Identification of Human

Pathogens

Gram Positive cocci

Staphylococcus
 Main pathogenic species (Coagulase-positive
staphylococci): Staphylococcus aureus.
 Other species (Coagulase-negative staphylococci):
S. albus gp. (S. epidermidis & S. saprophyticus).
 Non motile, non spore forming, & non capsulated.

A) Microscopical characters:
 Name of stain: Gram stain
 Type of stain: Differential stain
 Shape: cocci
 Size: small
 Arrangement: grape-like clusters
 Color: violet
 Gram reaction: gram positive
Staphylococci species
B) Culture characters
1. Nutrient agar
S. albus
Pigment Production S. citrus

S. aureus

1) S. aureus: golden yellow

Pigmentcolonies
Production on Nutrient aga
2) S. epidermidis: white coloniesDr. Hisham Esmat
3) S. citrus: lemon yellow colonies
2. Mannitol salt agar
Main Constituents:
• 7.5% NaCl (high conc.), mannitol & phenol red (acid-
base indicator)
• pH 7.5
Principle:
• Contains 7.5 % NaCl which inhibits the growth of
most microorganisms, except Staphylococcus species
(halophilic).
• Contains phenol red indicator which is red/pink
under neutral/basic conditions, but turns yellow
under acidic conditions.
• Mannitol when fermented, acid is produced which
changes the pH of medium to acidic.
• Staphylococcus aureus is able to ferment Mannitol
→ colonies surrounded by yellow zone, due to acid
production.
• Other Staph. sp. (non pathogenic): do not ferment
mannitol → colonies with red or purple zones.

Type: Selective & differential.

Use: Isolation of Staphylococcus sp. from clinical

specimens & differentiation between pathogenic S.
aureus and non pathogenic staphylococci.
 Staphylococcus aureus

Mannitol salt agar

3. CHROMagar MRSA
Main Constituents:
• Cefoxitin antibiotic
• specific chromogenic substrates

Principle:
• MRSA is resistant to cefoxitin antibiotic.
• MRSA hydrolyze the chromogenic substrates and
produce a rose to mauve-colored colony.
• Other organisms will hydrolyze various chromogenic
substances within the media, resulting in a variety of
colored colonies from white to blue to green.
Type:
selective and differential.
Use:
CHROMagar is a selective and differential
media for the identification of methicillin-
resistant Staphylococcus aureus.
methicillin-resistant Staphylococcus aureus on
CHROMagar
C- Biochemical reactions
1) Catalase
2) Blood hemolysis
3) Coagulase
4) Phosphatase
5) DNAse
6) Oxidation/Fermentation (O/F)
7) Novobiocin sensitivity
1. Catalase test
Constituents of test medium:
Nutrient agar + 1% glucose.

Principle:
H2O2 catalase H2O + O2 (froth)
Procedure: Inoculate the m.o. into the test medium
& incubate at 37oC for 18-24 hrs.
Reagent: H2O2 solution
Use: Differentiate between Staphylococcus (+ve) &
Streptococcus species (-ve).
Catalase
test +ve
 2. Blood hemolysis
•Medium: blood agar
•Principle:
β-hemolysis: bacteria cause complete blood
hemolysis producing clear zone around the growth.
γ-hemolysis: bacteria do not cause blood
hemolysis, thus no zones appear around the
growth.

•Use: to differentiate between pathogenic S. aureus

(β-hemolysis) and non pathogenic staphylococci (γ-
hemolysis)according to type of blood hemolysis.
Staphylococcus aureus showing β-hemolysis
on blood agar
3. Coagulase test
Constituents of test medium: Plasma + Na citrate
or Na oxalate (as anticoagulant).
Principle:
Fibrinogen (soluble) coagulase fibrin (clot or firm
gel)
Procedure: Inoculate the m.o. into citrated plasma &
incubate at 37oC for 4 hrs only.
The test should be compared with both +ve & -ve
control
Use: Identification & differentiation of Staphylococcus
aureus from non pathogenic Staph. sp.
4. Phosphatase test
Medium: Nutrient agar + Phenolphthalein
diphosphate
Reagent: 1% NH4OH solution
Principle:
Phosphatase enzyme splits ph. ph. diphosphate,
liberating free ph.ph. + NH3 vapor (pink color)
Use:
Test for the ability of Staphylococcus sp. to produce
phosphatase enzyme.
Phosphatase test
5. DNAse test
Constituents of test medium: DNA agar

Principle:
DNAse enzyme breaks DNA into smaller
nucleotides + 1N HCl ppt. DNA & turns the plate
cloudy, except a clear zone around the growth.
Procedure:
Inoculate the m.o. into the test medium & incubate
at 37oC for 18-24 hrs.
Use: Confirmatory test for S. aureus after coagulase
test.
DNAse test
6. Oxidation/Fermentation (O/F)
Medium: 1% Glucose +.5% agar + Bromocresol purple
N.B. Anaerobic tube contains the same medium covered
with liquid paraffin
Indicator: Bromocresol purple
Principle: Staphylococci are facultative anaerobe, they
utilize glucose oxidatively & fermentatively; changing the
color of the indicator from violet to yellow in both tubes.

Use:
1- Test for metabolic activity of m.o. according to O2
requirements.
2- Differentiation between Staphylococcus (Oxid. & Ferm.)
& Micrococcus sp. (Oxid. only)
 Micrococcus

+ve oxidation +ve oxidation

+ve fermentation -ve fermentation
7. Novobiocin sensitivity
Medium: Mueller-Hinton agar
Reagent :
30 ug novobiocin antibiotic disc applied to the
surface of the agar
Principle:
S. saprophyticus Resistant to novobiocin
S. epidermidis Sensitive to novobiocin
Use:
To distinguish S. epidermidis (S) from
S. saprophyticus (R)
 Novobiocin Susceptibility Test
Resistant Sensitive