5. Coagulase Test
Coagulase is an exoenzyme that causes fibrin of blood plasma to clot. Pathogenic S.aureus produces coagulase, while non-pathogenic strains are coagulase negative. Two forms of coagulase may be produced by S. aureus: free and/or bound. Bound coagulase, also known as clumping factor, is attached to the cell wall of the organism. Free Coagulase is an intracellular enzyme produced when the organism is cultured in broth.
The slide test is simple to perform and rapid, but detects bound coagulase only. Therefore, all negative slide coagulase must be followed by a tube test, which will detect both bound and free coagulase. The test tube test is more sensitive because it can pick up smaller quantities of coagulase. In the tube test procedure, free coagulase is liberated from the cell which acts on prothrombin in coagulase plasma. This product then acts on fibrinogen to form a clot. In this exercise perform a slide and tube coagulase test on both organisms, regardless of the results of the slide test.
a. Rapid Slide Test
i. For each organism, use a clean glass slide. Place one drop of coagulase plasma on each. Emulsify a loopful of the colonies( approximately 2-4) to be tested in the drop on the appropriate slide. Colonies should be from an 18-24 hour old culture.
ii. Mix with the loop or wood applicator stick to obtain a smooth suspension. Gently rock the slide.
iii. If the test is coagulase positive, visible clumps will appear within 1 to 2 minutes. It may be necessary to observe the mixture over a lamp to see the clumping appearance. A positive result is notated as a “positive,” no grading is required. A positive result would also be interpreted as S. aureus on the patient report form because S. aureus is the only Staphylococcus that is coagulase positive.
iv. If no agglutination is observed, it will be reported as “negative.”
v. Discard slides in a biohazard waste container and record results for each organism.
b. Tube Test
i. For each organism which had a negative rapid slide test, perform the tube test.
ii. For each organism, label a small test tube with patient name and ID number. Place approximately 0.5 ml of coagulase plasma into each tube.
iii. Inoculate the plasma with a large loopful of the colonies to be tested.
iv. Incubate the tubes in a 37°C incubator or waterbath. Coagulase positive organisms usually produce a visible clot within 1-4 hours. Examine the tubes periodically by gently tipping the tube. DO NOT shake or agitate the tube which would cause dissolution of the clot. A positive result is notated as a “positive,” no grading is required. A positive result would also be interpreted as S. aureus on the patient report form because S. aureus is the only Staphylococcus that is coagulase positive.
v. After the 1-4 hour incubation time, if the specimen is still negative, parafilming the tubes and reincubate for 24-48 hours and observe again for clot formation.
vi. After 24 hours, if the absence of a clot is observed, report as ‘negative”. A negative result would also be interpreted as Coagulase-negative Staphylococcus on the patient report.