Identification of Bacterial Pathogens

Identification of Bacterial Pathogens basic skills in diagnostic bacteriology: 

Identification of Bacterial Pathogens basic skills in diagnostic bacteriology Dr.T.V.Rao MD Dr.T.V.Rao MD 1

Identification of Microorganisms: 

2 Identification of Microorganisms For many students and professionals the most pressing topic in microbiology is how to identify unknown specimens . Why is this important? Labs can grow, isolate and identify most routinely encountered bacteria within 48 hrs of sampling. The methods microbiologist use fall into three categories : Phenotypic - morphology (micro and macroscopic) Immunological - serological analysis Genotypic - genetic techniques Dr.T.V.Rao MD

Microbe Identification: 

3 Microbe Identification The successful identification of microbe depends on: Using the proper aseptic techniques . Correctly obtaining the specimen. Correctly handling the specimen Quickly transporting the specimen to the lab. Once the specimen reaches the lab it is cultured and identified . Dr.T.V.Rao MD

Microbe Identification: 

4 Microbe Identification Identification measures include: Microscopy (staining) growth on enrichment, selective, differential or characteristic media specimen biochemical test (rapid test methods) immunological techniques molecular (genotypic) methods . After the microbe is identified for clinical samples it is used in susceptibility tests to find which method of control is most effective. Dr.T.V.Rao MD

Microbe Identification: 

5 Microbe Identification Dr.T.V.Rao MD

Specimen Collection: 

6 Specimen Collection Successful identification depends on how the specimen is collected , handled and stored . It is important that general aseptic procedures be used including sterile sample containers and sampling methods to prevent contamination of the specimen. E.g. Throat and nasopharyngeal swabs should not touch the cheek, tongue or salvia. What other precautions must be taken when collecting specimens? After collection the specimen must be taken promptly to the lab and stored appropriately (e.g. refrigeration). Dr.T.V.Rao MD

Specimen Collection: 

7 Specimen Collection Dr.T.V.Rao MD

Phenotypic Methods of Identification: 

8 Phenotypic Methods of Identification Microbiologists use 5 basic techniques to grow, examine and characterize microorganisms in the lab. They are called the 5 ‘I’s : inoculation, incubation, isolation, inspection and identification . Inoculation: to culture microorganisms a tiny sample (inoculum) is introduced into medium (inoculation). Isolation involves the separating one species from another. Dr.T.V.Rao MD

Phenotypic methods of Identification: 

9 Phenotypic methods of Identification Incubation : once the media is inoculated it is incubated which means putting the culture in a controlled environment (incubation) to allow for multiplication . After incubation the organisms are inspected and i dentified phenotypically, immunologically or genetically. Dr.T.V.Rao MD

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10 isolation inoculation incubation Specimen collection inspection identification 5 “I” s Dr.T.V.Rao MD

Phenotypic Methods: 

11 Phenotypic Methods ‘Old fashioned’ methods via biochemical, serological and morphological are still used to identify many microorganisms. Phenotypic Methods Microscopic Morphology include a combination of cell shape, size, Gram stain, acid fast rxn, special structures e.g. endospores, granule and capsule can be used to give an initial putative identification . Dr.T.V.Rao MD

Phenotypic Methods: 

12 Phenotypic Methods Macroscopic morphology are traits that can be accessed with the naked eye e.g. appearance of colony including texture, shape, pigment, speed of growth and growth pattern in broth. Physiology/Biochemical characteristic are traditional mainstay of bacterial identification. These include enzymes (catalase, oxidase, decarboxylase), fermentation of sugars, capacity to digest or metabolize complex polymers and sensitivity to drugs can be used in identification. Dr.T.V.Rao MD

Microscopy: 

Microscopy Magnification enhancement of size using ocular and objective lenses. Ocular: eyepiece (10X) Objective: 4X – 100X allows for visualization of bacteria, fungi, and parasites, not viruses Resolution ability to distinguish two objects as distinct resolving power is closest distance between two objects immersion oil is added when using 100X objective to prevent light scatter Contrast use stains to enhance visualization; allow organism to stand out from background Dr.T.V.Rao MD 13

Staining techniques by Grams Method: 

Staining techniques by Grams Method make slide by smear, drop, or cytocentrifuge dry, then fix by heat (flame, 10 min at 60ºC) or fix by methanol (95% 1min) Gram stain Crystal violet: primary stain Gram’s iodine: mordant/fixative Acetone-ethanol: decolorizer Safranin: counterstain Dr.T.V.Rao MD 14

Bacteria differ as per structure: 

http://www.sp.uconn.edu/~terry/229sp02/lectures/Lect2.html Bacteria differ as per structure Dr.T.V.Rao MD 15

Gram Staining Procedure: 

Gram Staining Procedure 16 Dr.T.V.Rao MD

Gm+ve cocci & Gm-ve bacilli: 

Gm+ve cocci & Gm-ve bacilli 17 Dr.T.V.Rao MD

Streptococcus: 

Streptococcus Dr.T.V.Rao MD 18

Bacilli: 

Bacilli Dr.T.V.Rao MD 19

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Neisseria gonorrhea - Gram stain http://www.cdc.gov/STD/LabGuidelines/default.htm Dr.T.V.Rao MD 20

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Wound specimen - Gram stain http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html Dr.T.V.Rao MD 21

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Oral specimen - Gram stain http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html Dr.T.V.Rao MD 22

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Sputum specimen Gram stain— Streptococcus pneumoniae http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html Dr.T.V.Rao MD 23

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Sputum—cystic fibrosis patient, encapsulated gram negative rods http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html Dr.T.V.Rao MD 24

Staining techniques for Mycobacterial spp: 

Staining techniques for Mycobacterial spp Acid-fast stains for staining of organisms with high degree of fatty (mycolic) acids—waxy render the cells resistant to decolorization: “acid-fast” Mycobacterium sp., Nocardia sp., Cryptosporidium sp. are acid-fast Procedure Ziehl-Neelsen: heat drives in primary stain (carbolfuchsin) Kinyoun: higher conc. of phenol does not require heat Decolorize with acid-alcohol Counterstain with methylene blue or malachite green Dr.T.V.Rao MD 25

Ziehl-Neelsen stain: 

Ziehl-Neelsen stain 4 5 6 7 1 2 3 26 Dr.T.V.Rao MD

How the Acid fast bacteria appear: 

How the Acid fast bacteria appear 27 Dr.T.V.Rao MD

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A Gram stain (left) of the abscess shows thin, gram positive rods in chains. An acid fast stain (right) was also positive. http://pathhsw5m54.ucsf.edu/overview/bacteria3.html Dr.T.V.Rao MD 28

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Sputum specimen—Acid fast stain http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html Dr.T.V.Rao MD 29

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Acid fast stain demonstrating chording Dr.T.V.Rao MD 30

Fluorochrome AFB Microscopy: 

Fluorochrome AFB Microscopy More rapid and sensitive Specificity : same with sufficient expérience Equipment cost , bulbs, technical demands for busy labs External quality assessment should be done if this method is performed 31 Dr.T.V.Rao MD

Direct Fluorescent Antibodies: 

32 Direct Fluorescent Antibodies

Indirect Fluorescent Antibodies: 

33 Indirect Fluorescent Antibodies

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Mycobacterium – auramine stain http://www.lung.ca/tb/abouttb/what/causes_tb.html Dr.T.V.Rao MD 34

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Yeast—calcofluor white http://www.med.sc.edu:85/mycology/mycology-3.htm Dr.T.V.Rao MD 35

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Mould—calcofluor white Dr.T.V.Rao MD 36

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Influenza virus infected cells, fluorescent antibody stain Dr.T.V.Rao MD 37

Better Use Microscopy: 

Better Use Microscopy Phase Contrast Microscopy shift in light allows visualization of organism; can visualize viable organisms Fluorescent Microscopy certain dyes (fluorochromes) give off light when excited (fluorescence) color of light depends on the dye and the filters used Staining techniques Fluorochroming: direct chemical interaction with organism Acridine orange: stains nucleic acid; useful for cell-wall deficient organisms Auramine-rhodamine: bind to mycolic acids in nearly all Mycobacteria Calcoflour white: binds to chitin in cell walls of fungi Immunofluorescence: fluorochrome is bound to an antibody; can detect/identify specific organisms Dr.T.V.Rao MD 38

Culture and isolation of bacteria: 

Culture and isolation of bacteria Principles of Cultivation Nutritional requirements General concepts non-fastidious: simple requirements for growth fastidious: complex, unusual, or unique requirements for growth Phases of growth media solid  agar; boil to dissolve, solidifies at 50ºC liquid, broth Dr.T.V.Rao MD 39

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Media classifications and functions Enrichment used to enhance growth of specific organisms Supportive support growth of most non-fastidious organisms Selective contains agents that inhibit the growth of all agents except that being sought (dyes, bile salts, alcohols, acids, antibiotics) Differential contains factor(s) that allow certain organisms to exhibit different metabolic characteristics Dr.T.V.Rao MD 40

According to Use: 

According to Use Enriched Medium – broth or solid, contains rich supply of special nutrients that promotes growth of a particular organism while not promoting growth of other microbes that may be present (e.g BAP & chocolate agar) Dr.T.V.Rao MD 41

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Types of artificial media Brain-heart infusion nutritionally rich supportive media used in broths, blood culture systems and susceptibility testing Sheep blood agar supportive media containing 5% sheep blood for visualization of hemolysis Chocolate agar same as sheep blood agar except blood has been “chocolatized” RBCs lysed by heating; releases X (hemin) and V (NAD) factors for Neisseria and Haemophilus MacConkey agar selective for Gram-negative rods (GNRs) because of crystal violet and bile salts; differential due to lactose, fermenters lower pH changing neutral red indicator pink/red Dr.T.V.Rao MD 42

According to Use: 

According to Use Selective Medium - contains inhibitors that discourage the growth of certain organisms & enhances the growth of the microbe sought (e.g. SSA, Mannitol Salt Agar ) Dr.T.V.Rao MD 43

According to Use: 

According to Use Differential Medium - contains dyes, indicators or other constituents that give colonies of particular organisms distinctive and easily recognizable characteristics (e.g. McConkey Agar) Dr.T.V.Rao MD 44

Hemolysis a guiding factor: 

Hemolysis a guiding factor Dr.T.V.Rao MD 45

Hemolysis a guiding factor: 

Hemolysis a guiding factor Dr.T.V.Rao MD 46

Enriched Media: 

47 Enriched Media Blood agar plate with bacteria from human throat. This media differentiates among different colonies by appearance Chocolate agar, a medium that gets brown from heated blood. Used for isolation of N. gonorrhea . Dr.T.V.Rao MD

Testing for Bacitracin and Optochin Sensitivity: 

Testing for Bacitracin and Optochin Sensitivity Dr.T.V.Rao MD 48

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Dr.T.V.Rao MD 49

Mac Conkey Agar a Minimal differentiating Medium: 

Mac Conkey Agar a Minimal differentiating Medium Dr.T.V.Rao MD 50

MacConkey Agar: 

MacConkey Agar Dr.T.V.Rao MD 51

General vs Selective Media: 

52 General vs Selective Media Dr.T.V.Rao MD

Differential Media: 

53 Differential Media Differential media grow several types of organisms and display visible differences among organisms. Differences may show up as colony size, media colour, gas bubble formation and precipitate formation. Dr.T.V.Rao MD

Salmonella-Shigella Agar: 

Salmonella-Shigella Agar Dr.T.V.Rao MD 54

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Dr.T.V.Rao MD 55

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Types of artificial media Hektoen enteric agar contains bile salts and dyes (bromothymol blue and acid fuchsin) to inhibit non-pathogenic GNRs; non pathogens ferment lactose changing BTB to orange; pathogens Salmonella and Shigella are clear; ferric ammonium citrate detects H 2 S production of Salmonella (black colonies) Columbia colistin-nalidixic acid (CNA) agar Columbia agar base, sheep blood, colistin and nalidixic acid; selective isolation of gram-positive cocci Dr.T.V.Rao MD 56

PowerPoint Presentation: 

Types of artificial media Hektoen enteric agar contains bile salts and dyes (bromothymol blue and acid fuchsin) to inhibit non-pathogenic GNRs; non pathogens ferment lactose changing BTB to orange; pathogens Salmonella and Shigella are clear; ferric ammonium citrate detects H 2 S production of Salmonella (black colonies) Columbia colistin-nalidixic acid (CNA) agar Columbia agar base, sheep blood, colistin and nalidixic acid; selective isolation of gram-positive cocci Dr.T.V.Rao MD 57

Types of artificial media : 

Types of artificial media Thayer-Martin agar CAP with antibiotics (colistin inhibits gram neg, vancomycin inhibits gram pos, nystatin inhibits yeast); for N. gonorrhoeae and N. meningitidis ; Martin-Lewis has similar function but different antibiotics Preparation of artificial media Sterilization autoclave: pressurized steam at 121ºC for 15-30 min. Dr.T.V.Rao MD 58

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Environmental requirements Oxygen and Carbon dioxide availability aerobic: room air facultative: aerobic or anaerobic microaerophilic: reduced oxygen tension anaerobic strict or aerotolerant capnophilic: increased C0 2 (5-10%) Temperature 35-37ºC 30ºC cold 42ºC Dr.T.V.Rao MD 59

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Bacterial Cultivation Isolation of bacteria from specimens streaking for isolation streaking for quantitation Evaluation of colony morphologies Type of media supporting growth Relative quantities of each colony type Colony characteristics colony form: pinpoint, circular, filamentous, irregular colony elevation: flat, raised, convex colony margin: smooth, irregular Gram stain and subcultures sterile loop, isolated colonies Dr.T.V.Rao MD 60

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http://science.nhmccd.edu/biol/wellmeyer/media/media.htm Dr.T.V.Rao MD 61

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Chocolate agar Uninoculated Haemophilus http://www.hardydiagnostics.com/catalog2/hugo/ChocolateAgar.htm Dr.T.V.Rao MD 62

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http://science.nhmccd.edu/biol/wellmeyer/media/media.htm MacConkey agar Dr.T.V.Rao MD 63

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http://medic.med.uth.tmc.edu/path/hekto.htm Hektoen agar Dr.T.V.Rao MD 64

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Nutritional requirements and metabolic capabilities Single enzyme tests Catalase: H 2 O 2 + catalase = O 2 and H 2 0; differentiates Staphylococcus v. Streptococcus , Listeria and Corynebacterium v. other non spore forming gram-positive bacilli Oxidase: detection of cytochrome oxidase that participates in nitrate metabolism; Pseudomonas , Aeromonas , Neisseria Indole: tryptophanase degrades tryptophan into pyruvic acid, ammonia, and indole; indole is detected by aldehyde indicator; presumptive id for E. coli Urease: hydrolyzes urea into ammonia, water and CO 2 ; increase pH changes causes bright pink color of indicator PYR: hydrolysis of PYR, indicator turns pink; Group A Strep and enterococci are + Dr.T.V.Rao MD 65

Biochemical Tests: 

66 Biochemical Tests The microbe is cultured in a media with a special substrate and tested for an end product . Prominent biochemical tests include carbohydrate fermentation, acid or gas production and the hydrolysis of gelatin or starch. Many of these test in rapid miniaturized system that can detect for 23 characteristics in small cups called Rapid test . The info from the rapid test are input into a computer to help in identification of the organisms. Dr.T.V.Rao MD

Carbohydrate Fermentation: 

67 Carbohydrate Fermentation . This medium show f ermentation ( acid production ) and gas formation . The small Durham tube for collecting gas bubbles. Left- right: Uninoculated negative control Centre, positive for acid (yellow) and gas (open space). Growth but no gas or acid. Dr.T.V.Rao MD

Methyl Red Test: 

68 Methyl Red Test This is a qualitative test for acid production . The bacteria is grown in MR-VP broth. After addition of several drops of methyl red solution a bright red colour is positive and yellow-orange negative. Dr.T.V.Rao MD

Nitrate Reduction: 

69 Nitrate Reduction After 24-48 hrs of incubation, nitrate reagents are added. Left to right : Gas formation (positive for nitrate reduction). positive for nitrate reduction to nitrite ( red colour). Negative control Dr.T.V.Rao MD

Biochemical Tests: 

70 Biochemical Tests Other biochemical tests of interest include: H 2 S production Indole test Oxidase test Oxidation fermentation Phenylalanine deaminase test Antibiotic susceptibility tests Principle, procedure, most common use. Dr.T.V.Rao MD

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Dr.T.V.Rao MD 71

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Dr.T.V.Rao MD 72

Coagulase Test : 

Coagulase Test Dr.T.V.Rao MD 73

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Tests for presence of metabolic pathways Oxidation and fermentation: oxidation of glucose requires oxygen, fermentation does not; pH decreases causing yellow color Amino acid degradation: detection of amino acid decarboxylase enzy mes Dr.T.V.Rao MD 74

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O-F glucose media http://academic.mwsc.edu/jcbaker/bio390sec01/bio390_laboratory_study_images.htm Dr.T.V.Rao MD 75

Differential Media Chromagar: 

76 Differential Media Chromagar Chromagar orientation uses colour-formation to distinguish at least 7 common urinary pathogens. This allow for rapid identification and treatment. The bacteria were streaked as to spell their names. Dr.T.V.Rao MD

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Principles of Phenotype-based ID schemes Selection and inoculation of ID test battery Type of bacteria to be identified Clinical significance of isolate Availability of reliable testing methods Incubation for substrate utilization Conventional ID Rapid ID Detection of metabolic activity Colorimetry: pH change of indicators Fluorescence: release of fluorophore from substrate or changes in fluorescence due to pH changes Turbiditiy: growth or no-growth Analysis of metabolic profiles ID databases Use of databases to ID unknowns Confidence in ID Dr.T.V.Rao MD 77

Rapid Tests: 

78 Rapid Tests Rapid test : a biochemical system for the identification of Enterobacteriaceae and other Gram –ve bacteria. It consist of plastic strips with 20 μl of dehydrated biochemical substrates used to detect biochemical characteristics. The biochemical substrates are inoculated with pure cultures and suspended in physiological saline. After 5 hrs-overnight the 20 tests are converted to 7-9 digital profile. Dr.T.V.Rao MD

Rapid Tests : 

79 Rapid Tests ONPG ( β galactosidase); ADH (arginine dihydrolase); LDC (lysine decarboxylase); ODC (ornithine decarboxylase); CIT (citrate utilization); H 2 S (hydrogen disulphide production); URE (urease); TDA ( tryptophan deaminase); IND (indole production); VP (Voges Proskauer test for acetoin); GEL ( gelatin liquefaction); the fermentation of glucose (GLU), mannitol (MAN), inositol (INO), sorbitol (SOR), rhamnose (RHA), sucrose (SAC); Melibiose (MEL), amygdalin (AMY), and arabinose (ARA); and OXI (oxidase). positive negative Dr.T.V.Rao MD

Bacteriophage Typing: 

80 Bacteriophage Typing What are bacteriophages? Bacteriophage typing is based on the specificity of phage surface receptor for the cell surface receptor. Only those phages that can attach to the surface receptors can cause lysis. The procedure involves: A plate is heavily inoculated so that there is no uninoculated areas. The plate is marked off in squares (15-20 mm) and each square inoculated with a drop of suspension for different phages . Dr.T.V.Rao MD

Heavily Inoculated Plate: 

81 Heavily Inoculated Plate Dr.T.V.Rao MD

Bacteriophage Typing: 

82 Bacteriophage Typing The plate is incubated for 24 hrs then observed for plaques. The phage type is reported as a specific genus and species followed by the types that can infect the bacterium. E.g. 10/16/24 means that the bacteria is sensitive to phages 10, 16 and 24. Phage tying remain a tool for research and reference labs . Dr.T.V.Rao MD

Uncultivable Organisms: 

Uncultivable Organisms Environmental researchers estimate that < 1% of microorganisms are culturable and therefore it is not possible to use phenotypic methods of identification. These microorganisms are called viable nonculturable (VNC). 83 Dr.T.V.Rao MD

Flow Cytometry: 

84 Flow Cytometry Classical techniques are not successful in identification of those microorganisms that cannot be cultured. What do is the name of microorganisms that cannot be cultured? Flow cytometry allows single or multiple microorganisms detection an easy, reliable and fast way. Inflow cytometry microorganisms are identified on the basis of the cytometry parameters or by means of certain dyes called fluorochromes that can be used independently or bound to specific antibodies. Dr.T.V.Rao MD

Flow Cytometry: 

85 Flow Cytometry The cytometer forces a suspension of cells through a laser beam and measures the light they scatter or the fluorescence the cell emits as they pass through the beam. The cytometer also can measure the cell’s shape, size and the content of the DNA or RNA. Dr.T.V.Rao MD

Recent Advances in Flowcytometry: 

Recent Advances in Flowcytometry Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometry parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way Dr.T.V.Rao MD 86

Immunological Methods: 

87 Immunological Methods Immunological methods involve the interaction of a microbial antigen with an antibody (produced by the host immune system). Testing for microbial antigen or the production of antibodies is often easier than test for the microbe itself. Lab kits based on this technique is available for the identification of many microorganisms. Dr.T.V.Rao MD

Principles of Serologic Test Methods : 

Principles of Serologic Test Methods Methods for antibody detection Direct whole pathogen agglutination assays pos patient sera causes organism to clump Particle agglutination tests latex beads or RBCs coated with Ag Flocculation tests RPR – precipitation of soluble Ag with Ab charcoal particles coated with cardiolipin-lecithin binds reagin ELISAs IFAs organism/antigen on slides; patient Ab detected with fluorescent secondary Ab Western blots Dr.T.V.Rao MD 88

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API strips – bioMerieux Dr.T.V.Rao MD 89

Commercial ID systems : 

Commercial ID systems Advantages and examples of commercial systems ARIS (Trek) MicroScan (Dade-Behring/Seimens) Phoenix (Becton-Dickinson) Vitek (bioM é rieux) Dr.T.V.Rao MD 90

Immunochemical methods for ID: 

Immunochemical methods for ID Principles of immunochemical methods Particle agglutination Latex agglutination Immunofloresecent assays Direct immunofluorescence assay (DFA) Indirect immunofluorescence assay (IFA) Enzyme immunoassays Solid-phase immunoassays Membrane bound immunoassays Immunochromatographic assays Optical immunoassays Dr.T.V.Rao MD 91

Serologic methods for diagnosis: 

Serologic methods for diagnosis Features of the Immune Response Characteristics of antibodies Features of immune response useful in diagnostic testing Acute v. anamnestic response IgM v. IgG IgM can’t cross placenta immunocompetent v. immunocompromised Interpretation of serologic tests single v. paired sera; rare pathogen 4-fold rise in titer v. qualitative testing cross reactivity (herpes viruses, heterophile Abs, pregnancy) Dr.T.V.Rao MD 92

Principles of Serologic Test Methods : 

Principles of Serologic Test Methods Methods for antibody detection Direct whole pathogen agglutination assays pos patient sera causes organism to clump Particle agglutination tests latex beads or RBCs coated with Ag Flocculation tests RPR – precipitation of soluble Ag with Ab charcoal particles coated with cardiolipin-lecithin binds reagin ELISAs IFAs organism/antigen on slides; patient Ab detected with fluorescent secondary Ab Western blots Dr.T.V.Rao MD 93

Genotypic methods: 

94 Genotypic methods Genotypic methods of microbe identification include the use of : Nucleic acid probes PCR (RT-PCR, RAPD-PCR) Nucleic acid sequence analysis rRNA analysis RFLP Plasmid fingerprinting.

Genotypic Methods: 

95 Genotypic Methods Genotypic methods involve examining the genetic material of the organisms and has revolutionized bacterial identification and classification. Genotypic methods include PCR (RT-PCR, RAPD-PCR),use of nucleic acid probes, RFLP and plasmid fingerprinting. Increasingly genotypic techniques are becoming the sole means of identifying many microorganisms because of its speed and accuracy . Dr.T.V.Rao MD

Real Time PCR and RT-PCR: 

96 Real Time PCR and RT-PCR Currently many PCR tests employ real time PCR . This involves the use of fluorescent primers . The PCR machine monitors the incorporation of the primers and display an amplification plot which can be viewed continuously thru the PCR cycle. Real time PCR yields immediate results . Another application of PCR is RT-PCR (reverse trancriptase PCR). During RT-PCR an RNA template is used to generate cDNA and from this dsDNA is generated. The enzyme used is reverse transciptase . RT-PCR is used to detect for HIV and to monitor the progress of the disease.

RAPD-PCR: 

97 RAPD-PCR Random amplified polymorphic DNA PCR uses a random primer (10-mer) to generate a DNA profile. What are random primers? The primer anneals to several places on the DNA template and generate a DNA profile which is used for microbe identification. RAPD has many advantages: Pure DNA is not needed Less labour intensive than RFLP. There is not need for prior DNA sequence data. RAPD has been used to fingerprint the outbreak of Listeria monocytogenes from milk.

Plasmid fingerprinting: 

98 Plasmid fingerprinting The procedure involves: The bacterial strains are grown , the cells lysed and harvested. The plasmids are separated by agarose gel electrophoresis The gels are stained with EtBr and the plasmids located and compared.

Computer and Bacteria Identification: 

Computer and Bacteria Identification Computers improve the efficiency of the lab operations and increase the speed and clarity with which results can be reported. Computers are also important for the result entry, analysis and preparation. 99

PowerPoint Presentation: 

Programme created by Dr.T.V.Rao MD for Medical Microbiologists in Developing world Email doctortvrao@gmail.com Dr.T.V.Rao MD 100