Identification of Bacterial        Pathogens basic skills in diagnostic bacteriology           Dr.T.V.Rao MD              ...
Identification of Microorganisms• For many students and professionals the most  pressing topic in microbiology is how to i...
Microbe Identification• The successful identification of microbe  depends on:   ♥ Using the proper aseptic techniques.   ♥...
Microbe Identification• Identification measures include:  ♣ Microscopy (staining)  ♣ growth on enrichment, selective, diff...
Microbe                IdentificationDr.T.V.Rao MD                5
Specimen Collection• Successful identification depends on how the specimen  is collected, handled and stored.• It is impor...
Specimen                CollectionDr.T.V.Rao MD                7
Phenotypic Methods of Identification• Microbiologists use 5 basic techniques to grow,  examine and characterize microorgan...
Phenotypic methods of Identification• Incubation: once the media is inoculated it is  incubated which means putting the cu...
Specimen collection                                   isolation                                                 5 “I” s   ...
Phenotypic Methods• ‘Old fashioned’ methods via biochemical,  serological and morphological are still used to  identify ma...
Phenotypic Methods• Macroscopic morphology are traits that can be  accessed with the naked eye e.g. appearance of colony  ...
Microscopy• Magnification  – enhancement of size using ocular and objective lenses.      • Ocular: eyepiece (10X)      • O...
Staining techniques by        Grams Method• make slide by smear, drop, or  cytocentrifuge• dry, then fix by heat (flame, 1...
Bacteria differ as per structurehttp://www.sp.uconn.edu/~terry/229sp02/lectures/Lect2.html                           Dr.T....
Gram Staining Procedure         Dr.T.V.Rao MD    16
Gm+ve cocci & Gm-ve bacilli           Dr.T.V.Rao MD      17
Streptococcus    Dr.T.V.Rao MD   18
Bacilli Dr.T.V.Rao MD   19
Neisseria gonorrhea - Gram stainhttp://www.cdc.gov/STD/LabGuidelines/default.htm                     Dr.T.V.Rao MD        ...
Wound specimen - Gram stainhttp://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html                           Dr.T.V.R...
Oral specimen - Gram stain                           Dr.T.V.Rao MD                       22http://www.healthsci.utas.edu.a...
Sputum specimen Gram stain—Streptococcus pneumoniaehttp://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html           ...
Sputum—cystic fibrosis patient, encapsulated gram negative rods http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.ht...
Staining techniques for           Mycobacterial spp• Acid-fast stains  – for staining of organisms with high degree of fat...
1   2                   34   5                   67        Ziehl-Neelsen            stain        Dr.T.V.Rao MD       26
How the Acid fast bacteria appear              Dr.T.V.Rao MD     27
A Gram stain (left) of the abscess shows thin, gram positive rods in chains.                An acid fast stain (right) was...
Sputum specimen—Acid fast stainhttp://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html                           Dr.T...
Acid fast stain demonstrating chording              Dr.T.V.Rao MD              30
Fluorochrome AFB Microscopy* More rapid and  sensitive* Specificity : same with  sufficient expérience* Equipment cost ,  ...
Direct Fluorescent Antibodies                                32
Indirect Fluorescent Antibodies                                  33
Mycobacterium – auramine stainhttp://www.lung.ca/tb/abouttb/what/causes_tb.html                    Dr.T.V.Rao MD          ...
Yeast—calcofluor whitehttp://www.med.sc.edu:85/mycology/mycology-3.htm                    Dr.T.V.Rao MD                  35
Mould—calcofluor white        Dr.T.V.Rao MD    36
Influenza virus infected cells, fluorescent antibody stain                       Dr.T.V.Rao MD                         37
Better Use Microscopy• Phase Contrast Microscopy  – shift in light allows visualization of organism; can    visualize viab...
Culture and isolation of bacteria•    Principles of Cultivation    – Nutritional requirements      •   General concepts   ...
• Media classifications and functions –Enrichment   • used to enhance growth of specific     organisms –Supportive   • sup...
According to Use• Enriched Medium –  broth or solid, contains  rich supply of special  nutrients that promotes  growth of ...
• Types of artificial media– Brain-heart infusion  • nutritionally rich supportive media used in broths,    blood culture ...
According to Use• Selective Medium -  contains inhibitors  that discourage the  growth of certain  organisms &  enhances t...
According to Use• Differential Medium -  contains dyes, indicators  or other constituents  that give colonies of  particul...
Hemolysis a guiding factor           Dr.T.V.Rao MD     45
Hemolysis a guiding factor           Dr.T.V.Rao MD     46
Enriched Media                                              Chocolate agar, a medium                                      ...
Testing for Bacitracin and Optochin             Sensitivity               Dr.T.V.Rao MD          48
Dr.T.V.Rao MD   49
Mac Conkey Agar a Minimal differentiating Medium          Dr.T.V.Rao MD     50
MacConkey Agar     Dr.T.V.Rao MD   51
General vs Selective Media           Dr.T.V.Rao MD     52
Differential Media                  • Differential media grow                    several types of                    organ...
Salmonella-Shigella Agar          Dr.T.V.Rao MD    54
Dr.T.V.Rao MD   55
• Types of artificial media– Hektoen enteric agar  • contains bile salts and dyes    (bromothymol blue and acid fuchsin) t...
• Types of artificial media– Hektoen enteric agar  •   contains bile salts and dyes (bromothymol      blue and acid fuchsi...
Types of artificial media    – Thayer-Martin agar      •    CAP with antibiotics (colistin inhibits gram           neg, va...
•   Environmental requirements– Oxygen and Carbon dioxide availability  •   aerobic: room air  •   facultative: aerobic or...
• Bacterial Cultivation– Isolation of bacteria from specimens  • streaking for isolation  • streaking for quantitation– Ev...
http://science.nhmccd.edu/biol/wellmeyer/media/media.htm                        Dr.T.V.Rao MD                      61
Chocolate agarUninoculated                                        Haemophilus     http://www.hardydiagnostics.com/catalog2...
MacConkey agarhttp://science.nhmccd.edu/biol/wellmeyer/media/media.htm                        Dr.T.V.Rao MD               ...
Hektoen agarhttp://medic.med.uth.tmc.edu/path/hekto.htm                   Dr.T.V.Rao MD              64
• Nutritional requirements and metabolic                 capabilities  – Single enzyme tests    • Catalase: H2O2 + catalas...
Biochemical Tests• The microbe is cultured in a media with a special  substrate and tested for an end product.• Prominent ...
Carbohydrate             . This medium showFermentation              fermentation (acid                          producti...
Methyl Red Test• This is a qualitative  test for acid  production.• The bacteria is grown  in MR-VP broth.• After addition...
Nitrate Reduction ♫After 24-48 hrs of                                 incubation, nitrate                                 ...
Biochemical Tests• Other biochemical tests of interest include:   H2S production   Indole test   Oxidase test   Oxidat...
Dr.T.V.Rao MD   71
Dr.T.V.Rao MD   72
Coagulase Test     Dr.T.V.Rao MD   73
–Tests for presence of     metabolic pathways• Oxidation and fermentation: oxidation  of glucose requires oxygen,  ferment...
O-F glucose mediahttp://academic.mwsc.edu/jcbaker/bio390sec01/bio390_laboratory_study_images.htm                          ...
DifferentialChromagar orientation                    Mediauses colour-formation to                                       ...
• Principles of Phenotype-based ID               schemes      – Selection and inoculation of ID test battery   • Type of b...
Rapid Tests• Rapid test: a biochemical system for the identification  of Enterobacteriaceae and other Gram –ve bacteria.• ...
Rapid Tests                                          positive                                         negativeONPG (β gala...
Bacteriophage Typing• What are bacteriophages?• Bacteriophage typing is based on the specificity of  phage surface recepto...
Heavily Inoculated Plate          Dr.T.V.Rao MD    81
Bacteriophage Typing• The plate is incubated for 24 hrs then observed  for plaques.• The phage type is reported as a speci...
Uncultivable Organisms• Environmental  researchers estimate  that < 1% of  microorganisms are  culturable and  therefore i...
Flow Cytometry• Classical techniques are not successful in identification  of those microorganisms that cannot be cultured...
Flow Cytometry• The cytometer forces a suspension of  cells through a laser beam and measures  the light they scatter or t...
Recent Advances in Flowcytometry• Flow cytometry (FCM) allows single- or multiple-  microbe detection in clinical samples ...
Immunological Methods• Immunological methods involve the interaction  of a microbial antigen with an antibody  (produced b...
Principles of Serologic Test Methods – Methods for antibody detection   • Direct whole pathogen agglutination assays      ...
API strips – bioMerieux         Dr.T.V.Rao MD    89
Commercial ID systems– Advantages and  examples of  commercial  systems  • ARIS (Trek)  • MicroScan (Dade-    Behring/Seim...
Immunochemical methods for ID• Principles of immunochemical methods  – Particle agglutination     • Latex agglutination  –...
Serologic methods for diagnosis• Features of the Immune Response  – Characteristics of antibodies    • Features of immune ...
Principles of Serologic Test Methods – Methods for antibody detection   • Direct whole pathogen agglutination assays      ...
Genotypic methods• Genotypic methods of microbe identification  include the use of :  Nucleic acid probes  PCR (RT-PCR, ...
Genotypic Methods• Genotypic methods involve examining the  genetic material of the organisms and has  revolutionized bact...
Real Time PCR and RT-PCR• Currently many PCR tests employ real time PCR.• This involves the use of fluorescent primers.• T...
RAPD-PCR• Random amplified polymorphic DNA PCR uses a  random primer (10-mer) to generate a DNA profile.•   What are rando...
Plasmid fingerprinting• The procedure involves:• The bacterial strains are  grown, the cells lysed and  harvested.• The pl...
Computer and Bacteria Identification• Computers improve  the efficiency of the  lab operations and  increase the speed  an...
• Programme created by Dr.T.V.Rao MD for MedicalMicrobiologists in Developing           world            • Email     • doc...
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Identification of bacterial pathogens

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Identification of bacterial pathogens

  1. 1. Identification of Bacterial Pathogens basic skills in diagnostic bacteriology Dr.T.V.Rao MD Dr.T.V.Rao MD 1
  2. 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 2
  3. 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 3
  4. 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 4
  5. 5. Microbe IdentificationDr.T.V.Rao MD 5
  6. 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 6
  7. 7. Specimen CollectionDr.T.V.Rao MD 7
  8. 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 8
  9. 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 identified phenotypically, immunologically or genetically. Dr.T.V.Rao MD 9
  10. 10. Specimen collection isolation 5 “I” s inoculation inspection identification incubation Dr.T.V.Rao MD 10
  11. 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 11
  12. 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 12
  13. 13. 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
  14. 14. 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
  15. 15. Bacteria differ as per structurehttp://www.sp.uconn.edu/~terry/229sp02/lectures/Lect2.html Dr.T.V.Rao MD 15
  16. 16. Gram Staining Procedure Dr.T.V.Rao MD 16
  17. 17. Gm+ve cocci & Gm-ve bacilli Dr.T.V.Rao MD 17
  18. 18. Streptococcus Dr.T.V.Rao MD 18
  19. 19. Bacilli Dr.T.V.Rao MD 19
  20. 20. Neisseria gonorrhea - Gram stainhttp://www.cdc.gov/STD/LabGuidelines/default.htm Dr.T.V.Rao MD 20
  21. 21. Wound specimen - Gram stainhttp://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html Dr.T.V.Rao MD 21
  22. 22. Oral specimen - Gram stain Dr.T.V.Rao MD 22http://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html
  23. 23. Sputum specimen Gram stain—Streptococcus pneumoniaehttp://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html Dr.T.V.Rao MD 23
  24. 24. 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
  25. 25. 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
  26. 26. 1 2 34 5 67 Ziehl-Neelsen stain Dr.T.V.Rao MD 26
  27. 27. How the Acid fast bacteria appear Dr.T.V.Rao MD 27
  28. 28. 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
  29. 29. Sputum specimen—Acid fast stainhttp://www.healthsci.utas.edu.au/hls/teaching/micro/mma.html Dr.T.V.Rao MD 29
  30. 30. Acid fast stain demonstrating chording Dr.T.V.Rao MD 30
  31. 31. 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 Dr.T.V.Rao MD 31
  32. 32. Direct Fluorescent Antibodies 32
  33. 33. Indirect Fluorescent Antibodies 33
  34. 34. Mycobacterium – auramine stainhttp://www.lung.ca/tb/abouttb/what/causes_tb.html Dr.T.V.Rao MD 34
  35. 35. Yeast—calcofluor whitehttp://www.med.sc.edu:85/mycology/mycology-3.htm Dr.T.V.Rao MD 35
  36. 36. Mould—calcofluor white Dr.T.V.Rao MD 36
  37. 37. Influenza virus infected cells, fluorescent antibody stain Dr.T.V.Rao MD 37
  38. 38. 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
  39. 39. 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
  40. 40. • 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
  41. 41. 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
  42. 42. • 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
  43. 43. 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
  44. 44. 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
  45. 45. Hemolysis a guiding factor Dr.T.V.Rao MD 45
  46. 46. Hemolysis a guiding factor Dr.T.V.Rao MD 46
  47. 47. Enriched Media Chocolate agar, a medium that gets brown from heated blood. Used for isolation of N. gonorrhea.Blood agar plate withbacteria from human throat.This media differentiatesamong different colonies byappearance Dr.T.V.Rao MD 47
  48. 48. Testing for Bacitracin and Optochin Sensitivity Dr.T.V.Rao MD 48
  49. 49. Dr.T.V.Rao MD 49
  50. 50. Mac Conkey Agar a Minimal differentiating Medium Dr.T.V.Rao MD 50
  51. 51. MacConkey Agar Dr.T.V.Rao MD 51
  52. 52. General vs Selective Media Dr.T.V.Rao MD 52
  53. 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 53
  54. 54. Salmonella-Shigella Agar Dr.T.V.Rao MD 54
  55. 55. Dr.T.V.Rao MD 55
  56. 56. • 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 H2S 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
  57. 57. • 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 H2S 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
  58. 58. 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
  59. 59. • Environmental requirements– Oxygen and Carbon dioxide availability • aerobic: room air • facultative: aerobic or anaerobic • microaerophilic: reduced oxygen tension • anaerobic – strict or aerotolerant • capnophilic: increased C02 (5-10%)– Temperature • 35-37ºC • 30ºC • cold • 42ºC Dr.T.V.Rao MD 59
  60. 60. • 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
  61. 61. http://science.nhmccd.edu/biol/wellmeyer/media/media.htm Dr.T.V.Rao MD 61
  62. 62. Chocolate agarUninoculated Haemophilus http://www.hardydiagnostics.com/catalog2/hugo/ChocolateAgar.htm Dr.T.V.Rao MD 62
  63. 63. MacConkey agarhttp://science.nhmccd.edu/biol/wellmeyer/media/media.htm Dr.T.V.Rao MD 63
  64. 64. Hektoen agarhttp://medic.med.uth.tmc.edu/path/hekto.htm Dr.T.V.Rao MD 64
  65. 65. • Nutritional requirements and metabolic capabilities – Single enzyme tests • Catalase: H2O2 + catalase = O2 and H20; 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 CO2; 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
  66. 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 66
  67. 67. Carbohydrate  . This medium showFermentation fermentation (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 67
  68. 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 68
  69. 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 69
  70. 70. Biochemical Tests• Other biochemical tests of interest include: H2S production Indole test Oxidase test Oxidation fermentation Phenylalanine deaminase test Antibiotic susceptibility tests• Principle, procedure, most common use. Dr.T.V.Rao MD 70
  71. 71. Dr.T.V.Rao MD 71
  72. 72. Dr.T.V.Rao MD 72
  73. 73. Coagulase Test Dr.T.V.Rao MD 73
  74. 74. –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 enzymes Dr.T.V.Rao MD 74
  75. 75. O-F glucose mediahttp://academic.mwsc.edu/jcbaker/bio390sec01/bio390_laboratory_study_images.htm Dr.T.V.Rao MD 75
  76. 76. DifferentialChromagar orientation Mediauses colour-formation to Chromagardistinguish at least 7common urinarypathogens.This allow for rapididentification andtreatment. The bacteria werestreaked as to spell theirnames. Dr.T.V.Rao MD 76
  77. 77. • 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
  78. 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 78
  79. 79. Rapid Tests positive negativeONPG (β galactosidase); ADH (arginine dihydrolase); LDC (lysine decarboxylase); ODC(ornithine decarboxylase); CIT (citrate utilization); H2S (hydrogen disulphide production); URE(urease); TDA ( tryptophan deaminase); IND (indole production); VP (Voges Proskauer test foracetoin); 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). Dr.T.V.Rao MD 79
  80. 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 80
  81. 81. Heavily Inoculated Plate Dr.T.V.Rao MD 81
  82. 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 82
  83. 83. 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 Dr.T.V.Rao MD 83 nonculturable (VNC).
  84. 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 84
  85. 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 85
  86. 86. 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
  87. 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 87
  88. 88. 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
  89. 89. API strips – bioMerieux Dr.T.V.Rao MD 89
  90. 90. Commercial ID systems– Advantages and examples of commercial systems • ARIS (Trek) • MicroScan (Dade- Behring/Seimens) • Phoenix (Becton- Dickinson) • Vitek (bioMérieux) Dr.T.V.Rao MD 90
  91. 91. 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
  92. 92. 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
  93. 93. 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
  94. 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. 94
  95. 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 95
  96. 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. 96
  97. 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. 97
  98. 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. 98
  99. 99. 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
  100. 100. • Programme created by Dr.T.V.Rao MD for MedicalMicrobiologists in Developing world • Email • doctortvrao@gmail.com Dr.T.V.Rao MD 100
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