Purpose

 The test is used to differentiate Staphylococcus aureus (positive) from coagulase-negative staphylococci (negative).

Principle

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 precipitation of fibrinogen on the staphylococcal cell, causing the cells to clump when a bacterial suspension is mixed with plasma. The presence of bound coagulase correlates with free coagulase, an extracellular protein enzyme that causes the formation of a clot when S. aureus colonies are incubated with plasma. The clotting mechanism involves activation of a plasma coagulase-reacting factor (CRF), which is a 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 Test (Detection of ?)

1. Place a drop of coagulase plasma (preferably rabbit plasma with ethylenediaminetetraacetic acid [EDTA]) on a clean, dry, glass slide.

2. Place a drop of distilled water or saline next to the drop of plasma as a 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.

Expected Results

Positive: Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping in saline or water drop (Figure 1, A, left side).

Negative: No clumping in either drop.

Note: All negative slide tests must be confirmed using the tube test (Figure 1, B, right side).

B. Tube Test

1. Emulsify several colonies in 0.5 mL of rabbit plasma (with EDTA) to give a milky suspension.

2. Incubate tube at 35°-37°C in ambient air for 4 hours.

 3. Check for clot formation.

Expected Results

 Positive: Clot of any size (Figure 1, A, left side).

 Negative: No clot (Figure 1, B, right side).

Limitations

 Slide Test

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

Tube Test

1. Test results can be positive at 4 hours and then revert to negative after 24 hours.

 2. If negative at 4 hours, incubate at room temperature overnight and check again for clot formation.

 Quality Control

 Positive: Staphylococcus aureus (ATCC25923)

Negative: Staphylococcus epidermidis (ATC12228)

Fig1. Coagulase test. A, Slide coagulase test for clumping factor. Left side is positive; right side is negative. B, Tube coagulase test for free coagulase. Tube on the left is positive, exhibiting clot. Tube on the right is negative.