TUBE COAGULASE TEST

 

AIM:

            To identify whether the organism is capable of producing the enzyme Coagulase. The test can also differentiate Staphylococcus from Coagulase negative Staphylococcus species (CoNS).

PRINCIPLE:

            Coagulase also known as Free coagulase is an enzyme present in the cells of bacteria will form plasma clot by reacting with Coagulase reacting factor (CRF) present in the plasma and converts fibrinogen into Fibrin (clot).

Free coagulase    +   CRF   ------->   binds to prothrombin

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Fibrinogen ------------> Fibrin (clot)

REAGENTS:

            Rabbit or Human plasma

PROCEDURE:

1.     Prepare 1 in 6 dilutions of plasma in physiological saline.

2.     Take 1ml of the diluted plasma in 3 tubess and emulsify the PC, test and NC colonies into the respective test tubes.

3.     Incubate at 37°C in water bath for 4 hours.

4.     Interpret the results in 4 hours. If negative, the tubes should be incubated at room temperature for overnight and interpreted on the next day.

INTERPRETATION:

            Positive control (ATCC 25923 S.aureus) – stiff gel colt (coagulum) formation is observed.

            Negative control (ATCC 12228 S.epidermidis) – liquid, flocculant or ropy precipitate is observed.

 If the controls were satisfactory, we can interpret the test results.

            Test – Stiff gel clot formation is observed.

OTHER METHOD:

1.     Inoculate the test strain in BHI broth/peptone water at 37°C overnight.

2.     Add 0.1ml of the suspension to 0.5ml of undiluted plasma.

3.     Incubate at 37°C in water bath for 4 hours.

4.     Interpret the results in 4 hours.

EXAMPLES :

            Tube coagulase positive – S.aureus, S.intermedius, S.hyicus

            Tube coagulase negative – S.lugdunensis, S.epidermidis, S.saprophyticus, S.schleiferi, etc.,

NOTE:

§  Human citrated plasma can produce false positive results hence should be avoided.

§  8 types of coagulase enzymes are present but there is only one type of clumping factor.

§  Most human strains of Staphylococcus produces Coagulase Type A.

§  Staphylokinase is an enzyme produced by Staphylococcus which can destroy the clot formation if the test tube is incubated for more than 4 hours at 37°C.