IDENTIFICATION OF PATHOGENIC BACTERIA IN CLINICAL MICROBIOLOGY

Identification of Gram Positive Cocci(I)
Staphylococcus species
Gram stain – Gram positive cocci in clusters
Motility – Non-motile
Catalase – Positive
Oxidase – Negative
So it is Staphylococcus
Coagulase Test
Staph. aureus Staph. epidermidis
Staph. saprophyticus

Identification of Gram Positive Cocci(I)
Staphylococcus species – cont.,
Novobiocin sensitivity
Resistant sensitive Staph. Saprophyticus Staph. epidermidis

Differentiation between Staphylococci and Micrococci also may be important because both may be similar in microscopic morphology.
Identification of Gram Positive Cocci(I)
Staphylococcus species- cont.,

(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.

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)

Bacitracin sensitivity
Sensitive Resistant
Strep. pyogenes Other Beta hemolytic
(PYR+ve) Streptococci
(Strep. agalactiae)
(Camp test +ve)
(Hippurate hydrolysis +ve)

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)

Optochin sensitivity test
(Sensitive) (Resistant)
Pneumococcus Viridans streptococci
(Capsulated) (Non-capsulated)
(Bile solubility test - Pos) (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.

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.

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

BILE ESCULIN HYDROLYSIS TEST
Positive Negative
Group D streptococcus Pneumococcus
Enterococcus Viridans streptococci
(Grow in MacConkey’s agar) (Not Grow in MacConkey’s agar)
(Tiny deep pink (LF) colonies)

Growth in 6.5% salt
(Salt tolerance test)
Positive (Growth) Negative (No growth)
Enterococcus Group D streptococcus
PYR (+ve) (-ve)
Sensitivity (R) (S)
to SXT
Ability to
grow at 450C Yes No

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

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 flavescens)
(Neisseria meningitidis) (Neisseria sicca)
(Neisseria subflava)

Growth on THAYER-MARTIN MEDIUM (selective medium)
Growth No growth
Neisseria gonorrhoeae Neisseria flavescens
Neisseria meningitidis Neisseria sicca
Neisseria subflava
Glucose (+ve) Glucose (+ve)
Maltose (-ve) Maltose (+ve)
N. gonorrhoeae N. meningitidis

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.

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.

Spore
(Present) (Absent)
Bacillus sp. Corynebacterium sp.
Clostridium sp. Listeria sp.
Ersipelothrix sp.
Lactobacillus sp.
Bacillus Clostridium
(Aerobic) (Anaerobic)

Bacillus species
Motile Non-motile
Other Bacillus sp. Bacillus anthracis
(McFadyean’s reaction +ve)

Catalase
(+) (-)
Corynebacterium Erysipelothrix
Listeria Lactobacillus
kurthia Actinomyces
Beta hemolytic on BA H2S Production
(+) (-)
(Yes) (No) Erysipelothrix Lactobacillus
(Listeria) (Kurthia) Actinomyces
(Corynebacterium)

Listeria
Corynebacterium
(Esculin Hydrolysis)
(+) (-)
Listeria Corynebacterium
(Motile at 250C) (Non-motile)
(Non-Motile at 370C)

Lactobacillus
Actinomyces
(Branching filaments)
(+) (-)
Actinomyces Lactobacillus

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.

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

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 Enterobacter
Citrobacter

Citrate test
(+) (-)
Citrobacter E.coli
Serratia
Citrobacter Serratia
(No red pigment) (Red pigment)

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.

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

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

Indole test
(+) (-)
Klebsiella oxytoca Klebsiella rhinoscleromatis
Klebsiella pneumoniae
Klebsiella ozanae
(Urease +ve) (Urease –ve)
Klebsiella pneumoniae Klebsiella rhinoscleromatis
Klebsiella ozanae

Malonate
(+) (-)
Klebsiella rhinoscleromatis Klebsiella ozanae
(VP POSITIVE) (VP POSITIVE)

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.

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

E.coli
Enterobacter
Citrobacter
Citrate test
(+) (-)
Citrobacter E. coli
Enterobacter

VP TEST
(+) (-)
Enterobacter Citrobacter
H2S Production
(-) (+) C. amalonauticus C. freundii
C. koseri/diversus

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 (+/-)
Remember citrobacter sometimes also may be Late lactose fermenter ( ONPG – +VE)
Remember Citrobacter always Citrobacter (+)

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

CITRATE TEST
(+) (-)
Citrobacter E.coli
Enterobacter
VP TEST (+) (-)
Enterobacter Citrobacter

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

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

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

Proteus species
Indole test
(+) (-)
Proteus vulgaris Proteus mirabilis
(Ornithine decarboxylase (+)) (Ornithine decarboxylase (-))

Morganella
Providencia
Ornithine decarboxylase
(+) (-)
Morganella morgani Prov. alcalifaciens Prov. rettgeri
Prov. stuartii

Providencia alcalifaciens
Providencia rettgeri
Providencia stuartii
Gas from glucose (-) (+)
Prov. rettgeri Prov. alcalifaciens
Prov. stuartii

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 – 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

Mannitol fermentation
(+) (-)
Shigella dysenteriae Shigella flexneri
Shigella boydii
Shigella sonnei

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

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.

Other biochemicals for Shigella species
Indole – (+/-)
MR – (+)
VP – (-)
Citrate – (-)
Urease – (-)
TSI – K/A, Gas (-), H2S (-)

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

PPA/PDA
(+) (-)
Proteus sp. Salmonella sp.
(Swarming on BA (+)) (Swarming on BA (-))
S. typhi
S. paratyphi A
S. paratyphi B

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

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.

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 (+)

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 – Positive
Oxidase – Positive
So it is one of the members of
Non-enterobacteriaeceae like
Pseudomonas species
Vibrio species

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

Other biochemicals for Pseudomonas aeruginosa
Indole – (-)
MR – (-)
VP – (-)
Citrate – (+)
Urease – (-)
TSI - K/K (or) K/no change, Gas (-), H2S (-)
Nitrate reduction test – (+)

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

Vibrio
Aeromonas (A. hydrophila)
Plesiomonas (Pl. shigelloides)
Lysine Ornithine
(+) (-)
Vibrio Aeromonas
Plesiomonas

Arginine
(+) (-)
Vibrio species Plesiomonas species
TCBS Medium
Yellow colonies Green colonies
V. Cholerae V. parahaemolyticus
V. alginolyticus

Vibrio cholerae
Vibrio alginolyticus
Swarming on Blood agar
(+) (-) V. alginolyticus V. cholerae
Two biotypes
Classical
ElTor

Vibrio Cholerae
Classical biotype ElTor biotype
VP – (-) VP – (+)
Non- haemolytic Haemolytic
Chick RBC agglutination (-) Chick RBC
agglutination (+)

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 (-)

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.