IDENTIFICATION OF
PATHOGENIC BACTERIA IN
CLINICAL MICROBIOLOGY
LABORATORY
G.HARIPRASAD M.Sc.,M.Phil.,Ph.D
Department of Mi...
Identification of Gram Positive Cocci(I)
Staphylococcus species
Gram stain
Motility
Catalase
Oxidase

– Gram positive cocc...
Identification of Gram Positive Cocci(I)
Staphylococcus species – cont.,
Novobiocin sensitivity
Resistant
Staph. Saprophyt...
Identification of Gram Positive Cocci(I)
Staphylococcus species- cont.,
• Differentiation between Staphylococci and Microc...
(contd..)
Note:
Other characteristic features to identify
Staphylococcus aureus
Mannitol fermentation – Positive
DNAase ...
IDENTIFICATION OF GRAM POSITIVE COCCI (II)
STREPTOCOCCUS
•
•
•
•

Gram stain – Gram positive cocci in chains
Motility – No...
Bacitracin sensitivity
Sensitive
Strep. pyogenes
(PYR+ve)

Resistant
Other Beta hemolytic
Streptococci
(Strep. agalactiae)...
IDENTIFICATION OF GRAM POSITIVE COCCI (III)
PNEUMOCOCCUS
• Gram stain – Gram positive cocci in pairs
(lanceolate shape)
• ...
Optochin sensitivity test
(Sensitive)
(Resistant)
Pneumococcus
(Capsulated)
(Bile solubility test - Pos)

Viridans strepto...
Note:
Pneumococcus – cause of Lobar pneumonia so it is most
likely to be present in sputum of infected person.
Remember pn...
IDENTIFICATION OF GRAM POSITIVE COCCI- IV
ENTEROCOCCUS
•
•
•
•

Gram stain – Gram positive cocci in pairs
Motility – Non-m...
BILE ESCULIN HYDROLYSIS TEST

Positive
Group D streptococcus
Enterococcus

Negative
Pneumococcus
Viridans streptococci

(G...
Growth in 6.5% salt
(Salt tolerance test)
Positive (Growth)

Negative (No growth)

Enterococcus
PYR
(+ve)
Sensitivity (R)
...
Note:
Enterococcus can be alpha or beta or gamma
hemolytic on blood agar
Enterococcus faecalis and Enterococcus
faecium ar...
IDENTIFICATION OF GRAM NEGATIVE COCCI (I)
NEISSERIA SPECIES
• Gram Stain – Gram negative diplococci
• Motility – Non- moti...
Growth on THAYER-MARTIN MEDIUM (selective medium)

Growth

No growth

Neisseria gonorrhoeae
Neisseria meningitidis

Glucos...
Note:
•Non-pathogenic Neisseria like N. flavescens,
N. sicca & N. subflava produce yellow pigment
•Non-pathogenic Neisseri...
IDENTIFICATION OF GRAM POSITIVE BACILLI
List of Gram positive Bacilli
Corynebacterium sp.
Listeria sp.
Erysipelothrix rhus...
Spore
(Present)

(Absent)

Bacillus sp.
Clostridium sp.

Bacillus
(Aerobic)

Corynebacterium sp.
Listeria sp.
Ersipelothri...
Bacillus species

Motile
Other Bacillus sp.

Non-motile
Bacillus anthracis
(McFadyean’s reaction +ve)
Catalase
(+)

(-)

Corynebacterium
Listeria
kurthia

Erysipelothrix
Lactobacillus
Actinomyces

Beta hemolytic on BA

H2S P...
Listeria
Corynebacterium
(Esculin Hydrolysis)
(+)
Listeria
(Motile at 250C)
(Non-Motile at 370C)

(-)
Corynebacterium
(Non...
Lactobacillus
Actinomyces
(Branching filaments)
(+)
Actinomyces

(-)
Lactobacillus
Note:
Other examples of anaerobic Gram positive bacilli –
Eubacterium, Propionibacterium, Bifidobacterium,
Mobilincus.
Rem...
•
•
•
•

IDENTIFICATION OF GRAM NEGATIVE BACILLI
(I)
ESCHERICHIA COLI /E.COLI
Gram stain – Gram negative bacilli
Motility ...
MacConkey’s agar – Dry, flat LF colonies
Motility – Motile
So it may be E.coli, Citrobacter, Enterobacter,
Serratia
Klebsi...
Citrate test
(+)
Citrobacter
Serratia

Citrobacter
(No red pigment)

(-)
E.coli

Serratia
(Red pigment)
IMVIC REACTIONS FOR E.COLI
•Indole – (+)
•Methyl red – (+)
•Voges-Proskauer – (-)
•Citrate - (-)
Other biochemical reactio...
IDENTIFICATION OF GNB (II)
KLEBSIELLA SPECIES

•
•
•
•

Gram stain – Gram Negative bacilli
Motility – Non-motile
Catalase ...
On MacConkey’s agar – Mucoid LF Colonies
Motility – Non-motile
So it may be Klebsiella species
But it may not be E.coli, C...
Indole test
(+)

(-)

Klebsiella oxytoca

Klebsiella rhinoscleromatis
Klebsiella pneumoniae
Klebsiella ozanae
(Urease +ve)...
Malonate
(+)
Klebsiella rhinoscleromatis
(VP POSITIVE)

(-)
Klebsiella ozanae
(VP POSITIVE)
IMVIC REACTIONS FOR KLEBSIELLA PNEUMONIAE
•Indole – (-)
•Methyl red – (-)
•Voges-Proskauer – (+)
•Citrate - (+)
Other bioc...
IDENTIFICATION OF GNB (III)
CITROBACTER
•
•
•
•

Gram stain – Gram negative bacilli
Motility – Motile
Catalase – Positive
...
E.coli
Enterobacter
Citrobacter
Citrate test
(+)
Citrobacter
Enterobacter

(-)
E. coli
VP TEST
(+)

(-)

Enterobacter

(-)
C. amalonauticus
C. koseri/diversus

Citrobacter
H2S Production
(+)
C. freundii
C. amalonauticus
C. koseri/diversus
MALONATE
ADONITOL
(+)
C. koseri/diversus

(-)
C. amalonauticus
Other reactions of Citrobacter
•Indole – (+/-)
•MR – (+)
•VP – (-)
•Urease – (weakly positive)
•TSI – A/A, Gas (+ve), H2S ...
IDENTIFICATION OF GNB (IV)
ENTEROBACTER
•
•
•
•

Gram stain – Gram Negative Bacilli
Motility – Motile
Catalase – Positive
...
CITRATE TEST
(+)

(-)

Citrobacter
Enterobacter

E.coli

VP TEST
(+)
Enterobacter

(-)
Citrobacter
Enterobacter cloacae
Enterobacter aerogenes
Lysine
(+)
Ent. aerogenes
(Arginine- Pos)

(-)
Ent. Cloacae
(Arginine- Neg)
IDENTIFICATION OF GNB-V
PROTEUS, MORGANELLA, PROVIDENCIA
•
•
•
•

Gram stain – Gram negative bacilli
Motility –Motile
Cata...
• It may be Salmonella or Proteus or
Morganella or Providencia
Being PPA/PAD test positive
It may not be Salmonella
It may...
Proteus species
Indole test
(+)
Proteus vulgaris
(Ornithine decarboxylase (+))

(-)
Proteus mirabilis
(Ornithine decarboxy...
Morganella
Providencia
Ornithine decarboxylase
(+)

(-)

Morganella morgani

Prov. alcalifaciens
Prov. rettgeri
Prov. stua...
Providencia alcalifaciens
Providencia rettgeri
Providencia stuartii
Gas from glucose
(-)
Prov. rettgeri
alcalifaciens
Prov...
Prov. rettgeri
Prov. Stuartii
Fermentation of trehalose
(+)
(-)
Prov. stuartii

Prov. rettgeri
Other biochemicals for Proteus species
MR – (+)
VP – (-)
Urease - (+)
Citrate – (-)
TSI – K/A, Gas(+), H2S (+)
IDENTIFICATION OF GNB – VI
SHIGELLA SPECIES
•
•
•
•

Gram stain – Gram negative bacilli
Motility – Non-motile
Catalase – P...
Mannitol fermentation
(+)
Shigella dysenteriae

(-)
Shigella flexneri
Shigella boydii
Shigella sonnei
Shigella flexneri
Shigella boydii
Shigella sonnei
ONGP
(-)
Shigella flexneri
sonnei
Shigella boydii

(+)
Shigella
Note:
Shigella dysenteriae type 1 is always catalase
negative.
Differentiation between
Sh. Flexneri and Sh. Boydii can be ...
Other biochemicals for Shigella species
Indole – (+/-)
MR – (+)
VP – (-)
Citrate – (-)
Urease – (-)
TSI – K/A, Gas (-), H2...
IDENTIFICATION OF GNB – VII
SALMONELLA SPECIES
•
•
•
•

Gram stain – Gram negative bacilli
Motility – Motile
Catalase – Po...
PPA/PDA
(+)
Proteus sp.
(Swarming on BA (+))

(-)
Salmonella sp.
(Swarming on BA (-))

S. typhi
S. paratyphi A
S. paratyph...
H2S Production
(+)
S. typhi
S. paratyphi B
Gas production
(-)
S. typhi

(-)
S. paratyphi A

(+)
S. paratyphi B
Another way of differentiation of Salmonella
species
•Remember S. paratyphi B alone is citrate
positive.
•Remember S. para...
Differentiation of Salmonella species
also may be possible by TSI reaction
S. typhi
K/A, Gas (-), speck of H2S (+)
S. para...
Other biochemicals for Salmonella species
Indole – (-)
MR – (+)
VP – (-)
Citrate – (only S. paratyphi B positive)
Urease –...
IDENTIFICATION OF GNB – VIII
PSEUDOMONAS SPECIES
•
•
•
•

Gram stain – Gram negative bacilli
Motility – Motile
Catalase – ...
On MAC – NLF colonies (irregular)
On NA – Bluish green (pyocyanin) pigmentation
OF –TEST – Oxidative reaction seen
It is m...
Other biochemicals for Pseudomonas aeruginosa
Indole – (-)
MR – (-)
VP – (-)
Citrate – (+)
Urease – (-)
TSI - K/K (or) K/n...
IDENTIFICATION OF GNB – IX
VIBRIO AND ASSOCIATED SPECIES
•
•
•
•

Gram stain – Gram negative bacilli
Motility – Motile (Da...
Vibrio
Aeromonas (A. hydrophila)
Plesiomonas (Pl. shigelloides)
Lysine
Ornithine
(+)
Vibrio
Plesiomonas

(-)
Aeromonas
Arginine
(+)
Vibrio species

(-)
Plesiomonas species

TCBS Medium

Yellow colonies
V. Cholerae
V. alginolyticus

Green col...
Vibrio cholerae
Vibrio alginolyticus
Swarming on Blood agar
(+)
V. alginolyticus

(-)
V. cholerae
Two biotypes
Classical
E...
Vibrio Cholerae

Classical biotype
VP – (-)
Non- haemolytic
Chick RBC agglutination (-)

ElTor biotype
VP – (+)
Haemolytic...
Specific tests for Vibrio cholerae
String test – (+)
Cholera Red Reaction – (+)
Gelatin liquefaction – (+)
____________...
Serotyping of Vibrio cholerae
Vibrio cholerae 01 antiserum helps to identify
Vibrio cholerae O1 strain. This is followed b...
• This presentation was created for those who
are working as a laboratory technician in
clinical microbiology diagnostics....
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  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 Gram stain Motility Catalase Oxidase – Gram positive cocci in clusters – Non-motile – Positive – Negative So it is Staphylococcus Coagulase Test Staph. aureus Staph. epidermidis Staph. saprophyticus
  3. 3. Identification of Gram Positive Cocci(I) Staphylococcus species – cont., Novobiocin sensitivity Resistant Staph. Saprophyticus sensitive Staph. epidermidis
  4. 4. Identification of Gram Positive Cocci(I) Staphylococcus species- cont., • Differentiation between Staphylococci and Micrococci also may be important because both may be similar in microscopic morphology. TEST MICROCOCCI STAPHYLOCOCCI OF TEST OXIDATIVE FERMENTATIVE MODIFIED OXIDASE TEST POSITIVE NEGATIVE BACITRACIN SUSCEPTIBILITY SENSITIVE RESISTANT FURAZOLIDONE SUSCEPTIBILITY RESISTANT SENSITIVE
  5. 5. (contd..) Note: Other characteristic features to identify Staphylococcus aureus Mannitol fermentation – Positive DNAase test – Positive Beta-hemolytic on blood agar Golden yellow pigment Mannitol Salt Agar (Selective medium) – Staph. aureus produce yellow color colonies due to mannitol fermentation.
  6. 6. IDENTIFICATION OF GRAM POSITIVE COCCI (II) STREPTOCOCCUS • • • • Gram stain – Gram positive cocci in chains Motility – Non-motile Catalase – Negative Oxidase – Negative So it is Streptococcus Pin-point colonies with wide zone of Beta hemolysis on blood agar (constant property) May be Beta-hemolytic streptococci like • Streptococcus pyogenes (Group A streptococci) • Streptococcus agalactiae (Group B streptococci)
  7. 7. Bacitracin sensitivity Sensitive Strep. pyogenes (PYR+ve) Resistant Other Beta hemolytic Streptococci (Strep. agalactiae) (Camp test +ve) (Hippurate hydrolysis +ve)
  8. 8. IDENTIFICATION OF GRAM POSITIVE COCCI (III) PNEUMOCOCCUS • Gram stain – Gram positive cocci in pairs (lanceolate shape) • Motility – Non-motile • Catalase – Negative • Oxidase – Negative On blood agar – ALPHA HEMOLYTIC It may be Streptococcus pneumoniae (or) Viridans streptococci (though it occur in chains predominantly sometimes may occur in pairs)
  9. 9. Optochin sensitivity test (Sensitive) (Resistant) Pneumococcus (Capsulated) (Bile solubility test - Pos) Viridans streptococci (Non-capsulated) (Bile solubility test - Neg) (Capsule swelling test – pos) (Capsule swelling test – Neg) (Bile Esculin test – Neg) (Bile Esculin Negative) 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.
  10. 10. Note: Pneumococcus – cause of Lobar pneumonia so it is most likely to be present in sputum of infected person. Remember pneumococcus is also cause of meningitis ( so also found in CSF) Along with sputum, viridans streptococcus, which is a normal flora in oral cavity, may be present when sputum contaminated with saliva. Viridans streptococcus, usually arranged in chains, may break into pairs looking like Streptococcus pneumoniae. Viridans streptococcus and Streptococcus pneumoniae are always alpha-haemolytic.
  11. 11. IDENTIFICATION OF GRAM POSITIVE COCCI- IV ENTEROCOCCUS • • • • Gram stain – Gram positive cocci in pairs Motility – Non-motile Catalase – Negative Oxidase – Negative So it may be Enterococcus (or) Pneumococcus (or) viridans streptococci
  12. 12. BILE ESCULIN HYDROLYSIS TEST Positive Group D streptococcus Enterococcus Negative Pneumococcus Viridans streptococci (Grow in MacConkey’s agar) (Not Grow in MacConkey’s agar) (Tiny deep pink (LF) colonies)
  13. 13. Growth in 6.5% salt (Salt tolerance test) Positive (Growth) Negative (No growth) Enterococcus PYR (+ve) Sensitivity (R) to SXT Group D streptococcus (-ve) (S) Ability to grow at 450C Yes No
  14. 14. Note: Enterococcus can be alpha or beta or gamma hemolytic on blood agar Enterococcus faecalis and Enterococcus faecium are important pathogenic members in Genus Enterococcus
  15. 15. IDENTIFICATION OF GRAM NEGATIVE COCCI (I) NEISSERIA SPECIES • Gram Stain – Gram negative diplococci • Motility – Non- motile • Catalase – Positive • Oxidase – Positive So it may be Pathogenic Neisseria (or) Non-pathogenic Neisseria (Neisseria gonorrhoeae) (Neisseria meningitidis) (Neisseria flavescens) (Neisseria sicca) (Neisseria subflava)
  16. 16. Growth on THAYER-MARTIN MEDIUM (selective medium) Growth No growth Neisseria gonorrhoeae Neisseria meningitidis Glucose (+ve) Maltose (-ve) N. gonorrhoeae Neisseria flavescens Neisseria sicca Neisseria subflava Glucose (+ve) Maltose (+ve) N. meningitidis
  17. 17. Note: •Non-pathogenic Neisseria like N. flavescens, N. sicca & N. subflava produce yellow pigment •Non-pathogenic Neisseria can grow on Nutrient agar but pathogenic Neisseria don’t. •Catalase test for Neisseria can be done by superoxol test using 30% Hydrogen peroxide. •N. gonorrhoeae – most likely to be present in urethral pus. •N. meningitidis – most likely to be present in CSF.
  18. 18. IDENTIFICATION OF GRAM POSITIVE BACILLI List of Gram positive Bacilli Corynebacterium sp. Listeria sp. Erysipelothrix rhusiopathiae Lactobacillus sp. Kurthia sp. Actinomyces sp. Bacillus sp. Clostridium sp.
  19. 19. Spore (Present) (Absent) Bacillus sp. Clostridium sp. Bacillus (Aerobic) Corynebacterium sp. Listeria sp. Ersipelothrix sp. Lactobacillus sp. Clostridium (Anaerobic)
  20. 20. Bacillus species Motile Other Bacillus sp. Non-motile Bacillus anthracis (McFadyean’s reaction +ve)
  21. 21. Catalase (+) (-) Corynebacterium Listeria kurthia Erysipelothrix Lactobacillus Actinomyces Beta hemolytic on BA H2S Production (+) (Yes) (Listeria) (Corynebacterium) (No) (Kurthia) (-) Erysipelothrix Lactobacillus Actinomyces
  22. 22. Listeria Corynebacterium (Esculin Hydrolysis) (+) Listeria (Motile at 250C) (Non-Motile at 370C) (-) Corynebacterium (Non-motile)
  23. 23. Lactobacillus Actinomyces (Branching filaments) (+) Actinomyces (-) Lactobacillus
  24. 24. Note: Other examples of anaerobic Gram positive bacilli – Eubacterium, Propionibacterium, Bifidobacterium, Mobilincus. Remember, Actinomyces and Lactobacillus also contains anaerobic species and microaerophilic species. Clostridium species (anaerobes) – another example of Gram positive bacilli 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.
  25. 25. • • • • IDENTIFICATION OF GRAM NEGATIVE BACILLI (I) ESCHERICHIA COLI /E.COLI Gram stain – Gram negative bacilli Motility – Motile Catalase – Positive Oxidase – Negative So it is one of the members of Enterobacteriaceae Enterobacteriaceae includes E.coli, Klebsiella, Citrobacter, Enterobacter, Serratia, Salmonella, Shigella, Proteus
  26. 26. MacConkey’s agar – Dry, flat LF colonies Motility – Motile So it may be E.coli, Citrobacter, Enterobacter, Serratia Klebsiella being non-motile, it is omitted Indole test Methyl-Red test (+) (-) E.coli Citrobacter Enterobacter
  27. 27. Citrate test (+) Citrobacter Serratia Citrobacter (No red pigment) (-) E.coli Serratia (Red pigment)
  28. 28. IMVIC REACTIONS FOR E.COLI •Indole – (+) •Methyl red – (+) •Voges-Proskauer – (-) •Citrate - (-) Other biochemical reactions: TSI – A/A, Gas (+), H2S (-) Urease – (-) Remember: 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.
  29. 29. IDENTIFICATION OF GNB (II) KLEBSIELLA SPECIES • • • • Gram stain – Gram Negative bacilli Motility – Non-motile Catalase – Positive Oxidase – Negative So it is one of the members of Enterobacteriaceae
  30. 30. On MacConkey’s agar – Mucoid LF Colonies Motility – Non-motile So it may be Klebsiella species But it may not be E.coli, Citrobacter, Enterobacter or Serratia ( because all are motile) Klebsiella species Klebsiella pneumoniae Klebsiella oxytoca Klebsiella rhinoscleromatis Klebsiella ozanae
  31. 31. Indole test (+) (-) Klebsiella oxytoca Klebsiella rhinoscleromatis Klebsiella pneumoniae Klebsiella ozanae (Urease +ve) (Urease –ve) Klebsiella pneumoniae Klebsiella rhinoscleromatis Klebsiella ozanae
  32. 32. Malonate (+) Klebsiella rhinoscleromatis (VP POSITIVE) (-) Klebsiella ozanae (VP POSITIVE)
  33. 33. IMVIC REACTIONS FOR KLEBSIELLA PNEUMONIAE •Indole – (-) •Methyl red – (-) •Voges-Proskauer – (+) •Citrate - (+) Other biochemical reactions: TSI – A/A, Gas (+), H2S (-) Urease – (+) Remember: 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.
  34. 34. IDENTIFICATION OF GNB (III) CITROBACTER • • • • Gram stain – Gram negative bacilli Motility – Motile Catalase – Positive Oxidase – Negative So it is one of the members of Enterobacteriaceae On Mac – LF colonies Being motile It may be E.coli or Enterobacter or Citrobacter
  35. 35. E.coli Enterobacter Citrobacter Citrate test (+) Citrobacter Enterobacter (-) E. coli
  36. 36. VP TEST (+) (-) Enterobacter (-) C. amalonauticus C. koseri/diversus Citrobacter H2S Production (+) C. freundii
  37. 37. C. amalonauticus C. koseri/diversus MALONATE ADONITOL (+) C. koseri/diversus (-) C. amalonauticus
  38. 38. Other reactions of Citrobacter •Indole – (+/-) •MR – (+) •VP – (-) •Urease – (weakly positive) •TSI – A/A, Gas (+ve), H2S (+/-) Remember citrobacter sometimes also may be Late lactose fermenter ( ONPG – +VE) Remember Citrobacter always Citrobacter (+)
  39. 39. IDENTIFICATION OF GNB (IV) ENTEROBACTER • • • • Gram stain – Gram Negative Bacilli Motility – Motile Catalase – Positive Oxidase – Negative So it is one of the members of Enterobacteriaceae On Mac- LF colonies (Less mucoid) Being motile It may be E.coli (or) Citrobacter (or) Enterobacter
  40. 40. CITRATE TEST (+) (-) Citrobacter Enterobacter E.coli VP TEST (+) Enterobacter (-) Citrobacter
  41. 41. Enterobacter cloacae Enterobacter aerogenes Lysine (+) Ent. aerogenes (Arginine- Pos) (-) Ent. Cloacae (Arginine- Neg)
  42. 42. IDENTIFICATION OF GNB-V PROTEUS, MORGANELLA, PROVIDENCIA • • • • Gram stain – Gram negative bacilli Motility –Motile Catalase – Positive Oxidase – Negative So it is one of the members of Enterobacteriaceae Being motile – Shigella is omitted On Mac – NLF – so E.coli, Citrobacter, Enterobacter are omitted
  43. 43. • It may be Salmonella or Proteus or Morganella or Providencia Being PPA/PAD test positive It may not be Salmonella It may be Proteus (or) Morganella (or) Providencia Swarming on Blood agar (+) Proteus (-) Providencia Morganella
  44. 44. Proteus species Indole test (+) Proteus vulgaris (Ornithine decarboxylase (+)) (-) Proteus mirabilis (Ornithine decarboxylase (-))
  45. 45. Morganella Providencia Ornithine decarboxylase (+) (-) Morganella morgani Prov. alcalifaciens Prov. rettgeri Prov. stuartii
  46. 46. Providencia alcalifaciens Providencia rettgeri Providencia stuartii Gas from glucose (-) Prov. rettgeri alcalifaciens Prov. stuartii (+) Prov.
  47. 47. Prov. rettgeri Prov. Stuartii Fermentation of trehalose (+) (-) Prov. stuartii Prov. rettgeri
  48. 48. Other biochemicals for Proteus species MR – (+) VP – (-) Urease - (+) Citrate – (-) TSI – K/A, Gas(+), H2S (+)
  49. 49. IDENTIFICATION OF GNB – VI SHIGELLA SPECIES • • • • Gram stain – Gram negative bacilli Motility – Non-motile Catalase – Positive Oxidase – Negative so it is one of the members of enterobacteriaceae On mac – NLF colonies Being non-motile It is not Salmonella (or) Proteus (being motile) It is Shigella species
  50. 50. Mannitol fermentation (+) Shigella dysenteriae (-) Shigella flexneri Shigella boydii Shigella sonnei
  51. 51. Shigella flexneri Shigella boydii Shigella sonnei ONGP (-) Shigella flexneri sonnei Shigella boydii (+) Shigella
  52. 52. Note: Shigella dysenteriae type 1 is always catalase negative. Differentiation between Sh. Flexneri and Sh. Boydii can be done only by serological method using specific antisera because of non-availability of suitable biochemical reactions.
  53. 53. Other biochemicals for Shigella species Indole – (+/-) MR – (+) VP – (-) Citrate – (-) Urease – (-) TSI – K/A, Gas (-), H2S (-)
  54. 54. IDENTIFICATION OF GNB – VII SALMONELLA SPECIES • • • • Gram stain – Gram negative bacilli Motility – Motile Catalase – Positive Oxidase – Negative So it is one of the members of Enterobacteriaeceae On Mac – NLF colonies Being motile It may not be Shigella (being non-motile) It may be Salmonella or Proteus
  55. 55. PPA/PDA (+) Proteus sp. (Swarming on BA (+)) (-) Salmonella sp. (Swarming on BA (-)) S. typhi S. paratyphi A S. paratyphi B
  56. 56. H2S Production (+) S. typhi S. paratyphi B Gas production (-) S. typhi (-) S. paratyphi A (+) S. paratyphi B
  57. 57. Another way of differentiation of Salmonella species •Remember S. paratyphi B alone is citrate positive. •Remember S. paratyphi A and S. paratyphi B both are xylose and arabinose positive •Remember S. typhi is both xylose and arabinose negative.
  58. 58. Differentiation of Salmonella species also may be possible by TSI reaction S. typhi K/A, Gas (-), speck of H2S (+) S. paratyphi A K/A, Gas (+), H2S (-) S. paratyphi B K/A, Gas (+), abundant H2S (+)
  59. 59. Other biochemicals for Salmonella species Indole – (-) MR – (+) VP – (-) Citrate – (only S. paratyphi B positive) Urease – (-)
  60. 60. IDENTIFICATION OF GNB – VIII PSEUDOMONAS SPECIES • • • • Gram stain – Gram negative bacilli Motility – Motile Catalase – Positive Oxidase – Positive So it is one of the members of Non-enterobacteriaeceae like Pseudomonas species Vibrio species
  61. 61. On MAC – NLF colonies (irregular) On NA – Bluish green (pyocyanin) pigmentation OF –TEST – Oxidative reaction seen It is more likely to be Pseudomonas aeruginosa (because it is only pseudomonas species produce such type of bluish green pigment) It may not be Vibrio species because it is fermentative in OF-TEST and not produce bluish green pigment
  62. 62. Other biochemicals for Pseudomonas aeruginosa Indole – (-) MR – (-) VP – (-) Citrate – (+) Urease – (-) TSI - K/K (or) K/no change, Gas (-), H2S (-) Nitrate reduction test – (+)
  63. 63. IDENTIFICATION OF GNB – IX VIBRIO AND ASSOCIATED SPECIES • • • • Gram stain – Gram negative bacilli Motility – Motile (Darting motility) Catalase – Positive Oxidase – Positive So it is one of the members of Non- Enterobacteriaeceae It may be Vibrio (or) Aeromonas (or) Plesiomonas It may not Pseudomonas because no bluish green pigment seen
  64. 64. Vibrio Aeromonas (A. hydrophila) Plesiomonas (Pl. shigelloides) Lysine Ornithine (+) Vibrio Plesiomonas (-) Aeromonas
  65. 65. Arginine (+) Vibrio species (-) Plesiomonas species TCBS Medium Yellow colonies V. Cholerae V. alginolyticus Green colonies V. parahaemolyticus
  66. 66. Vibrio cholerae Vibrio alginolyticus Swarming on Blood agar (+) V. alginolyticus (-) V. cholerae Two biotypes Classical ElTor
  67. 67. Vibrio Cholerae Classical biotype VP – (-) Non- haemolytic Chick RBC agglutination (-) ElTor biotype VP – (+) Haemolytic Chick RBC agglutination (+)
  68. 68. Specific tests for Vibrio cholerae String test – (+) Cholera Red Reaction – (+) Gelatin liquefaction – (+) _____________________________ Other reactions of Vibrio cholerae MR – (-) VP – (+/-) Citrate (+) Urease – (-) TSI – A/A, Gas (-), H2S (-)
  69. 69. Serotyping of Vibrio cholerae 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.
  70. 70. • This presentation was created for those who are working as a laboratory technician in clinical microbiology diagnostics. • Also may find useful for UG, PG, DMLT, PGDMLT in Microbiology. Mail ID: hprasad411@gmail.com

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