Coagulase Test: Slide and Tube Method

Principle:

This test is used to differentiate Staphylococcus aureus (positive) from coagulase- negative staphylococci (negative). S. aureus produces two forms of coagulase: bound and free. Bound coagulase, or “clumping factor”, is bound to the bacterial cell wall and reacts directly with fibrinogen. This results in an alteration of fibrinogen so that it precipitates on the staphylococcal cell, causing the cells to clump when a bacterial suspension is mixed with plasma. The presence of bound coagulase correlates well with free coagulase, an extracellular protein enzyme that causes the formation of clot when S. aureus colonies are incubated with plasma. The clotting mechanism involves activation of a plasma coagulase- reacting factor (CRF), which is modified or derived thrombin molecule, to form a coagulase- CRF complex. This complex in turn reacts with fibrinogen to produce the fibrin clot.

Method:

A. Slide Coagulase Test

1. Place a drop of coagulase plasma (preferably rabbit plasma with EDTA) on a clean, dry glass slide.
2.  Place a drop of distilled water or saline next to the drop of plasma as control.  
3. With a loop, straight wire, or wooden stick, emulsify a portion of the isolated colony being tested in each drop, inoculating the water or saline first. Try to create a smooth suspension. 
4. Mix well with a wooden applicator stick. 
5. Rock the slide gently for 5 to 10 seconds.

Results and Interpretation:

Positive: Macroscopic clumping in 10seconds or less in coagulated plasma drop and no clumping in saline or water drop.

Negative: No clumping in either drop. Note: All negative slide tests must be confirmed using the tube test.

Equivocal: Clumping in both drops indicates that the organism autoagglutinates and is unsuitable for the slide coagulase test.

B. Tube Coagulase Test 

1. Emulsify several colonies in a 0.5 ml of rabbit plasma (with EDTA) to give a milky suspension.
2. Incubate tube at 35 degree Celsius in ambient air for 4 hours. 
3. Check for clot formation. Note: Tests can be positive at 4 hours and then revert to negative after 24 hours. 
4. If negative at 4 hours, incubate at room temperature overnight and check again for clot formation.

Results and Interpretation:

Positive: Clot of any size

Negative: No clot