IDENTIFICATION OF PATHOGENIC BACTERIA IN CLINICAL MICROBIOLOGY LABORATORY  G.HARIPRASAD M.Sc.,M.Phil.,Ph.D Department of M...
Identification of Gram Positive Cocci(I) Staphylococcus species <ul><li>Gram stain  –  Gram positive cocci in clusters  </...
Identification of Gram Positive Cocci(I) Staphylococcus species – cont., <ul><li>Novobiocin sensitivity </li></ul><ul><li>...
<ul><li>Differentiation between Staphylococci and Micrococci also may be important because both may be similar in microsco...
<ul><li>(contd..) </li></ul><ul><li>Note :  </li></ul><ul><li>Other characteristic features to identify  Staphylococcus au...
IDENTIFICATION OF GRAM POSITIVE COCCI (II)  STREPTOCOCCUS  <ul><li>Gram stain  –  Gram positive cocci in chains  </li></ul...
<ul><li>Bacitracin sensitivity  </li></ul><ul><li>Sensitive   Resistant  </li></ul><ul><li>Strep. pyogenes  Other Beta hem...
IDENTIFICATION OF GRAM POSITIVE COCCI (III) PNEUMOCOCCUS <ul><li>Gram stain  –  Gram positive cocci in pairs  </li></ul><u...
<ul><li>Optochin sensitivity test  </li></ul><ul><li>(Sensitive)  (Resistant) </li></ul><ul><li>Pneumococcus  Viridans str...
<ul><li>Note: </li></ul><ul><li>Pneumococcus – cause of Lobar pneumonia so it is most likely to be present in sputum of in...
IDENTIFICATION OF GRAM POSITIVE COCCI- IV ENTEROCOCCUS <ul><li>Gram stain –  Gram positive cocci in pairs  </li></ul><ul><...
<ul><li>BILE ESCULIN HYDROLYSIS TEST </li></ul><ul><li>Positive  Negative  </li></ul><ul><li>Group D streptococcus  Pneumo...
<ul><li>Growth in 6.5% salt </li></ul><ul><li>(Salt tolerance test) </li></ul><ul><li>Positive (Growth)  Negative (No grow...
<ul><li>Note: </li></ul><ul><li>Enterococcus can be alpha or beta or gamma hemolytic on blood agar  </li></ul><ul><li>Ente...
IDENTIFICATION OF GRAM NEGATIVE COCCI (I) NEISSERIA SPECIES  <ul><li>Gram Stain  –  Gram negative diplococci  </li></ul><u...
<ul><li>Growth on THAYER-MARTIN MEDIUM (selective medium) </li></ul><ul><li>Growth  No growth  </li></ul><ul><li>Neisseria...
<ul><li>Note: </li></ul><ul><li>Non-pathogenic Neisseria like N. flavescens, N. sicca & N. subflava produce yellow pigment...
IDENTIFICATION OF GRAM POSITIVE BACILLI  <ul><li>List of Gram positive Bacilli  </li></ul><ul><li>Corynebacterium sp. </li...
<ul><li>Spore  </li></ul><ul><li>(Present)  (Absent) </li></ul><ul><li>Bacillus sp.   Corynebacterium sp. </li></ul><ul><l...
<ul><li>Bacillus species  </li></ul><ul><li>Motile  Non-motile </li></ul><ul><li>Other Bacillus sp.  Bacillus anthracis </...
<ul><li>Catalase  </li></ul><ul><li>(+)  (-) </li></ul><ul><li>Corynebacterium  Erysipelothrix </li></ul><ul><li>Listeria ...
<ul><li>Listeria </li></ul><ul><li>Corynebacterium </li></ul><ul><li>(Esculin Hydrolysis) </li></ul><ul><li>(+)  (-) </li>...
<ul><li>Lactobacillus  </li></ul><ul><li>Actinomyces </li></ul><ul><li>(Branching filaments)  </li></ul><ul><li>(+)  (-) <...
<ul><li>Note: </li></ul><ul><li>Other examples of anaerobic Gram positive bacilli – Eubacterium, Propionibacterium, Bifido...
IDENTIFICATION OF GRAM NEGATIVE BACILLI (I) ESCHERICHIA COLI /E.COLI <ul><li>Gram stain –  Gram negative bacilli  </li></u...
<ul><li>MacConkey’s agar – Dry, flat LF colonies </li></ul><ul><li>Motility – Motile </li></ul><ul><li>So it may be E.coli...
<ul><li>Citrate test </li></ul><ul><li>(+)  (-) </li></ul><ul><li>Citrobacter  E.coli  </li></ul><ul><li>Serratia  </li></...
<ul><li>IMVIC REACTIONS FOR E.COLI </li></ul><ul><li>Indole – (+) </li></ul><ul><li>Methyl red – (+) </li></ul><ul><li>Vog...
IDENTIFICATION OF GNB (II) KLEBSIELLA SPECIES <ul><li>Gram stain –  Gram Negative bacilli  </li></ul><ul><li>Motility –  N...
<ul><li>On MacConkey’s agar – Mucoid LF Colonies </li></ul><ul><li>Motility – Non-motile </li></ul><ul><li>So it may be Kl...
<ul><li>Indole test </li></ul><ul><li>(+)  (-) </li></ul><ul><li>Klebsiella oxytoca  Klebsiella rhinoscleromatis  </li></u...
<ul><li>Malonate  </li></ul><ul><li>(+)  (-)  </li></ul><ul><li>Klebsiella rhinoscleromatis  Klebsiella ozanae  </li></ul>...
<ul><li>IMVIC REACTIONS FOR KLEBSIELLA PNEUMONIAE </li></ul><ul><li>Indole – (-) </li></ul><ul><li>Methyl red – (-) </li><...
IDENTIFICATION OF GNB (III) CITROBACTER  <ul><li>Gram stain –  Gram negative bacilli </li></ul><ul><li>Motility –  Motile ...
<ul><li>E.coli </li></ul><ul><li>Enterobacter  </li></ul><ul><li>Citrobacter </li></ul><ul><li>Citrate test </li></ul><ul>...
<ul><li>VP TEST </li></ul><ul><li>(+)  (-)  </li></ul><ul><li>Enterobacter  Citrobacter  </li></ul><ul><li>H 2 S Productio...
<ul><li>C. amalonauticus  </li></ul><ul><li>C. koseri/diversus </li></ul><ul><li>MALONATE  </li></ul><ul><li>ADONITOL </li...
<ul><li>Other reactions of Citrobacter  </li></ul><ul><li>Indole – (+/-) </li></ul><ul><li>MR – (+) </li></ul><ul><li>VP –...
IDENTIFICATION OF GNB (IV) ENTEROBACTER <ul><li>Gram stain –  Gram Negative Bacilli  </li></ul><ul><li>Motility –  Motile ...
<ul><li>CITRATE TEST  </li></ul><ul><li>(+)  (-) </li></ul><ul><li>Citrobacter  E.coli </li></ul><ul><li>Enterobacter  </l...
<ul><li>Enterobacter cloacae  </li></ul><ul><li>Enterobacter aerogenes </li></ul><ul><li>Lysine  </li></ul><ul><li>(+)  (-...
IDENTIFICATION OF GNB-V PROTEUS, MORGANELLA, PROVIDENCIA <ul><li>Gram stain –  Gram negative bacilli  </li></ul><ul><li>Mo...
<ul><li>It may be Salmonella or Proteus or Morganella or Providencia  </li></ul><ul><li>Being PPA/PAD test positive  </li>...
<ul><li>Proteus species  </li></ul><ul><li>Indole test  </li></ul><ul><li>(+)  (-) </li></ul><ul><li>Proteus vulgaris  Pro...
<ul><li>Morganella  </li></ul><ul><li>Providencia  </li></ul><ul><li>Ornithine decarboxylase </li></ul><ul><li>(+)  (-) </...
<ul><li>Providencia alcalifaciens </li></ul><ul><li>Providencia rettgeri  </li></ul><ul><li>Providencia stuartii </li></ul...
<ul><li>Prov. rettgeri  </li></ul><ul><li>Prov. Stuartii </li></ul><ul><li>Fermentation of trehalose  </li></ul><ul><li>(+...
<ul><li>Other biochemicals for Proteus species  </li></ul><ul><li>MR – (+) </li></ul><ul><li>VP – (-) </li></ul><ul><li>Ur...
IDENTIFICATION OF GNB – VI SHIGELLA SPECIES <ul><li>Gram stain –  Gram negative bacilli  </li></ul><ul><li>Motility –  Non...
<ul><li>Mannitol fermentation  </li></ul><ul><li>(+)  (-) </li></ul><ul><li>Shigella dysenteriae  Shigella flexneri </li><...
<ul><li>Shigella flexneri  </li></ul><ul><li>Shigella boydii </li></ul><ul><li>Shigella sonnei </li></ul><ul><li>ONGP </li...
<ul><li>Note: </li></ul><ul><li>Shigella dysenteriae type 1 is always catalase negative. </li></ul><ul><li>Differentiation...
<ul><li>Other biochemicals for Shigella species  </li></ul><ul><li>Indole – (+/-) </li></ul><ul><li>MR – (+) </li></ul><ul...
IDENTIFICATION OF GNB – VII SALMONELLA SPECIES <ul><li>Gram stain –  Gram negative bacilli  </li></ul><ul><li>Motility –  ...
<ul><li>PPA/PDA </li></ul><ul><li>(+)  (-)  </li></ul><ul><li>Proteus sp.  Salmonella sp. </li></ul><ul><li>(Swarming on B...
<ul><li>H 2 S Production </li></ul><ul><li>(+)  (-)  </li></ul><ul><li>S. typhi  S. paratyphi A </li></ul><ul><li>S. parat...
<ul><li>Another way of differentiation of Salmonella species </li></ul><ul><li>Remember S. paratyphi B alone is citrate po...
Differentiation of Salmonella species also may be possible by TSI reaction  <ul><li>S. typhi  </li></ul><ul><li>K/A,  Gas ...
<ul><li>Other biochemicals for Salmonella species </li></ul><ul><li>Indole – (-) </li></ul><ul><li>MR – (+) </li></ul><ul>...
IDENTIFICATION OF GNB – VIII PSEUDOMONAS SPECIES <ul><li>Gram stain –  Gram negative bacilli  </li></ul><ul><li>Motility –...
<ul><li>On MAC – NLF colonies (irregular) </li></ul><ul><li>On NA – Bluish green (pyocyanin) pigmentation </li></ul><ul><l...
<ul><li>Other biochemicals for Pseudomonas aeruginosa </li></ul><ul><li>Indole – (-) </li></ul><ul><li>MR – (-) </li></ul>...
IDENTIFICATION OF GNB – IX VIBRIO AND ASSOCIATED SPECIES <ul><li>Gram stain –  Gram negative bacilli  </li></ul><ul><li>Mo...
<ul><li>Vibrio  </li></ul><ul><li>Aeromonas (A. hydrophila)  </li></ul><ul><li>Plesiomonas (Pl. shigelloides) </li></ul><u...
<ul><li>Arginine </li></ul><ul><li>(+)  (-) </li></ul><ul><li>Vibrio species  Plesiomonas species </li></ul><ul><li>TCBS M...
<ul><li>Vibrio cholerae  </li></ul><ul><li>Vibrio alginolyticus </li></ul><ul><li>Swarming on Blood agar </li></ul><ul><li...
<ul><li>Vibrio Cholerae  </li></ul><ul><li>Classical biotype  ElTor biotype </li></ul><ul><li>VP – (-)  VP – (+) </li></ul...
<ul><li>Specific tests for Vibrio cholerae </li></ul><ul><li>String test – (+) </li></ul><ul><li>Cholera Red Reaction – (+...
<ul><li>Serotyping of Vibrio cholerae  </li></ul><ul><li>Vibrio cholerae 01 antiserum helps to identify Vibrio cholerae O1...
<ul><li>This presentation was created for those who are working as a laboratory technician in clinical microbiology diagno...
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Identification of bacteria, Bacterial identification, Lab identification of bacteria, Medical bacteriology

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Identification of bacteria, Bacterial identification, Lab identification of bacteria, Medical bacteriology

  1. 1. IDENTIFICATION OF PATHOGENIC BACTERIA IN CLINICAL MICROBIOLOGY LABORATORY G.HARIPRASAD M.Sc.,M.Phil.,Ph.D Department of Microbiology Thoothukudi Govt. Medical College Thoothukudi
  2. 2. Identification of Gram Positive Cocci(I) Staphylococcus species <ul><li>Gram stain – Gram positive cocci in clusters </li></ul><ul><li>Motility – Non-motile </li></ul><ul><li>Catalase – Positive </li></ul><ul><li>Oxidase – Negative </li></ul><ul><li>So it is Staphylococcus </li></ul><ul><li>Coagulase Test </li></ul><ul><li>Staph. aureus Staph. epidermidis </li></ul><ul><li>Staph. saprophyticus </li></ul>
  3. 3. Identification of Gram Positive Cocci(I) Staphylococcus species – cont., <ul><li>Novobiocin sensitivity </li></ul><ul><li>Resistant sensitive </li></ul><ul><li>Staph. Saprophyticus Staph. epidermidis </li></ul>
  4. 4. <ul><li>Differentiation between Staphylococci and Micrococci also may be important because both may be similar in microscopic morphology. </li></ul>Identification of Gram Positive Cocci(I) Staphylococcus species- cont., TEST MICROCOCCI STAPHYLOCOCCI OF TEST OXIDATIVE FERMENTATIVE MODIFIED OXIDASE TEST POSITIVE NEGATIVE BACITRACIN SUSCEPTIBILITY SENSITIVE RESISTANT FURAZOLIDONE SUSCEPTIBILITY RESISTANT SENSITIVE
  5. 5. <ul><li>(contd..) </li></ul><ul><li>Note : </li></ul><ul><li>Other characteristic features to identify Staphylococcus aureus </li></ul><ul><li>Mannitol fermentation – Positive </li></ul><ul><li>DNAase test – Positive </li></ul><ul><li>Beta-hemolytic on blood agar </li></ul><ul><li>Golden yellow pigment </li></ul><ul><li>Mannitol Salt Agar (Selective medium) – </li></ul><ul><li>Staph. aureus produce yellow color colonies due to mannitol fermentation. </li></ul>
  6. 6. IDENTIFICATION OF GRAM POSITIVE COCCI (II) STREPTOCOCCUS <ul><li>Gram stain – Gram positive cocci in chains </li></ul><ul><li>Motility – Non-motile </li></ul><ul><li>Catalase – Negative </li></ul><ul><li>Oxidase – Negative </li></ul><ul><li>So it is Streptococcus </li></ul><ul><li>Pin-point colonies with wide zone of Beta hemolysis on blood agar (constant property) </li></ul><ul><li>May be Beta-hemolytic streptococci like </li></ul><ul><li>Streptococcus pyogenes (Group A streptococci) </li></ul><ul><li>Streptococcus agalactiae (Group B streptococci) </li></ul>
  7. 7. <ul><li>Bacitracin sensitivity </li></ul><ul><li>Sensitive Resistant </li></ul><ul><li>Strep. pyogenes Other Beta hemolytic </li></ul><ul><li>(PYR+ve) Streptococci </li></ul><ul><li>(Strep. agalactiae) </li></ul><ul><li>(Camp test +ve) </li></ul><ul><li>(Hippurate hydrolysis +ve) </li></ul>
  8. 8. IDENTIFICATION OF GRAM POSITIVE COCCI (III) PNEUMOCOCCUS <ul><li>Gram stain – Gram positive cocci in pairs </li></ul><ul><li>(lanceolate shape) </li></ul><ul><li>Motility – Non-motile </li></ul><ul><li>Catalase – Negative </li></ul><ul><li>Oxidase – Negative </li></ul><ul><li>On blood agar – ALPHA HEMOLYTIC </li></ul><ul><li>It may be Streptococcus pneumoniae </li></ul><ul><li>(or) Viridans streptococci (though it occur in chains predominantly sometimes may occur in pairs) </li></ul>
  9. 9. <ul><li>Optochin sensitivity test </li></ul><ul><li>(Sensitive) (Resistant) </li></ul><ul><li>Pneumococcus Viridans streptococci </li></ul><ul><li>(Capsulated) (Non-capsulated) </li></ul><ul><li>(Bile solubility test - Pos) (Bile solubility test - Neg) </li></ul><ul><li>(Capsule swelling test – pos) (Capsule swelling test – Neg) </li></ul><ul><li>(Bile Esculin test – Neg) (Bile Esculin Negative) </li></ul><ul><li>Note: Because the isolate is Gram positive cocci in pairs, we may also suspect Enterococcus sp., which may be alpha, beta or gamma hemolytic pattern on blood agar, hence Bile Esculin test, to which Enterococcus sp., is positive, can be used. </li></ul>
  10. 10. <ul><li>Note: </li></ul><ul><li>Pneumococcus – cause of Lobar pneumonia so it is most likely to be present in sputum of infected person. </li></ul><ul><li>Remember pneumococcus is also cause of meningitis ( so also found in CSF) </li></ul><ul><li>Along with sputum, viridans streptococcus, which is a normal flora in oral cavity, may be present when sputum contaminated with saliva. </li></ul><ul><li>Viridans streptococcus, usually arranged in chains, may break into pairs looking like Streptococcus pneumoniae. </li></ul><ul><li>Viridans streptococcus and Streptococcus pneumoniae are always alpha-haemolytic. </li></ul>
  11. 11. IDENTIFICATION OF GRAM POSITIVE COCCI- IV ENTEROCOCCUS <ul><li>Gram stain – Gram positive cocci in pairs </li></ul><ul><li>Motility – Non-motile </li></ul><ul><li>Catalase – Negative </li></ul><ul><li>Oxidase – Negative </li></ul><ul><li>So it may be Enterococcus (or) Pneumococcus (or) viridans streptococci </li></ul>
  12. 12. <ul><li>BILE ESCULIN HYDROLYSIS TEST </li></ul><ul><li>Positive Negative </li></ul><ul><li>Group D streptococcus Pneumococcus </li></ul><ul><li>Enterococcus Viridans streptococci </li></ul><ul><li>(Grow in MacConkey’s agar) (Not Grow in MacConkey’s agar) </li></ul><ul><li>(Tiny deep pink (LF) colonies) </li></ul>
  13. 13. <ul><li>Growth in 6.5% salt </li></ul><ul><li>(Salt tolerance test) </li></ul><ul><li>Positive (Growth) Negative (No growth) </li></ul><ul><li>Enterococcus Group D streptococcus </li></ul><ul><li>PYR (+ve) (-ve) </li></ul><ul><li>Sensitivity (R) (S) </li></ul><ul><li>to SXT </li></ul><ul><li>Ability to </li></ul><ul><li>grow at 45 0 C Yes No </li></ul>
  14. 14. <ul><li>Note: </li></ul><ul><li>Enterococcus can be alpha or beta or gamma hemolytic on blood agar </li></ul><ul><li>Enterococcus faecalis and Enterococcus faecium are important pathogenic members in Genus Enterococcus </li></ul>
  15. 15. IDENTIFICATION OF GRAM NEGATIVE COCCI (I) NEISSERIA SPECIES <ul><li>Gram Stain – Gram negative diplococci </li></ul><ul><li>Motility – Non- motile </li></ul><ul><li>Catalase – Positive </li></ul><ul><li>Oxidase – Positive </li></ul><ul><li>So it may be </li></ul><ul><li>Pathogenic Neisseria (or) Non-pathogenic Neisseria </li></ul><ul><li>(Neisseria gonorrhoeae) (Neisseria flavescens) </li></ul><ul><li>(Neisseria meningitidis) (Neisseria sicca) </li></ul><ul><li>(Neisseria subflava) </li></ul>
  16. 16. <ul><li>Growth on THAYER-MARTIN MEDIUM (selective medium) </li></ul><ul><li>Growth No growth </li></ul><ul><li>Neisseria gonorrhoeae Neisseria flavescens </li></ul><ul><li>Neisseria meningitidis Neisseria sicca </li></ul><ul><li>Neisseria subflava </li></ul><ul><li>Glucose (+ve) Glucose (+ve) </li></ul><ul><li>Maltose (-ve) Maltose (+ve) </li></ul><ul><li>N. gonorrhoeae N. meningitidis </li></ul>
  17. 17. <ul><li>Note: </li></ul><ul><li>Non-pathogenic Neisseria like N. flavescens, N. sicca & N. subflava produce yellow pigment </li></ul><ul><li>Non-pathogenic Neisseria can grow on Nutrient agar but pathogenic Neisseria don’t. </li></ul><ul><li>Catalase test for Neisseria can be done by superoxol test using 30% Hydrogen peroxide. </li></ul><ul><li>N. gonorrhoeae – most likely to be present in urethral pus. </li></ul><ul><li>N. meningitidis – most likely to be present in CSF. </li></ul>
  18. 18. IDENTIFICATION OF GRAM POSITIVE BACILLI <ul><li>List of Gram positive Bacilli </li></ul><ul><li>Corynebacterium sp. </li></ul><ul><li>Listeria sp. </li></ul><ul><li>Erysipelothrix rhusiopathiae </li></ul><ul><li>Lactobacillus sp. </li></ul><ul><li>Kurthia sp. </li></ul><ul><li>Actinomyces sp. </li></ul><ul><li>Bacillus sp. </li></ul><ul><li>Clostridium sp. </li></ul>
  19. 19. <ul><li>Spore </li></ul><ul><li>(Present) (Absent) </li></ul><ul><li>Bacillus sp. Corynebacterium sp. </li></ul><ul><li>Clostridium sp. Listeria sp. </li></ul><ul><li>Ersipelothrix sp. </li></ul><ul><li>Lactobacillus sp. </li></ul><ul><li>Bacillus Clostridium </li></ul><ul><li>(Aerobic) (Anaerobic) </li></ul>
  20. 20. <ul><li>Bacillus species </li></ul><ul><li>Motile Non-motile </li></ul><ul><li>Other Bacillus sp. Bacillus anthracis </li></ul><ul><li>(McFadyean’s reaction +ve) </li></ul>
  21. 21. <ul><li>Catalase </li></ul><ul><li>(+) (-) </li></ul><ul><li>Corynebacterium Erysipelothrix </li></ul><ul><li>Listeria Lactobacillus </li></ul><ul><li>kurthia Actinomyces </li></ul><ul><li>Beta hemolytic on BA H 2 S Production </li></ul><ul><li>(+) (-) </li></ul><ul><li>(Yes) (No) Erysipelothrix Lactobacillus </li></ul><ul><li>(Listeria) (Kurthia) Actinomyces </li></ul><ul><li>(Corynebacterium) </li></ul>
  22. 22. <ul><li>Listeria </li></ul><ul><li>Corynebacterium </li></ul><ul><li>(Esculin Hydrolysis) </li></ul><ul><li>(+) (-) </li></ul><ul><li>Listeria Corynebacterium </li></ul><ul><li>(Motile at 25 0 C) (Non-motile) </li></ul><ul><li>(Non-Motile at 37 0 C) </li></ul>
  23. 23. <ul><li>Lactobacillus </li></ul><ul><li>Actinomyces </li></ul><ul><li>(Branching filaments) </li></ul><ul><li>(+) (-) </li></ul><ul><li>Actinomyces Lactobacillus </li></ul>
  24. 24. <ul><li>Note: </li></ul><ul><li>Other examples of anaerobic Gram positive bacilli – Eubacterium, Propionibacterium, Bifidobacterium, Mobilincus. </li></ul><ul><li>Remember, Actinomyces and Lactobacillus also contains anaerobic species and microaerophilic species. </li></ul><ul><li>Clostridium species (anaerobes) – another example of Gram positive bacilli </li></ul><ul><li>Diptheroids, morphologically similar to C. diptheriae can grow on ordinary media like Nutrient agar. But Corynebacterium diptheriae can only grow on enriched media like Blood agar and Loeffler serum slope. </li></ul>
  25. 25. IDENTIFICATION OF GRAM NEGATIVE BACILLI (I) ESCHERICHIA COLI /E.COLI <ul><li>Gram stain – Gram negative bacilli </li></ul><ul><li>Motility – Motile </li></ul><ul><li>Catalase – Positive </li></ul><ul><li>Oxidase – Negative </li></ul><ul><li>So it is one of the members of Enterobacteriaceae </li></ul><ul><li>Enterobacteriaceae includes E.coli, Klebsiella, Citrobacter, Enterobacter, Serratia, Salmonella, Shigella, Proteus </li></ul>
  26. 26. <ul><li>MacConkey’s agar – Dry, flat LF colonies </li></ul><ul><li>Motility – Motile </li></ul><ul><li>So it may be E.coli, Citrobacter, Enterobacter, Serratia </li></ul><ul><li>Klebsiella being non-motile, it is omitted </li></ul><ul><li>Indole test </li></ul><ul><li>Methyl-Red test </li></ul><ul><li>(+) (-) </li></ul><ul><li>E.coli Enterobacter </li></ul><ul><li>Citrobacter </li></ul>
  27. 27. <ul><li>Citrate test </li></ul><ul><li>(+) (-) </li></ul><ul><li>Citrobacter E.coli </li></ul><ul><li>Serratia </li></ul><ul><li>Citrobacter Serratia </li></ul><ul><li>(No red pigment) (Red pigment) </li></ul>
  28. 28. <ul><li>IMVIC REACTIONS FOR E.COLI </li></ul><ul><li>Indole – (+) </li></ul><ul><li>Methyl red – (+) </li></ul><ul><li>Voges-Proskauer – (-) </li></ul><ul><li>Citrate - (-) </li></ul><ul><li>Other biochemical reactions: </li></ul><ul><li>TSI – A/A, Gas (+), H 2 S (-) </li></ul><ul><li>Urease – (-) </li></ul><ul><li>Remember: </li></ul><ul><li>E.Coli is the most commones cause of lower urinary tract infection. In this case, E.coli is most likely to be present in urine sample of infected persons. </li></ul>
  29. 29. IDENTIFICATION OF GNB (II) KLEBSIELLA SPECIES <ul><li>Gram stain – Gram Negative bacilli </li></ul><ul><li>Motility – Non-motile </li></ul><ul><li>Catalase – Positive </li></ul><ul><li>Oxidase – Negative </li></ul><ul><li>So it is one of the members of Enterobacteriaceae </li></ul>
  30. 30. <ul><li>On MacConkey’s agar – Mucoid LF Colonies </li></ul><ul><li>Motility – Non-motile </li></ul><ul><li>So it may be Klebsiella species </li></ul><ul><li>But it may not be E.coli, Citrobacter, </li></ul><ul><li>Enterobacter or Serratia ( because all are motile) </li></ul><ul><li>Klebsiella species </li></ul><ul><li>Klebsiella pneumoniae </li></ul><ul><li>Klebsiella oxytoca </li></ul><ul><li>Klebsiella rhinoscleromatis </li></ul><ul><li>Klebsiella ozanae </li></ul>
  31. 31. <ul><li>Indole test </li></ul><ul><li>(+) (-) </li></ul><ul><li>Klebsiella oxytoca Klebsiella rhinoscleromatis </li></ul><ul><li>Klebsiella pneumoniae </li></ul><ul><li>Klebsiella ozanae </li></ul><ul><li>(Urease +ve) (Urease –ve) </li></ul><ul><li>Klebsiella pneumoniae Klebsiella rhinoscleromatis </li></ul><ul><li>Klebsiella ozanae </li></ul>
  32. 32. <ul><li>Malonate </li></ul><ul><li>(+) (-) </li></ul><ul><li>Klebsiella rhinoscleromatis Klebsiella ozanae </li></ul><ul><li>(VP POSITIVE) (VP POSITIVE) </li></ul>
  33. 33. <ul><li>IMVIC REACTIONS FOR KLEBSIELLA PNEUMONIAE </li></ul><ul><li>Indole – (-) </li></ul><ul><li>Methyl red – (-) </li></ul><ul><li>Voges-Proskauer – (+) </li></ul><ul><li>Citrate - (+) </li></ul><ul><li>Other biochemical reactions: </li></ul><ul><li>TSI – A/A, Gas (+), H 2 S (-) </li></ul><ul><li>Urease – (+) </li></ul><ul><li>Remember: </li></ul><ul><li>Klebsiella pneumoniae is also the most commonest cause of lower urinary tract infection. In this case, it is most likely to be present in urine sample of infected persons. </li></ul>
  34. 34. IDENTIFICATION OF GNB (III) CITROBACTER <ul><li>Gram stain – Gram negative bacilli </li></ul><ul><li>Motility – Motile </li></ul><ul><li>Catalase – Positive </li></ul><ul><li>Oxidase – Negative </li></ul><ul><li>So it is one of the members of Enterobacteriaceae </li></ul><ul><li>On Mac – LF colonies </li></ul><ul><li>Being motile </li></ul><ul><li>It may be E.coli or Enterobacter or Citrobacter </li></ul>
  35. 35. <ul><li>E.coli </li></ul><ul><li>Enterobacter </li></ul><ul><li>Citrobacter </li></ul><ul><li>Citrate test </li></ul><ul><li>(+) (-) </li></ul><ul><li>Citrobacter E. coli </li></ul><ul><li>Enterobacter </li></ul>
  36. 36. <ul><li>VP TEST </li></ul><ul><li>(+) (-) </li></ul><ul><li>Enterobacter Citrobacter </li></ul><ul><li>H 2 S Production </li></ul><ul><li>(-) (+) </li></ul><ul><li>C. amalonauticus C. freundii </li></ul><ul><li>C. koseri/diversus </li></ul>
  37. 37. <ul><li>C. amalonauticus </li></ul><ul><li>C. koseri/diversus </li></ul><ul><li>MALONATE </li></ul><ul><li>ADONITOL </li></ul><ul><li>(+) (-) </li></ul><ul><li>C. koseri/diversus C. amalonauticus </li></ul>
  38. 38. <ul><li>Other reactions of Citrobacter </li></ul><ul><li>Indole – (+/-) </li></ul><ul><li>MR – (+) </li></ul><ul><li>VP – (-) </li></ul><ul><li>Urease – (weakly positive) </li></ul><ul><li>TSI – A/A, Gas (+ve), H 2 S (+/-) </li></ul><ul><li>Remember citrobacter sometimes also may be Late lactose fermenter ( ONPG – +VE) </li></ul><ul><li>Remember Citrobacter always Citrobacter (+) </li></ul>
  39. 39. IDENTIFICATION OF GNB (IV) ENTEROBACTER <ul><li>Gram stain – Gram Negative Bacilli </li></ul><ul><li>Motility – Motile </li></ul><ul><li>Catalase – Positive </li></ul><ul><li>Oxidase – Negative </li></ul><ul><li>So it is one of the members of Enterobacteriaceae </li></ul><ul><li>On Mac- LF colonies (Less mucoid) </li></ul><ul><li>Being motile </li></ul><ul><li>It may be E.coli (or) Citrobacter (or) Enterobacter </li></ul>
  40. 40. <ul><li>CITRATE TEST </li></ul><ul><li>(+) (-) </li></ul><ul><li>Citrobacter E.coli </li></ul><ul><li>Enterobacter </li></ul><ul><li>VP TEST </li></ul><ul><li>(+) (-) </li></ul><ul><li>Enterobacter Citrobacter </li></ul>
  41. 41. <ul><li>Enterobacter cloacae </li></ul><ul><li>Enterobacter aerogenes </li></ul><ul><li>Lysine </li></ul><ul><li>(+) (-) </li></ul><ul><li>Ent. aerogenes Ent. Cloacae </li></ul><ul><li>(Arginine- Pos) (Arginine- Neg) </li></ul>
  42. 42. IDENTIFICATION OF GNB-V PROTEUS, MORGANELLA, PROVIDENCIA <ul><li>Gram stain – Gram negative bacilli </li></ul><ul><li>Motility – Motile </li></ul><ul><li>Catalase – Positive </li></ul><ul><li>Oxidase – Negative </li></ul><ul><li>So it is one of the members of Enterobacteriaceae </li></ul><ul><li>Being motile – Shigella is omitted </li></ul><ul><li>On Mac – NLF – so E.coli, Citrobacter, Enterobacter are omitted </li></ul>
  43. 43. <ul><li>It may be Salmonella or Proteus or Morganella or Providencia </li></ul><ul><li>Being PPA/PAD test positive </li></ul><ul><li>It may not be Salmonella </li></ul><ul><li>It may be Proteus (or) Morganella (or) Providencia </li></ul><ul><li>Swarming on Blood agar </li></ul><ul><li>(+) (-) </li></ul><ul><li>Proteus Providencia </li></ul><ul><li>Morganella </li></ul>
  44. 44. <ul><li>Proteus species </li></ul><ul><li>Indole test </li></ul><ul><li>(+) (-) </li></ul><ul><li>Proteus vulgaris Proteus mirabilis </li></ul><ul><li>(Ornithine decarboxylase (+)) (Ornithine decarboxylase (-)) </li></ul>
  45. 45. <ul><li>Morganella </li></ul><ul><li>Providencia </li></ul><ul><li>Ornithine decarboxylase </li></ul><ul><li>(+) (-) </li></ul><ul><li>Morganella morgani Prov. alcalifaciens </li></ul><ul><li>Prov. rettgeri </li></ul><ul><li>Prov. stuartii </li></ul>
  46. 46. <ul><li>Providencia alcalifaciens </li></ul><ul><li>Providencia rettgeri </li></ul><ul><li>Providencia stuartii </li></ul><ul><li>Gas from glucose </li></ul><ul><li>(-) (+) </li></ul><ul><li>Prov. rettgeri Prov. alcalifaciens </li></ul><ul><li>Prov. stuartii </li></ul>
  47. 47. <ul><li>Prov. rettgeri </li></ul><ul><li>Prov. Stuartii </li></ul><ul><li>Fermentation of trehalose </li></ul><ul><li>(+) (-) </li></ul><ul><li>Prov. stuartii Prov. rettgeri </li></ul>
  48. 48. <ul><li>Other biochemicals for Proteus species </li></ul><ul><li>MR – (+) </li></ul><ul><li>VP – (-) </li></ul><ul><li>Urease - (+) </li></ul><ul><li>Citrate – (-) </li></ul><ul><li>TSI – K/A, Gas(+), H 2 S (+) </li></ul>
  49. 49. IDENTIFICATION OF GNB – VI SHIGELLA SPECIES <ul><li>Gram stain – Gram negative bacilli </li></ul><ul><li>Motility – Non-motile </li></ul><ul><li>Catalase – Positive </li></ul><ul><li>Oxidase – Negative </li></ul><ul><li>so it is one of the members of enterobacteriaceae </li></ul><ul><li>On mac – NLF colonies </li></ul><ul><li>Being non-motile </li></ul><ul><li>It is not Salmonella (or) Proteus (being motile) </li></ul><ul><li>It is Shigella species </li></ul>
  50. 50. <ul><li>Mannitol fermentation </li></ul><ul><li>(+) (-) </li></ul><ul><li>Shigella dysenteriae Shigella flexneri </li></ul><ul><li>Shigella boydii </li></ul><ul><li>Shigella sonnei </li></ul>
  51. 51. <ul><li>Shigella flexneri </li></ul><ul><li>Shigella boydii </li></ul><ul><li>Shigella sonnei </li></ul><ul><li>ONGP </li></ul><ul><li>(-) (+) </li></ul><ul><li>Shigella flexneri Shigella sonnei </li></ul><ul><li>Shigella boydii </li></ul>
  52. 52. <ul><li>Note: </li></ul><ul><li>Shigella dysenteriae type 1 is always catalase negative. </li></ul><ul><li>Differentiation between </li></ul><ul><li>Sh. Flexneri and Sh. Boydii can be done only by serological method using specific antisera because of non-availability of suitable biochemical reactions. </li></ul>
  53. 53. <ul><li>Other biochemicals for Shigella species </li></ul><ul><li>Indole – (+/-) </li></ul><ul><li>MR – (+) </li></ul><ul><li>VP – (-) </li></ul><ul><li>Citrate – (-) </li></ul><ul><li>Urease – (-) </li></ul><ul><li>TSI – K/A, Gas (-), H 2 S (-) </li></ul>
  54. 54. IDENTIFICATION OF GNB – VII SALMONELLA SPECIES <ul><li>Gram stain – Gram negative bacilli </li></ul><ul><li>Motility – Motile </li></ul><ul><li>Catalase – Positive </li></ul><ul><li>Oxidase – Negative </li></ul><ul><li>So it is one of the members of Enterobacteriaeceae </li></ul><ul><li>On Mac – NLF colonies </li></ul><ul><li>Being motile </li></ul><ul><li>It may not be Shigella (being non-motile) </li></ul><ul><li>It may be Salmonella or Proteus </li></ul>
  55. 55. <ul><li>PPA/PDA </li></ul><ul><li>(+) (-) </li></ul><ul><li>Proteus sp. Salmonella sp. </li></ul><ul><li>(Swarming on BA (+)) (Swarming on BA (-)) </li></ul><ul><li>S. typhi </li></ul><ul><li>S. paratyphi A </li></ul><ul><li>S. paratyphi B </li></ul>
  56. 56. <ul><li>H 2 S Production </li></ul><ul><li>(+) (-) </li></ul><ul><li>S. typhi S. paratyphi A </li></ul><ul><li>S. paratyphi B </li></ul><ul><li>Gas production </li></ul><ul><li>(-) (+) </li></ul><ul><li>S. typhi S. paratyphi B </li></ul>
  57. 57. <ul><li>Another way of differentiation of Salmonella species </li></ul><ul><li>Remember S. paratyphi B alone is citrate positive. </li></ul><ul><li>Remember S. paratyphi A and S. paratyphi B both are xylose and arabinose positive </li></ul><ul><li>Remember S. typhi is both xylose and arabinose negative. </li></ul>
  58. 58. Differentiation of Salmonella species also may be possible by TSI reaction <ul><li>S. typhi </li></ul><ul><li>K/A, Gas (-), speck of H 2 S (+) </li></ul><ul><li>S. paratyphi A </li></ul><ul><li>K/A, Gas (+), H 2 S (-) </li></ul><ul><li>S. paratyphi B </li></ul><ul><li>K/A, Gas (+), abundant H 2 S (+) </li></ul>
  59. 59. <ul><li>Other biochemicals for Salmonella species </li></ul><ul><li>Indole – (-) </li></ul><ul><li>MR – (+) </li></ul><ul><li>VP – (-) </li></ul><ul><li>Citrate – (only S. paratyphi B positive) </li></ul><ul><li>Urease – (-) </li></ul>
  60. 60. IDENTIFICATION OF GNB – VIII PSEUDOMONAS SPECIES <ul><li>Gram stain – Gram negative bacilli </li></ul><ul><li>Motility – Motile </li></ul><ul><li>Catalase – Positive </li></ul><ul><li>Oxidase – Positive </li></ul><ul><li>So it is one of the members of </li></ul><ul><li>Non-enterobacteriaeceae like </li></ul><ul><li>Pseudomonas species </li></ul><ul><li>Vibrio species </li></ul>
  61. 61. <ul><li>On MAC – NLF colonies (irregular) </li></ul><ul><li>On NA – Bluish green (pyocyanin) pigmentation </li></ul><ul><li>OF –TEST – Oxidative reaction seen </li></ul><ul><li>It is more likely to be Pseudomonas aeruginosa </li></ul><ul><li>(because it is only pseudomonas species produce such type of bluish green pigment) </li></ul><ul><li>It may not be Vibrio species because it is fermentative in OF-TEST and not produce bluish green pigment </li></ul>
  62. 62. <ul><li>Other biochemicals for Pseudomonas aeruginosa </li></ul><ul><li>Indole – (-) </li></ul><ul><li>MR – (-) </li></ul><ul><li>VP – (-) </li></ul><ul><li>Citrate – (+) </li></ul><ul><li>Urease – (-) </li></ul><ul><li>TSI - K/K (or) K/no change, Gas (-), H 2 S (-) </li></ul><ul><li>Nitrate reduction test – (+) </li></ul>
  63. 63. IDENTIFICATION OF GNB – IX VIBRIO AND ASSOCIATED SPECIES <ul><li>Gram stain – Gram negative bacilli </li></ul><ul><li>Motility – Motile (Darting motility) </li></ul><ul><li>Catalase – Positive </li></ul><ul><li>Oxidase – Positive </li></ul><ul><li>So it is one of the members of </li></ul><ul><li>Non- Enterobacteriaeceae </li></ul><ul><li>It may be Vibrio (or) Aeromonas (or) Plesiomonas </li></ul><ul><li>It may not Pseudomonas because no bluish green pigment seen </li></ul>
  64. 64. <ul><li>Vibrio </li></ul><ul><li>Aeromonas (A. hydrophila) </li></ul><ul><li>Plesiomonas (Pl. shigelloides) </li></ul><ul><li>Lysine </li></ul><ul><li>Ornithine </li></ul><ul><li>(+) (-) </li></ul><ul><li>Vibrio Aeromonas </li></ul><ul><li>Plesiomonas </li></ul>
  65. 65. <ul><li>Arginine </li></ul><ul><li>(+) (-) </li></ul><ul><li>Vibrio species Plesiomonas species </li></ul><ul><li>TCBS Medium </li></ul><ul><li>Yellow colonies Green colonies </li></ul><ul><li>V. Cholerae V. parahaemolyticus </li></ul><ul><li>V. alginolyticus </li></ul>
  66. 66. <ul><li>Vibrio cholerae </li></ul><ul><li>Vibrio alginolyticus </li></ul><ul><li>Swarming on Blood agar </li></ul><ul><li>(+) (-) </li></ul><ul><li>V. alginolyticus V. cholerae </li></ul><ul><li>Two biotypes </li></ul><ul><li>Classical </li></ul><ul><li>ElTor </li></ul>
  67. 67. <ul><li>Vibrio Cholerae </li></ul><ul><li>Classical biotype ElTor biotype </li></ul><ul><li>VP – (-) VP – (+) </li></ul><ul><li>Non- haemolytic Haemolytic </li></ul><ul><li>Chick RBC agglutination (-) Chick RBC </li></ul><ul><li>agglutination (+) </li></ul>
  68. 68. <ul><li>Specific tests for Vibrio cholerae </li></ul><ul><li>String test – (+) </li></ul><ul><li>Cholera Red Reaction – (+) </li></ul><ul><li>Gelatin liquefaction – (+) </li></ul><ul><li>_____________________________ </li></ul><ul><li>Other reactions of Vibrio cholerae </li></ul><ul><li>MR – (-) </li></ul><ul><li>VP – (+/-) </li></ul><ul><li>Citrate (+) </li></ul><ul><li>Urease – (-) </li></ul><ul><li>TSI – A/A, Gas (-), H 2 S (-) </li></ul>
  69. 69. <ul><li>Serotyping of Vibrio cholerae </li></ul><ul><li>Vibrio cholerae 01 antiserum helps to identify Vibrio cholerae O1 strain. This is followed by identification of specific subtypes like Inaba (or) Ogawa (or) Hikojima by using specific antisera. </li></ul>
  70. 70. <ul><li>This presentation was created for those who are working as a laboratory technician in clinical microbiology diagnostics. </li></ul><ul><li>Also may find useful for UG, PG, DMLT, PGDMLT in Microbiology. </li></ul><ul><li>Mail ID: </li></ul><ul><li>[email_address] </li></ul>
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