What is a Coagulase Test?
It is a procedure that differentiates Staphylococcus aureus from other Coagulase negative Staphylococci. A coagulase is an enzyme-like protein that converts fibrinogen to fibrin causing the plasma to clot.
The Staphylococcus aureus bacteria produce two forms of coagulase: the bound and free coagulase.
Picture 1 : The principles of coagulase test.
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What is the difference between them?
- Bound coagulase – It bounds to the cell wall of the organism and directly reacts with the fibrinogen causing the cells to clump when the bacterial suspension is mixed with plasma.
- Free coagulase – It uses a modified thrombin molecule in the form of plasma coagulase-reacting factor which reacts to fibrinogen thereby resulting in a fibrin clot. (1, 2, 3, and 4)
Coagulase test purpose
The primary purpose of the coagulase test is to find out if a particular bacterium produces coagulase, which is an enzyme that can coagulate rabbit plasma into a solid clot. (3, 4)
Coagulase test can be done in two ways: slide and tube coagulase test.
#1 – Slide Coagulase Test
Picture 2 : A slide coagulase test; one slide is negative and the other one has clumping which indicates a positive coagulase test.
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(The actual glass slide coagulase test; one is completely smooth while the other has visible clumping indicative of a positive coagulase test.)
It detects bound coagulase or clumping factor (cell-bound coagulase). The clumping factor is the one responsible for directly converting fibrinogen to fibrin causing agglutination.
If agglutination is present, it means that there is a positive Staphylococcus aureus. (4, 5)
How it is done?
- Emulsify the colony of Staphylococcus in a drop of water.
- Place on a clean and grease-free glass slide.
- Get an inoculating wire, dip in a flame and allow to cool.
- Dip the inoculating wire into the undiluted plasma.
- Withdraw the wire and stir the adhering traces of plasma into the glass slide with Staphylococcal suspension.
- Make a similar suspension of both controlled positive and negative strains, flame the inoculating wire, and repeat the process but this time in the control suspensions. (5, 6, and 7)
- Positive – The test is positive if you see a coarse clumping of cocci within 10 seconds.
- Negative – The test is negative if there is no clumping or any reactions within 10 seconds. (6, 7)
What to keep in mind?
- The slide coagulase test identifies S. aureus but it does have some limitations.
- Some strains of Staphylococcus aureus and MRSA may test negative for slide coagulase test.
- Some species of negative Staphylococcus may test positive. Therefore, coagulase negative slides must undergo another coagulase test but this time in the form of a tube to confirm the result. (6, 7, and 8)
#2 – Tube Coagulase Test
(It shows a coagulase test in a test tube with one test tube showing positive result and the other showing a negative result.)
(Two test tubes; one with no clot formation and the other has visible clot indicative of a positive coagulase test.)
It detects free coagulase; a Staphylococcus strain that reacts with coagulase-reacting factor; a thrombin-like molecule that converts fibrinogen to fibrin. (8, 9)
How it is done?
- A 1 in 6 dilutions of plasma in a saline water is needed.
- Place about 1 ml of the diluted plasma in a small tube.
- Emulsify the isolated colony of an organism to be tested in a 1 ml of diluted rabbit plasma. This will help you achieve a milky suspension.
- Incubate the tube at around 35 degree Celsius in a water bath for four hours. It can also be incubated in an ambient air of the same temperature.
- Tilt the test tube at around 90 degrees to check for clot formation.
- If no clot formation is found, leave the test tube at a room temperature overnight. Re-examine the following morning. This is important because some strains of Staphylococcus aureus and MRSA produce a delayed clot. (6, 8, 9, and 10)
- Positive – There will be a noticeable clot of any size.
- Negative – There will be no clot formed. If you check the plasma, you will notice that it remains wholly liquid.
- Coagulase positive bacteria
- Staphylococcus aureus
- S. intermedius
- S. pseudintermedius
- S. schleiferi
- S. hyicus
- S. hyicus
- S. delphini
- S. lutrae
- Coagulase Negative bacteria
- Staphylococcus epidermidis
- S. saprophyticus
- S. warneri
- S. hominis
- S. caprae
Are there any limitations?
- If there is noticeable clumping in both drops in the slide coagulase test, it means that the organism being tested for auto-agglutinates. This simply means that the organism is not suitable for the slide coagulase test.
- In the test tube coagulase test, some strain of bacteria test positive at four hours but will revert to negative after 24 hours. To avoid a false result, an organism that tests negative at four hours should be incubated at a room temperature overnight. Check for clot formation the following morning. (3, 6, 9, and 10)