BIO 171 Module 4 Exam Complete Solutions 2024

BIO 171 Module 4 Exam Growth media - ✔nutrient-rich environment for isolating and culturing microorganisms, includes sugars, amino acids, and vitamins, also called nutrient broth, grows microbes in a suspension Lysogeny broth - ✔most common nutrient broth, can grow a vast array of microbes, plates are yellow, non-selective and non-differential media, commonly used for growing e coli (easily produces recombinant proteins) Selective media - ✔suppress growth of unwanted bacteria and encourage growth of desired microbes, uses limited amounts of nutrients, varying degrees of pH, or various chemical additives like antibiotics that limit microbial growth, often used to grow Neisseria meningitides (fungi and mold outgrow this slow growing bacteria) Differiential media - ✔distinguishes between 2 unrelated microbes, e.g. E coli and salmonella are both gram negative but can be distinguished by the presence of lactose fermentation- e coli ferments lactose and turns the culture red, salmonella doesn't and culture remains white/tan enriched media - ✔contains complex organic substances such as blood, serum, hemoglobin, or special growth factors required by fastidious microbes, used to grow fastidious 2 forms of growth media - ✔liquid and solid solid growth media - ✔housed in a sterile petri dish, liquid growth media with hardening agent added Agar - ✔a gel-like polysaccharide compound used for culturing microbes; extracted from certain red algae, creates a solid smooth surface Colonies - ✔microbial growth in a media, appears as individual isolated dots- isolates merge into one another Lawn - ✔microbial growth of the entire petri dish Agar characteristics - ✔: Outside of the lab you've probably come across the 'cousin' of agar, gelatin. Gelatin is an animal-derived product that gives Jello its classic texture and form. Agar is simply the plant-based form and acts in a very similar way. In both cases, as you increase the amount of agar/gelatin the firmer the composition becomes. The dehydrated agar (and other necessary components) is mixed with water and heated to a high temperature to dissolve the agar into solution. The high temperature also ensures

sterility as it will kill most, if not all, foreign microbes. Once poured into a sterile petri dish and cooled, the agar plate is a sterile environment on which microbes can grow.

Agar plates are made by - ✔agar melted, sterilized, and poured into a sterile petri dish, solidifies at room temperature Trypticase Soy Agar - ✔grows a wide variety of microorganisms, non-selective and nondifferential, yellow in color, serves as a base when formulating specialized enriched medias Blood Agar - ✔BAP, derivative of TSA, blood usually from a sheep added to plate compensation as a concentration range of 5-10%, red in color, enriched non-selective but differential media BAP promotes the growth of - ✔fastidious microorganisms- strains of Streptococcus BAP detects - ✔hemolytic activity Hemolysis classification - ✔alpha, beta, gamma Alpha hemolysis - ✔incomplete lysis of red blood cells producing a greenish -brown halo around colonies because hemoglobin is oxidized to methemoglobin beta hemolysis - ✔complete lysis of red blood cells around a colony (results in a clear zone surrounding colonies) gamma hemolysis - ✔No hemolysis, and no change in the blood agar around the colony, white/tan in color Columbia CNA agar - ✔selective and differential media, enriched and red in color from blood enrichment (allows for differential based on hemolytic patterns) , has antimicrobial agents colistin and Nalidixic acid, suppresses growth of Gram-negative bacteria, used for isolation of Gram-positive microbes Chocolate agar CHOC or CBA - ✔enriched non-selective non-differential media derived of blood agar plates, contains RBC lysed by heat- gives appearance of dark brown, lysed RBC release various growth factors required for cultivation of fastidious pathogenic bacteria, e.g. haemophilus influenza, Neisseria meningitidis MacConkey Agar - ✔selective-differential, selects for gram negative enteric organisms, isolate intestinal pathogenic microbes belonging to the Enterobacteriaceae family such as Salmonella and Shigella species.

2 factors are present in MacConkey Agar - ✔crystal violet and bile salts, restricts the growth of Gram + bacteria 2 factors in MacConkey Agar to differentiate Gram - bacteria - ✔lactose and the pH indicator neutral red—it is only red when under acidic conditions—are added to differentiate between lactose fermenters (lac+; red colonies) and non-fermenters (lac-; white/tan colonies) T/F: The base color of the agar plate also often changes color as the microbes consume the nutrients within the MacConkey Agar plate - ✔True Color of MacConkey Agar pH >8.0 - ✔agar can turn yellow (pH > 8.0) within the surrounding area of a growing non-fermenting microbe.

Color of MacConkey Agar pH<6.8 - ✔agar may turn pink or even a darker red within the region surrounding a lac+ microbe given its level of acid production during fermentation, e.g. Gram-negative/lac+ bacteria Escherichia coli- vibrant pink (pH < 6.8) Sorbitol MacConkey Agar (SMAC) - ✔variant of MacConkey agar specifically formulated to detect the presence of the pathogenic strain of Escherichia coli O157:H7 (unable to ferment sorbitol), under enteric conditions E coli is able to ferment lactose and sorbitol SMAC plates contain - ✔sorbitol (instead of lactose) in order to differentiate nonpathogenic strains of Escherichia coli (red colonies; acidic conditions) from pathogenic O157:H7 strains (white colonies; neutral to basic conditions).

Eosin Methylene Blue Agar (EMB) - ✔red in color and is classified as both a selective and differential form of media, contains eosin and methylene blue that restricts the growth of Gram-positive bacteria EMB differentiation - ✔based on their ability to ferment the lactose present in the media, microbes grow as white/tan colonies, while lactose-fermenting microbes produce dark (purple to black) colonies on the surface of the agar Color of e coli on EMB plate - ✔metallic green sheen Plating - ✔process of spreading a bacterial culture onto a petri dish filled with agar 3 sterile devices used for plating - ✔loop, swab, wire loop, spreads the bacteria on the plate using a back-forth motion Advantage of placing sample onto agar - ✔cells are held in place, unlike in a nutrient broth where bacterial cells can multiply but are free to move around in solution, bacteria plated onto agar are fixed in such a way as to support the formation and visualization of

colonies. Colonies are visible to the naked eye but only after the bacterial cell has multiplied, often a million times over. Thus, each colony is derived from a single cell and should be free from outside contaminants (other microbes) Quadrant Streak Method - ✔allows sequential diluation of the original microbial material over the entire surface of a fresh plate. The original sample is diluted by streaking it over successive quadrants, the number of organisms decreases. This produces single, discrete colonies. It can be further examined for its size and shape, motility, Gram status, biochemical properties, etc.

4-phase dilution gradient process - ✔sample is spread into 4 regions where each new region is perpendicular to the previous region, such that each region will become more diluted than the previous first phase. (P1), a sterile loop is first used to streak a sample back-and-forth across a small area of the plate. The first region will contain the highest concentration of microbial growth. Next, a new sterile loop will be used to pull some of the sample from the first region into the second phase (P2), where it is further streaked.

The microbial concentration is now diluted because only a small portion of the bacterial sample from phase one was moved and spread out into P2. The same process is repeated from P2 to phase 3 (P3) and then from P3 to phase four (P4), each time using a new sterile loop. Thus, each phase should be systematically diluted such that the bacterial concentration of each phase will be established as P1 > P2 > P3 > P4.

T/F a new (or sterilized) loop must be used each time a new phase is started. - ✔True, Failure to do so would prevent the establishment of a dilution gradient, as the same bacterial concentration would be spread across both phase regions Subsequent phases should contain - ✔lower concentration of microbes such that an individual colony will be eventually produced. Although individual colonies are most often seen in P4, depending on the starting concentration in P1 individual colonies may also begin to appear in P3 or even in P2.

What should you do after streaking a sample? - ✔plates are inverted and placed in an incubator at 37 C for 12-24 hrs, after 12 hrs of incubation the phases are examined and individual colonies identified. Individual colonies are then isolated, picked and reinoculated in nutrient broth (or onto nutrient agar) for the expansion of the now pure culture to be used for laboratory testing.

Temp of yeast growth - ✔30 C T/F pathogenic bacteria grow faster than non-pathogenic strains at 37 C - ✔True, incubators may be set at 25 C to restrict pathogenic growth Expanding microbial population - ✔inoculate a simple growth media with the collected sample or streak it onto an agar plate. The culture (or plate) is then grown at 37°C to

encourage microbial growth. Once a bacterial population has been expanded, samples can be taken and re-streaked onto selective and/or differential media for further analysis Scenario #1 - ✔theinitial test results indicate a motile Gram-negative rod. Now, if the physician remembers his microbiology he will likely consider the causative agent for abdominal pain as either salmonella or E. coli (both are common pathogens of foodborne illnesses). However, the initial test results are not enough. The physician then orders the isolated bacterium to be streaked out on EMB agar. The next day he receives a report of a distinct metallic green growth pattern and immediately knows the causative agent is E. coli and not salmonella.

Scenario #2 - ✔although workers were already being treated for staph aureus it is unclear if one, both or neither of the cleaning companies were responsible for its spread. Initial test results indicated both companies could potentially be the source as both sets of samples contained non-motile, Gram (+) cocci—key characteristics of staph aureus. Unfortunately, the test results came back with an inconclusive result regarding the exact morphology of the bacteria isolated from each company. (As a trained microbiologist would know, if any results came back as decisively Gram-positive cocci chains this immediately rules out staph aureus as it forms Gram-positive clusters). As an alternative strategy, a researcher then streaks the isolated bacterial samples from each company onto MSA agar. After incubating the samples overnight at 37°C, Company 1 samples showed only colorless colonies growing on red agar indicating the presence of non-pathogenic bacteria while the samples collected from Company 2 (floors and their cleaning mops and sponges) showed a distinct yellow agar indicating the presence of staph aureus. fastidious microbes - ✔require growth factors and complex nutrients