- Identify the presence of the enzyme coagulase
The enzyme coagulase, produced by a few of the Staphylococcus species, is a key feature of pathogenic Staph. The enzyme produces coagulation of blood, allowing the organism to "wall " its infection off from the host's protective mechanisms rather effectively. There are 2 methods: slide test and tube test. The tube test is more accurate, but the slide test is faster. If you have a Staph unknown, you might consider running the slide test, and, if negative, running the tube test. The tube method is THE definitive test.
- 0.5 ml rabbit plasma per unknown
Pathogenic Staph species (e.g. Staph aureus) can be confirmed using the coagulase test. Since there are 2 kinds of coagulase enzymes—bound and free---there are 2 different tests that can be used to identify these enzymes. Both of the enzymes activate fibrinogen in plasma, in different ways. Your instructor will direct you as to which procedure to use, but the tube test is the definitive test: if you get a negative test result for the slide test, run the tube test.
Be sure that you have a Staph before running this test: the plasma is expensive. It is a waste to run the test on a non-Staph isolate. Run this test on the G+ (make sure that your organisms are catalase +, and gram + cocci in clusters).
SLIDE AGGLUTINATION TEST
- Make a 1 inch diameter circle on a clean glass slide using a wax pencil.
- Place two drops of thawed rabbit plasma into the circle, using a wooden pick or a clean loop.
- Add a HEAVY inoculum and emulsify it in the plasma (should be milky-looking).
- Fibrin threads form between the cells, causing them to agglutinate, or clump.
- There will a visible clumping of cells within 10-15 seconds.
- This test is for the bound coagulase enzyme.
TUBE AGGLUTINATION TEST
- Inoculate a tube containingg ½ ml of rabbit plasma with the bacterial inoculum.
- Place at 37º C and check at ½ hour or at next lab period (some strains will give a + reaction in a few hours, other strains take longer) by tipping the slide at an angle.
- Any degree of coagulation is considered a positive test for the free coagulase enzyme.
IMAGES FOR COAGULASE TEST
- Why take the inoculum from an agar plate or agar slant?
- The presence of the coagulase enzyme is an indicator of:
Jackie Reynolds, Professor of Biology (Richland College)