- Describe what coagulase is and what this enzyme does.
- Tell that bacterial species that produce coagulase are pathogenic.
- Identify that the coagulase test is useful for differentiating between Staphylococcus sp.
- Tell how the two types of coagulase tests work and which one is more definitive.
- Successfully conduct and interpret the coagulase test.
The enzyme coagulase, produced by a few of the Staphylococcus species (but not all of them), is a key feature of pathogenic Staphylococcus species. The coagulase enzyme produces coagulation of blood, allowing the microorganism to "wall" its infection off from the host's protective mechanisms rather effectively. As with other enzymes, whether or not a species can produce an enzyme is dependent upon whether that species has the gene for the enzyme. Therefore, testing for coagulase activity tells us about the genes and DNA of a bacterial species.
This test determines the ability of the bacteria to produce bound coagulase. This exoenzyme will cause the conversion of fibrinogen in blood plasma to fibrin and form a clot. The fibrin covers the surface of the bacteria, making it resistant to phagocytosis.
There are 2 methods to test for coagulase: (1) slide agglutination test and (2) tube agglutination test. The tube agglutination test is more accurate, but the slide agglutination test is faster. If you have an unknown Staphylococcus species, you might consider running the slide test, and, if negative, running the tube test. The tube method is the definitive test.
Pathogenic Staphylococcus species (e.g. Staphylococcus 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.
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.
Figure 1: The slide agglutination coagulase test is coagulase positive when clots (clumps) occur with a clear solution in the background (left). The slide coagulase test is coagulase negative when there is a smooth mixture with a milky background (right).
Tube Agglutination Test
If the slide coagulase test reaction is negative, inoculate a tube of rabbit plasma overnight and check for a clot in the tube. This is considered a positive reaction for free coagulase.
- Inoculate a tube containing 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.
Figure 2: The tube agglutination coagulase test. When the test tube is tilted, if the fluid is not clumped and does not contain clumps (clotting), the result is coagulase negative (top). When the test tube is tilted, the fluid either exhibits a clump or contains clumps in it (clotting), the result is coagulase positive (bottom).
Results & Questions
|coagulase (+/-)||species produces a coagulase enzyme (+/-)||species contains a coagulase gene (+/-)|
- Complete the table above to indicate results of the coagulase test.
- A coagulase positive result indicates what about the bacterial species' pathogenicity?
- When a bacterial species living inside of a host (such as a human) produces coagulase, what does coagulase do? Be specific.
- Can testing for coagulase activity be useful for identifying and characterizing bacterial species? Explain your answer.
- The coagulase test is useful for differentiation bacterial species with the genus ____________________.