d. Transformation Answer: b. Each of these occurs in just prokaryotic cells; however, transposition or the transfer of transposons from one DNA area to another, occurs in both eukaryotes and prokaryotes. 148.

In which phase of the cell growth curve in a fixed culture is the maximum

culture density reached? a. Log phase b. Stationary phase c. Lag phase d. Decline phase Answer: b. In the stationary phase, the log phase has already occurred and the maximum culture density is reached. 149.

What is not true of using the Petroff-Hausser chamber for detecting

bacterial cell concentrations? a. It does not work for very dilute solutions. b. It cannot easily tell the difference between dead and live cells. c. It involves a microscopic analysis of cell counts. d. Fluorescence staining cannot be used. Answer: d. Each of these is a true statement with regard to PetroffHausser chambers except that fluorescence staining will be able to tell the difference between dead and live cells.

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150.

What is the main advantage of the plating method for detecting the

number of bacteria in a colony? a. It only detects living cells. b. It can count organisms that normally form chains or clusters. c. It can detect very low or very high concentrations. d. It is the fastest method of detection of cells. Answer: a. The biggest advantage is that it can detect only living cells that can thrive on a culture plate. It is not able to accurately count cells that grow in chains or clusters and is not a fast method of detection because the colonies must be allowed to grow on the plate. 151.

In a biofilm, what is quorum sensing?

a. It is the formation of an extracellular matrix. b. It is the formation of waste, nutrient, and water channels. c. It is the ability of the organisms to share nutrients. d. It is the ability of the biofilm organisms to sense their own density. Answer: d. Quorum sensing can be important to virulence of the organisms in the biofilm. It is the ability of the organisms to sense their own density and to increase virulence when the density is high enough. 152.

Which organism in a thioglycolate tube will grow throughout the tube’s

length? a. Strict aerobes b. Facultative anaerobes c. Aerotolerant anaerobes d. Strict anaerobes Answer: c. Aerotolerant anaerobes rely on anaerobic metabolism but are completely tolerant of the presence of oxygen.

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153.

Which organisms in a thioglycolate culture tube will only grow at the

bottom of the tube? a. Strict aerobes b. Facultative anaerobes c. Aerotolerant anaerobes d. Strict anaerobes Answer: d. Strict anaerobes do not like oxygen so they preferentially grow where the oxygen concentration is the lowest, which is at the bottom of the tube. 154.

Capnophiles grow best when what is present in the environment?

a. Sulfur b. Oxygen c. Carbon dioxide d. Nitrogen Answer: c. Capnophiles grow best in low oxygen and high carbon dioxide environments. 155.

What do acidophile bacteria do to tolerate the low pH environments they

live in? a. They pump hydrogen ions outside the cell membrane. b. They have specialized enzymes that do not denature in an acidic environment. c. They create a mucus coat that repels hydrogen ions. d. They have an especially thick peptidoglycan layer in their cell wall. Answer: a. These organisms actively pump hydrogen ions outside of their cell membrane in order to tolerate an acidic environment.

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156.

Which organisms prefer temperatures below freezing temperatures?

a. Alkaliphiles b. Psychrotrophs c. Acidophiles d. Psychrophiles Answer: d. Psychrophiles are organisms that specifically like to live in temperatures that are below freezing, such as would be seen in Antarctica. 157.

Which biological safety level involves organisms that are generally lethal

and have no cure, such as Ebola? a. BSL-1 b. BSL-2 c. BSL-3 d. BSL-4 Answer: d. BSL-4 organisms are the most dangerous and cause lethal infections for which there is no cure. The highest level of protection is required for these organisms. 158.

What is the highest degree applied to a technique that will definitely rid an

item from microorganisms and endospores? a. Sterilization b. Disinfecting c. Antisepsis d. Degerming Answer: a. Sterilization will get rid of all organisms and endospores from something, providing the greatest degree of protection from bacterial contamination.

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159.

What will least likely kill an organism with a chemical agent?

a. High concentrations of organisms b. Short contact times with the agent c. The presence of endospores d. The agent being bactericidal Answer: b. Short contact times will least likely kill an organism with a chemical agent, regardless of the organism or the actual agent involved. 160.

Which substance for getting rid of bacteria is not an antiseptic?

a. Surfactant b. Heavy metals c. Halogens d. Phenolics Answer: a. Surfactants are not antiseptics but work to degerm an area of bacteria. These include both detergents and soaps. 161.

What chemical is used to make a supercritical fluid in order to kill many

pathogens? a. Oxygen b. Carbon dioxide c. Nitrogen d. Hydrogen peroxide Answer: b. Carbon dioxide is used as a supercritical fluid in order to sterilize things under high pressures and sometimes high temperatures.

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162.

What is least likely to affect the dosage of an antimicrobial drug given to a

patient? a. The patient’s mass b. The presence of liver disease c. The presence of kidney disease d. The presence of a drug allergy Answer: d. Each of these will cause a problem with the dosage given to the patient except for the presence of a drug allergy, which is not dosedependent. 163.

Which type of infection is best treated with an oral drug, even if the drug is

poorly absorbed? a. Sepsis b. Gastrointestinal infection c. Skin infection d. Liver infection Answer: b. GI infections often can be treated with oral antibiotics, including those that are not easily absorbed by the gastrointestinal tract. These drugs can decontaminate the bowels. 164.

Which drug classification is not a beta-lactam antibiotic?

a. Polymyxin B b. Penicillins c. Carbapenems d. Cephalosporins Answer: a. Each of these is a beta-lactam drug belonging to a class of drugs that started with the discovery of penicillin. Polymyxin B is not one of these drugs.

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165.

Which drug affects the synthesis of the cell wall by preventing

peptidoglycan subunits from getting from the inside to the outside of the cell membrane? a. Vancomycin b. Bacitracin c. Penicillin d. Aztreonam Answer: b. Bacitracin is unique in that it prevents peptidoglycan subunits from exiting the cell in order to prevent cell wall synthesis. 166.

What is least likely to be a part of the body damaged by the use of

aminoglycosides? a. Nerves b. Ears c. Kidneys d. Liver Answer: d. Aminoglycosides are mainly toxic to the nerves, ears, and kidneys. They are not generally considered toxic to the liver. 167.

What is least likely to be affected by using the tetracycline class of

antibiotics? a. Skin b. Liver c. Ears d. Teeth Answer: c. Tetracyclines can cause sunburn, liver damage, and teeth discoloration. They are not ototoxic and do not affect hearing.

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168.

Which part of the bacterium is affected with drugs like daptomycin and

polymyxin B? a. Cell wall b. Cell membrane c. DNA synthesis d. Protein synthesis Answer: b. These drugs inhibit the synthesis of the cell membrane, sometimes affecting the outer membrane of gram-negative drugs. 169.

What part of the cell is affected by fluoroquinolone drugs?

a. Cell membrane b. Cell metabolism c. DNA synthesis d. Protein synthesis Answer: c. These drugs inhibit DNA gyrase so they prevent replication of DNA in the cells of bacteria—both gram-negative and gram-positive. 170.

What part of the fungal organism is most effectively treated with imidazole

drugs? a. Cell membrane synthesis b. Cell wall synthesis c. Protein synthesis d. DNA replication Answer: a. Because the fungal cell membrane contains ergosterol not seen in humans, these drugs selectively target ergosterol synthesis, affecting the fungal cell membrane.

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171.

What is the most common activity of antiviral drugs?

a. They inhibit protein synthesis b. They block virus attachment c. They are nucleoside analogs d. The cause breakage of DNA linkages Answer: c. The majority of antiviral drugs act as nucleoside analogs that mimic certain nucleosides, affecting nucleic acid synthesis. 172.

What virus is affected with drugs like Tamiflu and Relenza?

a. Influenza b. Hepatitis C c. Herpesviruses d. HIV Answer: a. These drugs will specifically treat influenza, which will be prevented from releasing outside of the host cell it infects. 173.

What least likely contributes to antimicrobial drug resistance?

a. Patient noncompliance b. Overprescribing antimicrobials c. Using the wrong antimicrobial d. Giving doses that are too high Answer: d. Each of these will contribute to antimicrobial drug resistance except for giving doses that are too high. In fact, giving doses that are too low will more likely contribute to drug resistance.

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174.

What is true of the MBC and the MIC of an antimicrobial drug?

a. It will not depend on the organism being studied. b. The MBC and the MIC should be roughly the same. c. The MIC will be greater than the MBC. d. The MBC will be greater than the MIC. Answer: d. The MBC is the concentration of drug that will kill 99.9 percent of the organism, while the MIC is the minimum inhibitory concentration. The MBC will be greater than the MIC. 175.

What least likely affects the length of the incubation period in an

infectious disease? a. The organism’s virulence b. The size of the inoculum c. Host immunity level d. Each of these affects the incubation period Answer: d. There are many factors involved in disease incubation periods, including the route of entry and the type of organism involved. 176.

Transmission and contagiousness of a pathogen during which phase of the

infectious disease confers the greatest advantage to the pathogen? a. Incubation b. Prodromal c. Illness d. Convalescence Answer: b. The ability of a pathogen to transmit during the prodromal period confers an advantage to the pathogen because it means that the organism can transmit before the person can be quarantined in order to prevent further disease spread.

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177.

During which phase of an infectious disease are the symptoms nonspecific

but noticeable? a. Incubation b. Illness c. Convalescence d. Prodromal Answer: d. In the prodromal phase, there will be nonspecific symptoms, such as fever, malaise, or inflammation but the true syndromal symptoms are not seen yet. 178.

Which disease is more likely to be a chronic disease rather than a latent

disease? a. Hepatitis C b. Herpes simplex c. Epstein-Barr virus d. Varicella virus Answer: a. Each of these will cause a latent disease except for hepatitis C, which lends itself to chronic infections rather than latent disease. 179.

What is not true of virulence of an organism?

a. An organism is either virulent or not virulent. b. Virulence occurs in pathogens, while not all pathogens are very virulent. c. Virulence involves the median lethal dose of an organism. d. Virulence can be determined in animal models. Answer: a. Virulence is actually a continuum rather than an all or none phenomenon. The rest of the statements on virulence are all true.

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180.

The difference between a primary pathogen and an opportunistic pathogen

depends mostly on what factor or factors? a. Virulence of the organism b. Host factors c. Type of organism d. Incubation period of the infection Answer: b. Opportunistic pathogens differ from primary pathogens because of host factors, that will protect the healthy person from an opportunistic infection but not necessarily a primary pathogenic infection. 181.

What portal of entry is not related to the mucous membranes?

a. Gastrointestinal tract b. Vaginal c. Needle injection d. Respiratory tract Answer: c. Each of these is a mucous membrane portal of entry except for a needle injection, which is a parenteral portal of entry. 182.

Which is not considered a TORCH infection, capable of vertical

transmission from mother to fetus in utero? a. Rubella b. HIV c. Hepatitis B d. Hepatitis A Answer: d. The diseases listed include TORCH infections, except for hepatitis A, which is not transmitted this way.

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183.

What type of infection will involve most or all of the person’s organs and

tissues? a. Primary infection b. Secondary infection c. Focal infection d. Systemic infection Answer: d. A systemic infection involves most or all of the person’s organs or tissues, leading to what becomes a systemic disease. 184.

What type of organism will have a protein or glycoprotein adhesin on

fimbriae as part of its virulence factor? a. Virus b. Bacteria c. Helminth d. Protozoan Answer: b. Bacteria will have adhesins made from proteins or glycoproteins on fimbriae that bind to receptors on the host cell. 185.

What is the operative definition of toxemia?

a. The presence of bacterial toxins in the bloodstream b. The presence of bacteria in the bloodstream c. The state of shock that comes with a severe infection d. The multiplication of an organism in the blood Answer: a. Toxemia in particular means that toxins are present in the bloodstream. It does not necessarily mean there are bacteria, shock, or organism multiplication in the blood itself.

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186.

What is the major difference between an endotoxin and an exotoxin?

a. Endotoxins are heat labile and exotoxins are heat stable b. Exotoxins cause inflammatory responses and endotoxins cause specific host responses c. Exotoxins are far more lethal than endotoxins d. Endotoxins are made from proteins and exotoxins are made from lipids Answer: c. Each of these statements is false except that exotoxins are far more lethal in smaller quantities when compared to endotoxins. 187.

Which virulence factor in bacteria will break down antibodies in order to

prevent phagocytosis? a. Capsules b. Fimbriae c. Proteases d. Phospholipases Answer: c. Bacteria can contain proteases that break down antibodies, which in turn prevents the phagocytic process. 188.

What happens in viruses most often to help the virus be more infective to

the host? a. Toxin production b. Formation of capsules around the virus c. Protease synthesis d. Antigenic variation Answer: d. Viruses undergo antigenic variation in order to change themselves in order to be able to infect a host cell. They do not make toxins, capsules, or proteases.

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189.

What is not a toxin produced by Aspergillus as part of its virulence factors?

a. Elastase b. Ergot toxin c. Aflatoxin d. Gliotoxin Answer: b. Each of these, plus catalase, is made by Aspergillus as part of its virulence; however, ergot toxin is made by another fungus that grows on rye. 190.

What represents the percent of people who die of a disease compared to a

standard number in the population? a. Mortality rate b. Morbidity rate c. Prevalence d. Incidence Answer: a. The mortality rate is a percentage that is the number of patients who die of a disease compared to a standard number in the population. 191.

What is the responsibility of goblet cells in the innate immune system?

a. They make mucus b. They secrete immunoglobulins into the GI and respiratory tract c. They cause increased inflammation if there are pathogens present d. They contain cilia that drive off debris and pathogens Answer: a. Goblet cells make mucus by secreting it from secretory vesicles. The mucus helps trap debris and pathogens before they can invade.

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192.

What is not a mechanical defense system in the eyes?

a. Blinking b. Tears c. Cilia d. Eyelashes Answer: c. Each of these is a mechanical defense mechanism in the eyes, except there are no cilia in the eyes that act as a defense system in this area. 193.

What aspect of the innate immune system is disrupted by taking an

antibiotic? a. Cilia movement b. Chemical barriers c. Immunoglobulins d. Microbiome Answer: d. The microbiome is disrupted when antibiotics are taken, which keeps these organisms from preventing an opportunistic infection. 194.

What is least likely a factor with the sebum produced by sebaceous glands

to prevent bacterial contamination of the skin? a. It supports the growth of a healthy microbiome. b. It contains antibodies that are directed at pathogens. c. It seals off the hair follicle. d. It is the source of acidic oleic acid. Answer: b. Each of these is a reason why sebum is protective against pathogens but it does not contain any antibodies directed at pathogens.

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195.

What is not something that antimicrobial peptides do in the innate

immune system? a. The destroy nucleic acids. b. They damage the cell membrane. c. They bind iron. d. The prevent cell wall synthesis. Answer: c. Each of these is something that antimicrobial peptides do but they do not bind iron as one of their properties. 196.

Where are most of the antimicrobial peptides made and used?

a. Skin b. Blood c. Lymph glands d. Mouth Answer: a. Most of the antimicrobial peptides are made and used by the skin. These act as chemical protection against different pathogens. 197.

Where can you find acute-phase proteins in the innate immune system?

a. Bone marrow b. Lymph nodes c. Epithelial surfaces d. Blood plasma Answer: d. Acute-phase proteins have antimicrobial properties and are found in blood plasma as part of the innate immune response.

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198.

The coating of bacteria with a substance to help kill it is called what?

a. Forming a membrane attack complex b. Phagocytosis c. Opsonization d. Complement activation Answer: c. The coating of bacteria with a substance that assists in its recognition by the phagocytic cells is called opsonization. 199.

The MAC or membrane attack complex is active against what type of

pathogen? a. Viruses b. Gram-positive bacteria c. Gram-negative bacteria d. Fungi or yeast Answer: c. Gram negative bacteria are the only pathogens affected by the MAC because it causes pores to be made into the membranes of these cells. 200. What type of proteins are interleukins, interferons, and chemokines? a. Enzymes b. Structural antibody proteins c. Complement proteins d. Cytokines Answer: d. These proteins are cytokines that have different effects on the immune system, most of which are stimulatory in nature.

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201.

Which molecules are directly responsible for increasing the person’s body

temperature in an infection? a. Bradykinins b. Prostaglandins c. Histamines d. Interferons Answer: b. Prostaglandins are most involved in raising body temperature, which impacts the growth of microorganisms. 202. Which is not a type of granulocyte? a. Lymphocyte b. Neutrophil c. Eosinophil d. Basophil Answer: a. Each of these is a granulocyte because they contain granules, except for lymphocytes, which do not contain granules. 203.

What is not something that natural killer cells do to kill off damaged cells?

a. Trigger target cell apoptosis b. Phagocytize target cells c. Cause pores in the target cell d. Release granzymes to damage cells Answer: b. NK cells are active against damaged cells. They do each of these things except they do not participate in phagocytosis.

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204. What chemical mediator is responsible for the vasodilation that happens because of the inflammatory response? a. Histamine b. Interferon c. Cytokines d. Interleukin Answer: a. The release of histamine from mast cells causes vasodilation that ultimately dilutes the bacteria or toxins and allows for inflammatory cells to reach the site of the infection. 205.

Where does extravasation of white blood cells occur?

a. Arteries b. Lymph nodes c. Capillaries d. Veins Answer: c. Extravasation or diapedesis occurs only in the capillaries because they have very thin walls and also have low levels of turbulence so the white blood cells can easily attach to the endothelial lining and pass between adjacent cells. 206. What least likely attaches to the pathogen as part of opsonization? a. Complement proteins b. Cytokines c. Lectin d. Antibodies Answer: b. Opsonization or the addition of opsonin to a pathogen involves lectin, antibodies, and some complement proteins. These will increase the chances of phagocytosis.

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207.

Which substance does not participate in destroying a pathogen inside a

phagocyte? a. Hydrogen peroxide b. Reactive oxygen species c. Complement proteins d. Lysosomal enzymes Answer: c. Inside the phagocyte, there are lysosomal enzymes, hydrogen peroxide, and reactive oxygen species that participate in digesting the pathogen. Complement proteins are not involved. 208. What molecule type is too small to initiate an immune response in the body by itself but is able to cause an immune response if bound to something bigger? a. Hapten b. Epitope c. Antigen d. Conjugate antigen Answer: a. A hapten is a small molecule that is too small to initiate an immune response by itself. If bound to a larger protein, it forms a conjugate antigen that will be antigenic. 209. What part of the antibody is directly involved in binding to the antigen? a. Fc region b. Constant region c. Disulfide bond d. Variable region Answer: d. The variable region is part of the Fab region that actually does the binding of the antigen to the antibody.

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210.

What is not true of an antigen presenting cell?

a. They are all capable of making antibodies. b. They can be macrophages, B cells, or dendritic cells. c. They are the only cells that express MHC II. d. They engulf antigens and re-present them on their surface. Answer: a. Each of these is true of an antigen presenting cell, which may or may not produce antibodies, depending on which type of cell it is. Only the B cells make antibodies; dendritic cells and macrophages do not do this.

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College Level Microbiology

Articles inside

Using Fluorescent Antibody Methods

EIAs and ELISA Testing

Agglutination Assays

Detection of Antigen-Antibody Complexes

Quiz

B Lymphocyte Function

Major Histocompatibility Complexes and Antigen Presentation

T Lymphocyte Function

Quiz

Vaccinations

Key Takeaways

Pathogen Recognition and Phagocytosis

Inflammatory Processes

Chemical Defense Systems

Key Takeaways

Tracking Infectious Diseases

Quiz

Virulence Factors for Eukaryotic Pathogens

Cellular Defense

Virulence Factors for Viruses and Prokaryotes

Pathogens

Quiz

Identifying New Antimicrobials and Drug Sensitivities

Key Takeaways

Drug Resistance

Other Antimicrobial Therapies

Antibacterial Therapy

Quiz

Key Takeaways

Controlling Microbial Growth

Effects of the Environment on Microbial Growth

Media and Microbial Growth

Antiseptics

Quiz

Key Takeaways

Genetic Diversity in Prokaryotes

Operons and Gene Regulation

Mutations

Translation and Protein Synthesis

Quiz

Key Takeaways

RNA Transcription

Cellular Genomes

RNA Structure and Function

Quiz

Biogeochemical Cycles

Key Takeaways

Photosynthesis

Catabolism of Proteins and Lipids

Cellular Respiration

Fermentation

Catabolism of Carbohydrates

Quiz

Key Takeaways

Biochemical Principles in Microbiology

Proteins

Carbohydrates

Lipids

Quiz

Lichens

Algae

Helminths

Fungi

Quiz

Key Takeaways

Gram-negative Bacteria

Quiz

Isolation and Identification of Viruses

Key Takeaways

Proteobacteria

Viroids and Prions

Viral Life Cycle

Quiz

The Prokaryotic Cell

The Eukaryotic Cell

Key Takeaways

Quiz

Staining of Microorganisms