Identification of Gram Positive Bacteria
Catalase Test
Positive Negative
Staphylococcus aureus Streptococcus pyogens
Micrococcus luteus Streptococcus pneumoniae
Enterococcus faecalis
➢ To distinguish Staphylococcus from micrococcus after both are catalase
positive, use modified oxidase (microdase) test or bacitracin sensitivity.
• Staphylococcus : modified oxidase negative, bacitracin resistant.
• Micrococcus : modified oxidase positive, Bacitracin sensitive.
➢ Once, we know an isolates is Staphylococcus (Catalase positive, not
Micrococcus), the next important test is Coagulase test.
• Coagulase Positive: Staphylococcus aureus
• Coagulase Negative: CONS such as Staphylococcus epidermidis or
Staphylococcus saprophyticus.
A common flow
• Gram positive cocci in clusters Staphylococcus, Gram positive in tetrade
than micrococcus.
• Bacitracin or microdase test separates Micrococcus from Staphylococcus.
If Staphylococcus do coagulase test.
If coagulase negative, novobiocin sensitivity can further separate:
• Staphylococcus epidermidis: novobiocin sensitive.
• Staphylococcus saprophyticus: novobiocin resistant.
, COAGULASE TEST
▪ Two types of coagulase test are:
I. Tube Coagulase
II. Slide Coagulase
Tube Coagulase (Free coagulase)
Principle:
Detects free coagulase released by bacteria.
Free coagulase reacts with a plasma component called coagulase reacting
factor, this complex acts like thrombin and converts fibrinogen to fibrin clot.
Procedure:
1. Take 0.5 ml of plasma in a sterile test tube.
2. Inoculate with a heavy suspension of the test organism.
3. Mix gently without shaking vigorously.
4. Incubate at 370C.
5. Examine for clot formation at 1 hour, 2 hours and 4 hours.
6. If negative, leave at room temperature and recheck at 18-24 hours.
Interpretation:
• Positive: Clot formation (plasma does not flow when tube is tilted):
Staphylococcus aureus
• Negative: No clot: Coagulase Negative Staphylococci (CONS)
Slide Coagulase (BOUND COAGULASE)
Principle:
Detects bound coagulase (clumping factor) present on the bacterial cell wall
This clumping factor directly reacts with fibrinogen in plasma and convert
fibrinogen to fibrin on the bacteria surface this cause visible agglutination
(clumping).
Procedure:
1. Place two separate drops of normal saline in a clean glass slide.
2. Emulsify a small portion of the test organism in both drops to make
smooth suspension.
Catalase Test
Positive Negative
Staphylococcus aureus Streptococcus pyogens
Micrococcus luteus Streptococcus pneumoniae
Enterococcus faecalis
➢ To distinguish Staphylococcus from micrococcus after both are catalase
positive, use modified oxidase (microdase) test or bacitracin sensitivity.
• Staphylococcus : modified oxidase negative, bacitracin resistant.
• Micrococcus : modified oxidase positive, Bacitracin sensitive.
➢ Once, we know an isolates is Staphylococcus (Catalase positive, not
Micrococcus), the next important test is Coagulase test.
• Coagulase Positive: Staphylococcus aureus
• Coagulase Negative: CONS such as Staphylococcus epidermidis or
Staphylococcus saprophyticus.
A common flow
• Gram positive cocci in clusters Staphylococcus, Gram positive in tetrade
than micrococcus.
• Bacitracin or microdase test separates Micrococcus from Staphylococcus.
If Staphylococcus do coagulase test.
If coagulase negative, novobiocin sensitivity can further separate:
• Staphylococcus epidermidis: novobiocin sensitive.
• Staphylococcus saprophyticus: novobiocin resistant.
, COAGULASE TEST
▪ Two types of coagulase test are:
I. Tube Coagulase
II. Slide Coagulase
Tube Coagulase (Free coagulase)
Principle:
Detects free coagulase released by bacteria.
Free coagulase reacts with a plasma component called coagulase reacting
factor, this complex acts like thrombin and converts fibrinogen to fibrin clot.
Procedure:
1. Take 0.5 ml of plasma in a sterile test tube.
2. Inoculate with a heavy suspension of the test organism.
3. Mix gently without shaking vigorously.
4. Incubate at 370C.
5. Examine for clot formation at 1 hour, 2 hours and 4 hours.
6. If negative, leave at room temperature and recheck at 18-24 hours.
Interpretation:
• Positive: Clot formation (plasma does not flow when tube is tilted):
Staphylococcus aureus
• Negative: No clot: Coagulase Negative Staphylococci (CONS)
Slide Coagulase (BOUND COAGULASE)
Principle:
Detects bound coagulase (clumping factor) present on the bacterial cell wall
This clumping factor directly reacts with fibrinogen in plasma and convert
fibrinogen to fibrin on the bacteria surface this cause visible agglutination
(clumping).
Procedure:
1. Place two separate drops of normal saline in a clean glass slide.
2. Emulsify a small portion of the test organism in both drops to make
smooth suspension.
