Microbiology World Issue 4

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Microbiology World

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Chief Editor Mr. Sagar Aryal Medical Microbiology (M.Sc), Nepal (Founder) Reviewers Mr. Samir Aga Department of Immunological Diseases Medical Technologist, Iraq Mr. Saumyadip Sarkar ELSEVIER Student Ambassador South Asia, Reed Elsevier (UK) Ph.D Scholar (Human Genetics), India Editors Dr. Sao Bang Hanoi Medical University Dean of Microbiology Department (Provincial Hospital) Microbiology Specialist, Vietnam Mr. Tankeshwar Acharya Lecturer: Patan Academy of Health Sciences (PAHS) Medical Microbiology, Nepal Mr. Avishekh Gautam Ph.D Scholar, Hallym University, South Korea Mr. Manish Thapaliya Lecturer: St. Xavier’s College Food Microbiology, Nepal Mr. Sunil Pandey Medical Microbiology, Nepal www.microbiologyworld.com

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Table of Content Page No.

Who are Microbiologist?

4-7

DNA Microarray Technique

8-10

Animal Vaccination

11-15

Microbiology in Pharmaceuticals Industry

16-19

Need of Advocacy and awareness about cancer in Nepal

20-21

How to Microorganism contribute to body odor?

22-23

Embryonic Stem cell research

24-28

Staphylococcus aureus And MRSA

29-32

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Who are Microbiologist? Microbiologists are biological scientists who study organisms so small that, generally, they can only be seen with a microscope. These microorganisms include bacteria, algae, yeasts, fungi, protozoa, viruses, and other microscopic forms of life. Microbiologists isolate and make cultures of microorganisms, identify their characteristics, and observe their reactions to chemicals and other kinds of stimuli. They also study how microorganisms develop and reproduce as well as their distribution in nature. Many microbiologists work for universities, where they teach and do research. Others work at medical centers or in private industry. Some work for government agencies. Although their jobs have different aspects and responsibilities, most microbiologists do some research or laboratory work. They use special equipment to study microorganisms including light microscopes, electron microscopes, centrifuges, glass tubes, slides, and computers. They are often assisted by biological technicians. Microbiology is a broad field that includes the study of viruses as well as microscopic organisms found in all kingdoms of life: plants, animals, protists, fungi, and bacteria. Some microbiologists specialize in one type of microorganism. For example, bacteriologists concentrate on bacteria and virologists study viruses. Microbiologists work in several areas. Many do basic research to increase knowledge about the life processes common to microbes. Their work helps to answer basic questions such as those pertaining to the use of food and oxygen in cells. Other microbiologists are employed in medicine. Medical microbiologists study the relationship between microorganisms and disease. They isolate and identify disease-producing organisms and study their distribution. They also study the ways that the organisms enter the bodies of humans and animals, establish themselves, and cause disease. Immunologists, for example, study the body's defensive responses to microorganisms. Microbiologists are also employed in the related field of public health. They work to combat problems such as outbreaks of epidemics, food poisoning, and the pollution of air and water. For example, public health microbiologists test blood samples sent in by physicians to see whether patients have a communicable disease. They also test drinking water, milk supplies, and other substances that can affect the health of the general public. Other fields in which microbiologists work include agriculture, marine microbiology, and industry. Agricultural microbiologists study the microorganisms found in soil and their effects on plant growth. Marine microbiologists seek ways to control the growth of harmful bacteria in oceans and rivers. Industrial microbiologists work in a variety of industries, including food processing, chemicals, and drugs. They may work to control the activities of microorganisms in such processes as the tanning of leather and the fermentation of wine. www.microbiologyworld.com

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Working Conditions Working conditions for microbiologists vary. Most spend at least part of their time in clean, well-lighted laboratories. Some microbiologists have to collect samples of soil, seawater, and other substances that contain microorganisms. Some microbiologists spend part of their time in classrooms and offices. The workweek for many microbiologists in medical centers and private industry is generally forty hours. Those who work in universities and other research centers may have more flexible hours, but their workweeks generally total more than forty hours. Some overtime or shift work may be necessary when a project must be completed or when an experiment must be monitored around the clock. Microbiologists usually spend some time reading and studying to keep up with the newest findings of other scientists. Microbiologists must take precautions to prevent specimens from being contaminated and to keep harmful microorganisms from reproducing uncontrollably. They should have skill in scientific experimentation and mathematics and be willing to do the precise, detailed work required in microbiology. Microbiologists should be able to work either independently or as part of a team. They must be able to keep careful records and to communicate their ideas and findings to others.

Description, Areas and Scope of Medical Microbiologist Medical microbiology course is branch within the field of medicine which focuses on micro organisms of medical interest which include the bacteria, viruses, fungi and parasites which are of medical importance and are capable of causing infectious diseases in human beings. Microbiologists can rather be called as detectives who investigate in the microscopic world - a world that holds much wonder and mystery, the world which much people cannot see. Medical Microbiology Course description includes the study of microbial pathogenesis and epidemiology and is related to the study of disease pathology and immunology. Microbiologists study micro organisms which can cause disease in people, looking into life cycles of such organisms, on how they cause infection, how they spread, and cause disease, the means to treat the diseases and irradiate the disease causing microbes. This field of microbiology is constantly engaged with identifying new micro organisms, monitoring changes in rapidly mutating species and dealing with the ongoing challenges in medicine, ranging from the development of resistance to antibiotics in bacteria to contamination of water supplies with protozoans. They work in all ways to contribute to mankind and improve humans and environmental quality of life and living.

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Scope and career prospectus of microbiologist TEACHING : If you are interested in teaching as a profession and if you are interested to pursue your masters degree, and you are basically a science student, then microbiology is the best suited to pursue a masters in. Teaching is a noble profession, and the returns after completing your study is tremendous. And again the scope is vast as it is the basic subject of study in all medical colleges as well as Para medical colleges like dental science, physiotherapy, pharmaceutical science, nursing science, radiography, lab technology etc. It is the most highly paid among the teaching profession in science, even at the initial stage as a beginner. As well you get an opportunity to work among the dignified doctors and amongst the highly esteemed medical professionals. Academic microbiologists are employed by universities, schools and teaching hospitals. RESEARCH SCIENTIST: Research is a never ending field and thus one can take up research. As a research scientist a microbiologist can work in universities, institutions, industries, hospitals, government organizations and carry out research in laboratories. Many work as associate or assistant scientists or researchers doing the routine work of conducting experiments, others are senior scientists or project managers who lead experiments, supervise lab workers, interpret data and develop new theories and experiments. CLINICAL ASSOCIATE: One can get into clinical research as clinical coordinator or clinical trials administrator and later on generally qualify to move on to a full clinical research associate position. CLINICAL MICROBIOLOGIST: Clinical microbiologist assist the treating team of doctors with the investigations, diagnosis and treatment of disease. That analyze and interpret data related to patient samples. They work for the betterment of the society by engaging in a wider range for complex work in laboratories especially against incurable, life threatening diseases like cancer, AIDS etc. They advise clinicians on the use of tests, diagnosis of disease and planning and progress of the treatment. They work to combat problems such as outbreaks of epidemics, food poisoning, pollution of air and water, for eg., they check blood samples sent in by physicians to check for any communicable diseases. TOXICOLOGIST: Microbiologists are employed as toxicologists in investigatory laboratories. They plan and carry out laboratory and field studies to identify, monitor and evaluate the impact of toxic materials and radiations on human and animal health, and on the health and current status of the environment, as well as the impact of future technologies. www.microbiologyworld.com

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FORENSIC SCIENTISTS: Microbiologists with appropriate skills are also considered for posts in forensic science. BIOMEDICAL SCIENTISTS: Following basic training, most biomedical scientists specialize in one aspect of medical laboratory science. The main areas are microbiology, clinical chemistry, transfusion science, hematology, histopathology, cytology, immunology and virology. BIOSTATISTICIAN: Biostatisticians are statisticians who work in the health related fields. They design research studies and collect and analyze data on problems such as how disease progress, how safe is the treatment, or the impact of certain risk factors associated with medical conditions. Microbiologists have enough and more jobs as quality assurance specialists in the pharmaceutical companies, food processing industry, diary and milk products, beverages, hotels, biotechnology companies, agriculture, forestry etc.

Microbiology in Nepal Nepal is a least developed and land locked country. Ecologically it is divided in to Mountain region, Hilly region and Terai region. Majority of population living in remote and village area and these areas are lacking many facilities and there is unplanned urbanization in developed part resulting in lack of health care facilities, places and water supply. Most of them have poor economic status and have little or no knowledge about proper sanitation so that infectious can be prevented. About 70% of all health problems are attributed by infectious diseases in nepal. Many children are dying from easily preventable and treatable diseases such as diarrhea and/or dysentery, and acute respiratory infections. So, Knowledge of microbiology is important in Nepal .

- Sunil Pandey Nobel College, Kathmandu, Nepal

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DNA Microarray Technique It is DNA homology analysis for detecting polymorphisms and mutations in both prokaryotic and eukaryotic genomic DNA. A microarray is a compact device which contains a large number of well defined immobilized capture molecules in the form of spots assembled in a known pattern on a solid support, usually glass slides, nylon membranes or silicon chips. These molecules can be PCR products, proteins, antibodies, oligos.

PRINCIPLE The basic principle of microarray is based on the hybridization of complimentary probe and target cells. Each DNA spot contains a small amount of a specific DNA sequence in picomoles (10−12 moles), which are known as probes. These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called target) under high-stringency conditions. The complementary nucleic acid sequences specifically pair with each other by making hydrogen bonds between complementary nucleotide base pairs. Probe-target hybridization is usually detected and quantified by detection of fluorophore, silver, or chemo-iluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target. Thus by using an array containing many DNA samples, scientists can determine the expression level of thousands of genes in a cell by measuring the amount of mRNA bound to each spot on the array.

HISTORY Microarray technology is evolved from Southern Blotting, in Southern blotting technique DNA fragments are attached to a substrate and then they are probed with a known DNA sequence segment. In 1987 the arrayed DNA were used for the identification of genes whose expression is regulated by interferon. These early gene arrays where developed by spotting cDNA on filter papers with the help of automated pin spotting device. In 1995 quantitative monitoring of gene expression with a complimentary DNA microarray was done.

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In 1996 for the first time microarrays were commercialized by an American company AFFYMETRIX. In 1997 a complete eukaryotic genome of a yeast Saccharomyces cerevisiae was published on an microarray. From 2000-2003 the use of microarray in clinics was introduced for the detection of cancerous cells. In 2004 the whole human genome was published on a microarray.

TYPES OF MICROARRAYS DNA microarrays such as cDNA microarray, oligonucleotide microarrays and SNP microarrays . MMchips, for surveillance of microRNA populations. Peptide microarrays, for detailed analysis or optimization of protein protein interactions. Tissue microarray. Chemical compound microarray. Cellular microarray (also called transfection microarrays) Antibody microarrays. Carbohydrate microarrays (glycoarrays) Phenotype microarrays.

BASIC STEPS INVOLVED IN MICROARRAY TECHNIQUE 1. Obtain DNA microarray chip 2. Extraction of RNA from the samples 3. Cy3 and Cy5 labeling of the RNA 4. Production of cDNA by RT-PCR 5. Hybridization with cDNA probes to DNA array 6. Detection of hybridization signal 7. DNA microarray data analysis

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APPLICATIONS OF MICROARRAY DNA microarray are used to examine gene expression patterns under diseases such as cancer. Tumor profiling using DNA microarrays allows the analysis of development and progression of such complex diseases. DNA microarrays are used for virus detection from blood and other samples thus it is used for pathogen detection. DNA microarrays more recently have been used to indentify inheritable markers, and there used as genotyping tool. Used for single nucleotide polymorphism (SNP) detection.

ADVANTAGES OF MICROARRAYS Microarrays are better than other profiling techniques because: Easier to use Can analyze thousand of genes or markers at a time Generate large amount of data in little time Do not require large scale sequencing Allow the quantization of thousand of genes from many samples Compare two different population like: wild type vs wild, normal tissues vs cancerous tissue, study specific chromosomal regions.

- Muhammad Hamza Sana (D.V.M) +923436247467 hamzasana46@yahoo.com Institute of Microbiology, University of Agriculture, Pakistan.

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Animal Vaccination: low cost insurance against disease Introduction It is always good to prevent rather than to cure a disease and vaccination is the most effective way of preventing diseases. Beginning with the variolation the method of prophylaxis reached a new era with the advent of cell culture techniques in 1950’s which led to the development of many live attenuated and inactivated virus vaccines. More recently, the field of vaccinology has witnessed the introduction of novel ‘new generation vaccines’ produced through various form of recombinant DNA and related techniques which offer significant improvements and potential advantages in terms of both their safety and their efficacy. It’s all because of vaccination that we were able to eradicated smallpox and rinderpest globally. Like people, animals also need vaccines. The importance of vaccination is when you are aware of the vaccines of your animal and their schedule and more importantly getting your animal vaccinated regularly at prescribed time interval. This article provides information to animal owners and pet lovers on routine vaccination programs to prevent common diseases.

When to vaccinate Animals should be vaccinated for a disease before they will encounter the microorganisms causing it. It takes from 10 to 21 days after vaccination to mount a protective immune response. The exact length of time depends on the animal, its age and health status, the vaccine itself, and whether it has been vaccinated before. We have to emphasize the fact that no single schedule is suitable for every situation. An effective vaccination schedule depends on the type of operation, maternal antibody levels, disease challenge on the farm, age, level of hygiene and many other factors. It is best to consult a veterinary specialist to advice a schedule based on your specific circumstances. Here are the vaccination schedule charts for different animals mentioned below which can serve as a guideline only.

Vaccination of Cattle, Buffalo, Sheep and Goats A herd health management plan is vital to profitable production. Some producers, however, do not vaccinate until they experience a loss. The investment in disease prevention is less than the cost of disease treatment. Don’t wait until a disease outbreak occurs. For animals to reach their performance potential, they must be healthy. Many animal health problems can be controlled with good management, proper nutrition and vaccination against infectious diseases. www.microbiologyworld.com

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Here is the table for the vaccination schedule for cattle, buffalo, sheep and goat: Diseases

Animal

Primary Vaccination

Re-Vaccination

F.M.D.

All cloven footed animals Cattle, Buffalo, Sheep,Goat

4 months

Twice in a year

6 months

Annually before monsoon (twice a year in endemic areas)

Anthrax

All species of Animals

6 months

Once Annually (In Affected area only)

B.Q.

Cattle,Buffalo,Sheep,Goat

6 months

Annually before monsoon

Brucellosis

Female cattle & buffalo Calf

4-8 months Only

Only once i.e. at 4-8 months of age in females in problem herds

Theileriosis

Cattle

above 2 months

Annually

Enterotoxaemia

Sheep,Goat

Before monsoon (preferably in May). Booster vaccination after 15 days of first vaccination

P.P.R.

Sheep,Goat

4 months for kid or lamb (If dam is vaccinated). At the age of 1st week for kid or lamb (If dam is not vaccinated) 3 months for kid or lamb

Sheep Pox, Goat Pox, C.C.P.P.

Sheep,Goat

At the age of 3 month & above for lamb or kid

Once Annually (December month)

Rabies

All species of Animals

H.S.

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Once in three years

0, 3,7,14,28 & 90 days post exposure only

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Vaccination of Dog There are three major vaccines used in dogs. The vaccination schedule is given below: Diseases

Primary Vaccination

Secondary Vaccination

ReVaccination

Canine Distemper, Canine Hepatitis (CanineAdenovirus 2), Corona Viral Enteritis, Canine Parainfluenza, Parvo Virus Infection, Leptospirosis (L.canicola, L.icterohaemorrhagiae)

6-8 weeks of age

2-3 weeks later upto 16 weeks of age

Annual

3 months of age

After 3 months

Rabies Bordatella

After 1 year

Annual Annual

Vaccination of Cat Diseases

Primary Vaccination

Secondary Vaccination

Re-Vaccination

Panleukopenia, Rhinotracheitis, Calicivirus

6 weeks of age and repeat every 3-4 weeks until 16 weeks of age

1 year after completion of initial series

Every 3 years

Feline leukemia

8 weeks of age and repeat in 3-4 weeks

1 year after completion of initial series

Annual

Rabies

Single dose as early as 8 weeks of age, depending on the product.

2 doses, 12 months apart

Annually or every 3 years, depending on vaccine used

Bordetella

8 weeks, then 2-4 weeks later

2 doses, 2-4 weeks apart

Annually

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Vaccination of Horse Vaccinate your horse against all diseases he may be exposed to, including at home or if you travel with your horse. The chart below is a guideline of the vaccines suggested for some general ages and classes of horses. Diseases

Re-Vaccination

Anthrax

Primary Vaccination From non-vaccinated mare: First dose: 3-4 months Second dose: 4-5 months From vaccinated mare: First dose: 6 months Second dose: 7 months Third dose: 8-9 months 6 months

Rabies

3 months or above

Annually. Post exposure : 3,7,14,28 & 90 days

Tetanus toxoid

Annually

Once Annually (In Affected area only)

Some important points to remember: Practice good management. 1. Before any vaccination deworming should be compulsory to get better results. 2. Read and follow vaccine labels carefully. 3. Always follow the manufacturer’s recommendations for dosage, method of administration, number of times given and proper storage. 4. Vaccinate to prevent diseases which have a high risk of occurring, not diseases with a low risk. 5. Herd-specific vaccination programs should always be developed in consultation with a veterinarian. 6. The best way to stay on schedule with vaccinations for your dog or cat is to follow the recommendations of a veterinarian you trust.

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Reference and Readings 1. Greene, C.E., Schultz, R.D. and Ford, R.B. 2001.Canine vaccination. Vet Clin North Am Small Anim Pract.31:473-492. 2. Scherk, M.A., Ford, R.B., Gaskell, R.M., Hartmann, K., Hurley, K.F., Lappin, M.R., Levy, J.K., Little, S.E., Nordone, S.K. and Sparkes, A.H. 2013.AAFP Feline Vaccination Advisory Panel Report. Journal of Feline Medicine and Surgery.15(9):785 3. Gaskell, R.M., Gettinby, G., Graham, S.J. and Skilton, D.2002. Veterinary Products Committee working group report on feline and canine vaccination. Vet Rec.150:126-134. 4. Callan, R.J. 2001. Fundamental considerations in developing vaccination protocols. AABP Proceedings.34:14-22. 5. Hunsaker, B.D. and Tripp, S.P. 2007. Vaccine field efficacy: A review of field efficacy reported for vaccine antigens used in beef cattle and dairy practice,1996 to present. AAPB Proceedings.40:26-32. 6. Perino, L.J., and Hunsaker, B.D. 1997. A review of bovine respiratory disease vaccine field efficacy. Bovine Practitioner.31(1):59-66. 7. Radostits, O.M. 2001. Herd Health: Food Animal Production Medicine, 3rd ed.WB Saunders, Philadelphia. 8. Singh, S.N. 2011. Foot And Mouth Disease Control Strategies Globalframe Work. International Journal of Life Science and Pharma Research.288:9-42. 9. Giggins T., Soorej, K. 2010.Current Indian Veterinary Index. VETads Publications, Kerala, pp.375-396. http://www.naro.go.ug. National Agriculture Research System http://www.ndri.res.in. National Dairy research Institute http://www.cirg.res.in. Central Institute for Research on Goats. http://www.indg.in. India development gateway.

Vinod Kumar Singh1*, Vipin Kumar Upadhayay1, Sakshi Bhadouriya 1, Vikas Gupta 1, Utkarsh Kumar Tripathi2, Yogender Singh3, Jobin Jose Kattor1, Dash Prakash M.1 1. Indian Veterinary Research Institute, Izatnagar, Bareilly (U.P.) 2. National Dairy Research Institute, Karnal (Haryana) 3. Nanaji Deshmukh Pashu Chikitsa Vigyan Vishvavidyalaya, Jabalpur (M.P.) www.microbiologyworld.com

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Microbiology in Pharmaceuticals Industry Microbiology is one of the important disciplines of science applied in Pharmaceutical industry in order to ensure the sterility of the products such as injections, eye drops, ear drops and infusions. These products must be free from all kinds of microbes particularly bacteria and fungi. To ensure this, a number of procedures are applied which are mentioned below:

1. Sterilization Protocol for working Area/Room The purpose of this practice is to minimize contamination during filling of the injection vials and ampoules or performing a sterility test to get accurate results. The requirements for this procedure are antiseptic such as 3% chlorhexadine solution, 70% Isopropyl alcohol (IPA), distilled water and 0.5% Phenol. As a first step, all types of wastes are removed from the area followed by cleaning with a vacuum cleaner. Then the whole area is washed with 3% chlorhexadine and autoclaved water. Doors and windows are cleaned with wet cloth. The laminar flow hood (LFH), filling machine and other equipments placed in the area are then cleaned with 70% v/v Isopropyl alcohol (IPA). The room’s floor, roof and walls are cleaned with 0.5% Phenol.

2. Bio Burden/Bio Impression Test (General) To confirm that the area/lab is sterilized, bio-impression test is carried out. This test can be done by two methods. Taking swab from floor and walls Exposure of media plates to the area to identify the presence of microorganis ms For taking swab samples, touching walls, equipments or any other items present in the sterile area should be avoided. One must enter the area in a sterile autoclaved uniform in order to avoid contamination. The tools should be rinsed with alcohol inside the area. Separate swab should be used for each area such as floor, walls, ceiling and other indoor installations. The swab should carefully be brought into the LFH. The media plates should be streaked with the swabs and put in the incubator on 35°C for 48 hours. The results should be recorded through colony counter.

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The plates with sterile media (Generally Nutrient agar) should be placed uncovered in the area for 3-4 hours. Plate No.

Filling Area Under Laminar

01

Acceptance Criteria Bacteria

Molds

Conveyer Belt

<1

<1

02

Machine Belt

<1

<1

03 04

Filling Area Sealing Area

<1 <1

<1 <1

05 Sealing End <1 <1 06 Filling Area Floor <1 <1 07 Buffer Area <5 <1 08 Compounding Area <5 <1 nd 09 2 Change Room <10 <1 10 1st Change Room <10 <1 Figure 2: Show the normal acceptance level of organisms in the sterile area

3. Bio Burden/Bio Impression Test (Personnel) This test is used to find out the microbial load on the uniform of working staff in the sterile area. This is done by the microbiologist by taking a swab sample form the clothes i.e. chest, fore arms and finger tips on separate swabs. The swab is streaked on the media (Nutrient agar) and incubated at 35 °C for 48 hours. The results are recorded through colony counter. Response Level/Colony Description of the sample side Bacteria

Molds

Chest

<5

<1

Fore Arm

<5

<1

Finger tips (All Five)

<3

<1

Figure1: Shows the normal acceptable level of bacteria and fungi on the body www.microbiologyworld.com

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4. Sterility Test of the Product by Membrane Filtration Method The purpose of this test is to check the sterility of the vials and ampoules of injections, eye drops and Ear drops. Chemicals required for this test is Peptone water (Buffer), Fluid Thioglycolate Media (FTM), Tryptone Soya Broth (TSB), Isopropyl alcohol (IPA). The test is done in a sterilized microbiology laboratory with a laminar flow hood. Two tubes of both TSB and FTM are taken to the lab along with a sterilized filtration assembly and a membrane filter with 0.2 ¾m pore size. The filtration assembly is unwrapped inside the LFH and is connected with the suction pump. The sample ampoules/vials are opened and a small quantity of it is put into the 100 ml peptone water and then into the cup of filtration assembly. By switching on the suction pump, the entire liquid pass through the membrane filter and moves to the bottle attached to the suction pump. The cup is removed from the assembly and filter is cut through a sterilized scissors into two pieces. The half of filter paper is placed in one bottle of FTM and other half in the other bottle of TSB. FTM is incubated 32°C ¹2.5°C and TSB is at 22°C ¹2.5°C for 14 days. The result is dependent on the turbidity of the media in bottles. Anaerobic bacteria grow in the bottom and aerobic on the surface. FTM is used for Bacteria and TSB is used for fugal culture.

5. Pyrogen/Endotoxine Detection Test Pyrogen is the substance which causes fever. The purpose of this test is to find endotoxin in injection vials and ampoules. For this, a technique known as Limulus Amebocyte Lysate (LAL) test is performed in the laboratory. In the procedure of this test 1mg/ml dilution of the sample is prepared and put in a test tube marked as Test and the two other test tubes with Blank and Standard marks. Depyrogenated water (0.05ml) is added to each tube with the help of sterilized pipette. A 1:10 dilution is made of the test tube marked as Test. Then 0.05 ml of lysate is added to each tube. The tube is then incubated at 37°C for 30 minutes. After incubation the tubes are immediately transferred to chilled water. A 0.250 ml of diazo coupling reagents 8,9,10 (available with the kit) is transferred to the reaction. The intensity of color formed as a result of the reaction between Endotoxin and Lysate is seen. The absorbance of sample test and standard is measured by spectrophotometer with a wave length of 545nm. If 0.4ml of 8M acetic acid is used instead of Diazo Coupling Reagent, then the absorbance is measured by 405nm wave length. The following formula is used for calculation. đ??śđ?‘œđ?‘›đ?‘? .đ?‘œđ?‘“ đ?‘ đ?‘Ąđ?‘‘.Ă—( đ?‘ đ?‘Žđ?‘šđ?‘?đ?‘™đ?‘’ đ?‘Žđ?‘?đ?‘ đ?‘œđ?‘&#x;đ?‘?đ?‘Žđ?‘›đ?‘?đ?‘’ −đ??ľđ?‘™đ?‘Žđ?‘›đ?‘˜ đ?‘Žđ?‘?đ?‘ đ?‘œđ?‘&#x;đ?‘?đ?‘Žđ?‘›đ?‘?đ?‘’ ) EU/ml = (đ?‘†đ?‘Ąđ?‘Žđ?‘›đ?‘‘đ?‘Žđ?‘&#x;đ?‘‘ đ?‘Žđ?‘?đ?‘ đ?‘œđ?‘&#x;đ?‘?đ?‘Žđ?‘›đ?‘?đ?‘’ −đ??ľđ?‘™đ?‘Žđ?‘›đ?‘˜ đ??´đ?‘?đ?‘ đ?‘œđ?‘&#x;đ?‘?đ?‘Žđ?‘›đ?‘?đ?‘’

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6. Air Sampling This technique is used for the identification of microorganisms in the air of sterilized area. For this purpose a machine named as air sampler is used. A sterilized media plate of nutrient agar is placed inside it. The machine sucks the air and throws it on the plate. It can be run for 5 to10 minutes. The plate is then incubated at 35 °C for 48 hours to see the presence of any microbes.

References 1. Handbook of Microbiological Quality Control in Pharmaceuticals and Medical 11 New Fitter Lane, London EC4P 4EE: Taylor & Francis; 2000. 2. Williams RL. United States Pharmacopeia; Microbiological & Biological Tests and Assays 1, 2000 ed2000. p. 1809-23.

- Abid Jan Institute of Basic Medical Sciences (IBMS), Khyber Medical University, Peshawar, Pakistan

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Need of Advocacy and awareness about cancer in Nepal Many people in Nepal are ignorant about cancer. This is because unlike infectious disease, chronic diseases like cancer comes under least priority area of governmental health policy in Nepal. Very few national programs are conducted to educate and aware people about cancer. Education program are conducted sporadically and only few people have access to such program. In the recent time, Healthy Nepal Foundation (HNF), an organization dedicated to increase awareness about cancer has done some remarkable work to increase understanding of cancer to Nepalese population. Since 2012, HNF has developed several education modules to educate students and public about common cancer such as cervical, breast, oral and lung cancer in Nepal. With the help of volunteers from different colleges, this organization conducts program at colleges and sometime in the community. Volunteers is the main working pillars of HFN. “With the help of volunteers, we are able to conduct education programs at around twenty schools to date and our main goal is to educate student and the public about cancer and its prevention”, said Kiran Sapkota, co-founder of the organization. “We are dedicated to educate and inform public about risks of cancer and promote healthy life style to reduce cancer burden in the society”, Sapkota said. Members of healthy Nepal foundation have conducted several education program at secondary schools, higher secondary colleges and universities around the nation. HN College Coordinator Mr. Sunil Pandey, who is also a student of Medical Microbiology at Nobel College, said “we have got very good response and appreciation from both teachers and guardians from respective schools where we conduct cancer education program”. Mr. Pandey was so passionate to assist HNF that he allocate his tight time schedule out of his college to conduct programs around the nation. Mr. Pandey contact schools, recruit volunteers, communicate with teacher and conduct education awareness program at different schools around the country. “With the help of experts we were able to develop education material for only few cancers and we are still developing additional cancer programs in the future” said Pandey. Healthy Nepal Foundation is operated by physicians, public health officials, and community and school volunteers. Till date it has conducted programs in Parwat, Myagdi, Baglung, Pokhara, Kathmandu, Chitwan and several other districts. “Our main aim is to make a disease free healthy society” said Dr. Kalyan Sapkota, another co-founder of the organization. Dr. Sapkota and his team have conducted cancer screening programs at several communities in Myagdi, Parwat and Baglung in the past.

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Currently Healthy Nepal has concentrated more of its activities on educating students and public about cervical and breast cancer. “These two cancers are the significant cause of mortality and morbidity in women in Nepal” said Dr. Sapkota. With the available funding, Healthy Nepal Foundation had organized free Pap smear screening camp at different places in Nepal. A pap test is the best way to detect cell changes that may be early sign of precancerous of the cervix. Dr. Sapkota added that, Breast cancer affects one in eight women during their lives. No one knows why some women get breast cancer, but there are a number of risk factors. “If we reduce these risk factors, we can minimize the number of cancer patients in Nepal’, said Dr. Sapkota. Sharing his experience while conducting cervical cancer awareness program, Mr. Sunil Pandey said, we were so shocked to know that many students do not know much about common cancer, and many have misconception about it. After we conduct program, we do posttest and found that our program really changed how students perceive about cancer. Their knowledge remarkably changes just after an hour or two education session, added Mr. Pandey. Many women do not know about the screening test available to detect cancer at early age. This include Pap smear screening and self-breast examination in Nepal. Mr. Sunil Pandey said there are several opportunities available for youth to be involved in cancer education projects. He is recruiting many volunteers to initiate oral and lung cancer awareness in the near future. For more information or to get involved, email healthynepalfoundation@gmail.com, or visit the organization’s Facebook page at https://www.facebook.com/healthynepal

- Sunil Pandey Medical Microbiology Nobel College, Kathmandu, Nepal

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How to Microorganism contribute to body odor? More than 100 trillion bacteria live in the human body, according to the results of the first DNAsequence of human microbiome. Humans are an important ecosystem for a wide variety of microorganisms. The human body is a diverse environment in and on which specific niches are formed. The average adult carries 10 times more microbial cells (10^14) than human cells (average about 10^3). The adult human is covered with approximately 2m^2 of skin. It has been considered that this surface area supports 10^12 bacteria. A body odor can be an unpleasant smell generated from the skin. It is as a result of bacteria that lives on the skin which break down sweat into acid. The bacteria break chemical bond of protein in the sweat and convert it to acid. In this case we can say that microorganisms and the body exhibit a type of relationship called Ammensalism (ammensalism is a type of relationship in which the product of one organism has a negative effect on the other organism). Most skin bacteria are found on superficial cells, colonizing dead cells or closely associated with the oil and sweat glands. Secreations from the sweet glands provide the water, amino acids, urea, electrolytes and specific fatty acids that serve as nutrients primary for S. epidermidis and aerobic corynebacteria. The oil glands secrete complex lipids that may be partially degraded by the enzymes from certain gram-positive bacteria (e.g, Propionibacterium acne). These bacteria usually are harmless; however, they are associated with the skin disease acne vulgaris. They can change the lipids secreted by the oil glands to unsaturated fatty acid such as oleic acid that have strong antimicrobial activity against gram negative bacteria and some fungi. Some of these fatty acids are volatile and may have a strong odor. Sweat interacting with microbes can produce body odor. Examples of microbes that interact with sweat are Propionobacteria, Staphylococci, Minococci, Corneforms and Pityrosporum. There are two types of sweat; Ecrine and apocrine, which are secreated by different types of glands. Bacteria only interact with apocrine sweat, which is fattier and richer and usually occurs in areas with body hair such as groin and armpits. The acids produced depends on the species of bacteria; *Propionic acid (propanoic acid) is commonly found in sweat-propionibacteria break amino acids down into propionoic acid. Propionicbacteria live in the ducts of sebaceous glands. *Isovaleric acid (3-methylbutanoic acid) is as a result of the action of bacteria staphyloccoccus epidermidis, which are present in several strong cheese type.

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TREATMENT/PREVENTION Antibacterial soap and frequent washing can keep bacteria down to a manageable level. Avoid wearing thick clothe as thick clothes absorb sweat and allow the multiplication of bacteria instead wear clothe that allows sweat to evaporate and this prevent bacteria from breaking down the sweat. Deodorants that contain antibacterial substances that act selectively against grampositive bacteria can be used to reduce production of volatile unsaturated fatty acids and body odor.

- Ayplux

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Embryonic Stem cell research - an ethical dilemma, current scenario & how we can overcome this Now-a-days we talk a lot about Stem cells. Undoubtedly human stem cells have opened new era of medical research and applications by empowering to treat diseases like Parkinson's disease, Arthritis, Type I diabetes, Burn victims, cardiovascular diseases and many more. These also give the opportunity to unlock the secret of life, how life starts, how a fertilized egg get transformed by cell division and cell differentiation into the diverse range of specialized cells that make a full grown baby by following the human developmental pathway. Because diseases like cancer or conditions as birth defects are thought to occur due to problems in the differentiation process, a more complete understanding of the genetic and molecular controls of these processes of the development that happens in normal cells will help scientists treat the developmental errors that can occur and suggest new strategies for therapy. For all of these the key is Embryonic Stem cells, and here the debate begins—an ethical dilemma. Different countries have chosen different regulatory measures in embryonic stem cell research. Mentioning embryonic stem cells in the pub still divides opinion on the topic. But what exactly are the ethical arguments and why are they so tricky to resolve? Research on Embryonic stem cell poses a moral and ethical conflict between two views. It forces us to choose between one of these two moral principles: • •

The duty to prevent or alleviate suffering of thousands of human beings The duty to respect and save the value of human life

In this case of embryonic stem cell research, it's virtually not possible to take care of each ethical principles. As embryonic stem cells are derived from the inner cell mass of a blastocyst, an earlystage pre implantation embryo, the embryo has got to be destroyed first. This implies the destruction of a possible human life. However embryonic stem cell study could cause the invention of recent medical treatments and therapies that may save lifetime of many human beings. Therefore during this state of affairs, which of these ethical principle ought to have the higher hand? The solution lies on our tendency to view the embryo. Does it have the status of a person at all? 1. The embryo has full ethical value from fertilization onwards: An embryo can be viewed as a person or a potential person while it is still an embryo. Because different people gives different definition to be a person, the criteria for personhood is not clear yet.

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• Arguments supporting this view: Development of a zygote into full grown baby is a continuous process. A human embryo is nothing but a human being in the embryonic stage. Though the embryo does not have the characteristics of a person right now, it will become a person once and that is why should be given the minimum respect and dignity of a person. • Arguments against this view: An early embryo that has not yet implanted into the uterus can never have the psychological, emotional or physical properties that we associate with being a person. It therefore does not have any interests to be protected and we can use it for the benefit of patients (who ARE persons). 2. There is a cut-off point at 14 days after fertilization: Some people argue that a human embryo deserves special protection from around day 14 after fertilization because: • Arguments supporting this view: After 14 days the embryo can no longer split to form twins. Before this point, the embryo could still be split to become two or more babies, or it might fail to develop at all. • Argument against this view: Before day 14, the embryo has no central nervous system and therefore no senses. If we can take organs from patients who have been declared brain dead and use them for transplants, then we can also use hundred-cell embryos that have no nervous system. 3. The embryo has increasing status as it develops: • Arguments supporting this view: If a life is lost, we tend to feel differently about it depending on the stage of the lost life. A fertilized egg before implantation in the uterus could be granted a lesser degree of respect than a human fetus or a born baby. More than half of all fertilized eggs are lost due to natural causes. If the natural process involves such loss, then using some embryos in stem cell research should not worry us either. • Argument against this view: If we judge the moral status of the embryo from its age, then we are making arbitrary decisions about who is human. For example, even if we say formation of the nervous system marks the start of personhood, we still would not say a patient who has lost nerve cells in a stroke has become less human. If we are not sure whether a fertilized egg should be considered a human being, then we should not destroy it. A hunter does not shoot if he is not sure whether his target is a deer or a man.

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4. The embryo has no moral status at all: An embryo is organic material with a status no different from other body parts. • Arguments supporting this view: Fertilized human eggs are just parts of other people’s bodies until they have developed enough to survive independently. The only respect due to blastocysts is the respect that should be shown to other people’s property. If we destroy a blastocyst before implantation into the uterus we do not harm it because it has no beliefs, desires, expectations, aims or purposes to be harmed. • Argument against this view: By taking embryonic stem cells out of an early embryo, we prevent the embryo from developing in its normal way. This means it is prevented from becoming what it was programmed to become – a human being.

Attitude of Different Countries towards this Issue: 1. USA: In March 2009, President Obama issued an executive order intended to bolster human embryonic stem (hES) cell research in the United States. Obama's move overturns an order signed by President Bush in 2001 that barred the National Institutes of Health from funding research on embryonic stem cells beyond using 60 cell lines that existed at that time. Obama also signed a presidential memorandum establishing greater independence for federal science policies and programs. "In recent years, when it comes to stem cell research, rather than furthering discovery, our government has forced what I believe is a false choice between sound science and moral values," President Obama said at the White House.( Ref:3)

2. England: Britain, who many consider to be the leader in stem cell research, opened the world’s first stem cell bank in May of 2004 which currently stores and distributes these cells. This bank serves as a repository for all human stem cell lines produced under conditions of Good Manufacturing Practice. At the opening of the UK stem cell bank Lord Warner, Minister of Health, addressed many of the issues surrounding embryonic stem cell research and did an excellent job of representing the views of the British government and the majority of the British people. He recognized that there are concerns over what this kind of controversial research could lead to in the future, but that the benefits outweigh the risks. He also made assurances that there are safety measures in place to prevent embryonic stem cell research from leading to human reproductive cloning and other scientific technologies that are globally rejected and seen as being morally unacceptable. www.microbiologyworld.com

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3. Sweden: The Swedish government, much like that of Great Britain, recognizes the impact that stem cell research will have in the next century not only on medicine, but in the way people think about disease and injury. Sweden has therefore been another forerunner in breakthroughs made in the past decade in developing stem cell research. As of 2001, 24 of the 64 embryonic stem cell lines available globally were originated in Sweden (at both the both the Karolinska Institute and Göteborg University).

4. Germany: The use of embryos for research is heavily restricted in Germany under the Embryo Protection Act (Embryonenschutzgesetz) 1991, which makes the derivation of embryonic stem cell lines a criminal offence. The embryo is also protected under the German Constitution (Grundgesetz). The Basic Law states that “human dignity is inviolable” and that “everyone has the right to life and inviolability of his person.” Nonetheless, it also states the freedom to pursue science and research. The derivation of embryonic stem cells is banned but embryonic stem cell lines can be imported specifically for research if the line was generated before a defined cut-off date. 5. Elsewhere, Japan, India, Iran, Israel, South Korea, and China are supportive, Australia is partially supportive (exempting reproductive cloning yet allowing research on embryonic stem cells that are derived from the process of IVF); however New Zealand, most of Africa (excepting South Africa) and most of South America (excepting Brazil) are restrictive.

How to solve this critical issue: The possible options for resolving the conflict is to search for the replacement of hES and hopefully there are options. Life is a journey of gaining some new attributes whereas losing some old ones. For long time scientists have studied the human development pathway from a single unspecialized cell to a full grown specialized baby. They have seen that in this peculiar process as the zygote divides and differentiate some classes of genes are switched off and some are switched on. There are very much sophisticated information and special routes for every cell to become specialized from unspecialized state. In the specialized adult cells, the earlier genes (when it was a stem cell) are switched off forever. Then scientists thought if somehow they can again turn on those genes in an adult cell, they can reverse the route to make a stem cell from an adult cell. Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. The iPSC technology was pioneered by Shinya Yamanaka’s lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes could convert adult cells to pluripotent stem cells. He was awarded the www.microbiologyworld.com

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2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent." Pluripotent stem cells hold great promise in the field of regenerative medicine. Because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic, and liver cells), they represent a single source of cells that could be used to replace those lost to damage or disease. Since iPSCs can be derived directly from adult tissues, they not only bypass the need for embryos, but can be made in a patient-matched manner, which means that each individual could have their own pluripotent stem cell line. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. While the iPSC technology has not yet advanced to a stage where therapeutic transplants have been deemed safe, iPSCs are readily being used in personalized drug discovery efforts and understanding the patient-specific basis of disease. No need to say it is very difficult to come to a conclusion on the ethical dilemma of stem cell research. But, as iPS cells have the potential to develop into a human embryonic stem cell, the debate over stem cell research is becoming increasingly irrelevant.

References: 1. Nature Cell Biology (http://www.nature.com/ncb/journal/v12/n7/full/ncb0710-627.html ) 2. EuroStem Cell : Embryonic stem cell research: an ethical dilemma , 23 Mar 2011 ( http://www.eurostemcell.org/factsheet/embyronic-stem-cell-research-ethical-dilemma ) 3. CNNPolitics.com March 9, 2009 -- Updated 1643 GMT (0043 HKT) Obama overturns Bush policy on stem cells (http://edition.cnn.com/2009/POLITICS/03/09/obama.stem.cells/ ) 4. Stem Cell Research : International Policies (http://www.mtholyoke.edu/~amhunsak/europe.htm ) 5. Regulation of stem cell research in Germany — Kate Doherty (http://www.eurostemcell.org/regulations/regulation-stem-cell-research-germany ) 6. Wikipedia: Induced pluripotent stem cell (http://en.wikipedia.org/wiki/Induced_pluripotent_stem_cell )

- Bharat Manna B.Tech Student, Department of Biotechnology, Bengal College of Engineering & Technology West Bengal, India. www.microbiologyworld.com

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Staphylococcus aureus And MRSA Staphylococci are spherical gram-positive cocci arranged in irregular grapelike clusters. All staphylococci produce catalase, whereas no streptococci do (catalase degrades H2O2 into O2 and H2O). Catalase is an important virulence factor because H2O2 is microbicidal and its degradation limits the ability of neutrophils to kill. Although S. aureus is not always pathogenic, it is a common cause of skin infections (e.g. boils), respiratory disease (e.g. sinusitis), and food poisoning. Disease-associated strains often promote infections by producing potent protein toxins, and expressing cell-surface proteins that bind and inactivate antibodies. The emergence of antibiotic-resistant forms of pathogenic S. aureus (e.g. MRSA) is a worldwide problem in clinical medicine. Staphylococcus was first identified in 1880 in Aberdeen, United Kingdom, by the surgeon Sir Alexander Ogston in pus from a surgical abscess in a knee joint. This name was later appended to Staphylococcus aureus by Rosenbach who was credited by the official system of nomenclature at the time. It is estimated that 20% of the human population are long-term carriers of S. aureus which can be found as part of the normal skin flora and in anterior nares of the nasal passages. S. aureus is the most common species of staphylococcus to cause Staph infections and is a successful pathogen due to a combination of nasal carriage and bacterial immuno-evasive strategies. S. aureus can cause a range of illnesses, from minor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis, carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), bacteremia, and sepsis. Its incidence ranges from skin, soft tissue, respiratory, bone, joint, endovascular to wound infections. It is still one of the five most common causes of nosocomial infections and is often the cause of postsurgical wound infections. Each year, some 500,000 patients in American hospitals contract a staphylococcal infection.

Methicillin-Resistant Staphylococcus aureus (MRSA) Staphylococcus aureus (S. aureus) is the leading cause of gram positive bacterial infections and produces a wide spectrum of diseases, ranging from minor skin infections to fatal necrotizing pneumonia. Although S. aureus infections were historically treatable with common antibiotics, emergence of drug-resistant organisms is now a major concern. Methicillin-resistant Staphylococcus aureus (MRSA) was endemic in hospitals by the late 1960s, but it appeared rapidly and unexpectedly in communities in the 1990s and is now prevalent worldwide. S. aureus is notorious for its ability to become resistant to antibiotics. Infections that are caused by antibiotic-resistant strains often occur in epidemic waves that are initiated by one or a few www.microbiologyworld.com

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successful clones. MRSA features prominently in these epidemics. Historically associated with hospitals and other health care settings, MRSA has now emerged as a widespread cause of community infections. Community or community-associated MRSA (CA-MRSA) can spread rapidly among healthy individuals. Outbreaks of CA-MRSA infections have been reported worldwide. The frequency of MRSA infections continues to grow in hospital-associated settings, and more recently, in community settings globally. The increase in the incidence of infections due to S. aureus is partially a consequence of advances in patient care and also of the pathogen's ability to adapt to a changing environment. Infection due to S. aureus imposes a high and increasing burden on health care resources. A growing concern is the emergence of MRSA infections in patients with no apparent risk factors. The growing problem in the Indian scenario is that MRSA prevalence has increased from 12% in 1992 to 80.83% in 1999. MRSA in tonsils may serve as a potential source for the spread of these organisms to other body sites as well to other individuals. MRSA is prevalent in many hospitals and often reflects the difficulties in hospitals and the health service generally, in terms of the control and prevention of healthcare-associated infection. Multidrug-resistant bacteria, such as MRSA, are endemic in healthcare settings in the United States and many other countries of the world. Nosocomial transmission of MRSA serves as a source of hospital outbreaks, and recent reports of vancomycin resistant S. aureus strains in the United States emphasize the need for better control of MRSA and other resistant bacteria within healthcare settings.

Pathogenesis: Staph.aureus present in the nose of 30% of healthy people and may be found on the skin. It causes infection most commonly at sites of lowered host resistance, such as damaged skin (e.g., surgical site infection) or mucous membranes (e.g. ventila tor-associated pneumonia).

Staphylococcal Toxins: Enterotoxins: Enterotoxins, types A-E,G, H, I & J are commonly produced by up to 65% of strains of Staph. aureus, sometimes singly & sometimes in combination. These toxic proteins withstand exposure to 100c for several minutes. When ingested as performed toxins in contaminated food, microgram amounts of toxins, within a minute, induce the symptoms of staphylococcal food poisoning: nausea, vomiting, & diarrhea. However, enterotoxins, which are super antigens, probably also, play an important role in other serious staphylococcal infections, e.g., bloodstream infections, especially accompanied by septic shock. www.microbiologyworld.com

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Toxic Shock Syndrome Toxin (TSST-1): This was discovered in early 1980s as result of epidemiological and microbiological investigations in USA of toxic shock syndrome, a multi system disease caused by staphylococcal TSST-1 or enterotoxins or both. A link was established with the use of highly absorbent tampons in menstruating women, although non-menstrual cases are now as common. The absence of circulating antibodies of TSST-1 is a factor in the pathogenesis of this syndrome. TSST-1 and the enterotoxins are now recognized as super antigens, that is, they are potent activators, of T lymphocytes resulting in the liberation of cytokines such as tumor necrosis factor, and they bind with high affinity to mononuclear cells. These characteristics partly explain the florid & multisystem nature of the clinical conditions associated with these toxins. Epidermolytic Toxin: Two kinds of Epidermolytic toxins (types A & B) are commonly produced by strains that cause blistering diseases. These toxins induce intraepidermal blisters at the granular cell layer. Such blisters of pemphigus neonatarum. The most dramatic manifestation of Epidermolytic toxin is scalded skin syndrome in small children, where the toxin spreads automatically in individuals who lack neutralizing antitoxin. Panton-Valentine Leukocidin (PVL): This toxin was recognized some decades back but its potential contribution to clinical manifestations and outcome have been increasingly described in the context of communityacquired MRSA (CA-MRSA). As the name suggests PVL can adversely affect cells, resulting in leucopenia, but animals studies do not suggest high virulence. Nonetheless, epidemiological data in many countries reveal an association between necrotizing pneumonia and some complicated skin and soft tissue infections caused by PVL-positive strains of CA-MRSA.

Laboratory Diagnosis: One or more of the following specimens should be collected to confirm diagnosis;     

Pus from abscesses, wounds, burns etc. is much preferred swabs. Sputum from patients with pneumonia, bronchoscopic lavage, is increasingly used in critically ill patients. Faeces or vomit from patients with suspected food poisoning, or the remains of implicated foods. Blood from patients with suspected BSI such as septic shock, osteomyelitis, or endocarditis. Mid-stream urine from patients with suspected cystitis or pyelonephritis.

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Anterior nasal and perineal swabs (moistened in saline or sterile water) from suspected carriers, nasal swabs should be rubbed in turn over the anterior walls of both nostrils.

The characteristic clusters of Gram-positive cocci can be often be demonstrated by microscopy, and the organisms cultured readily on blood agar and the most other media within 24 hrs. or less. The tube or slide coagulase test is performed to distinguish Staph. aureus from coagulase negative species and AST with ceofoxitin to confirm MRSA using standard methods. Commercially, available molecular methods using PCR have been developed to reduce the time to the detection of MRSA from 48-72 h with culture to less than 12 h facilitate earlier preventative measures. However, the results from trails with PCR to date mixed and do not clearly indicate that this more expensive approach can assist in reducing MRSA rates in acute hospitals.

- Anil Bhujel Bsc Microbiology, Pokhara Bigyan Thata Prabidhi Campus Nayabazzar-9, Pokhara. Nepal Email: grgaryan147@gmail.com Website: www.microbiollogy.blogspot.com

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You can also send your articles to info@microbiologyworld.com or broneps1@gmail.com Selected ones will be published in our next issue of May-June 2014. Thanks, Microbiology World Team

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