Coagulase Test

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.

Image source : slideplayer.com

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.

Image source : blogspot.com

(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)}

Results

• 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)}

Results

• 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.

Examples

• 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)}

References

1. https://microbeonline.com/diagnostic-tests-biochemical-tests-coagulase-test/
2. https://microbiologyinfo.com/coagulase-test-principal-procedure-types-interpretation-and-examples/
3. https://en.wikipedia.org/wiki/Coagulase
4. http://staff.um.edu.mt/amce1/Stains%20and%20Tests%20PAT2322/The%20Coagulase%20Test.htm
5. https://microbenotes.com/coagulase-test-principle-procedure-and-result-interpretation/
6. http://microbesinfo.com/2013/05/coagulase-test-a-definitive-test-for-identification-of-staphylococcus-aureus/
7. https://www.sigmaaldrich.com/catalog/product/sial/75832?lang=en&region=PH&gclid=Cj0KCQjwguDeBRDCARIsAGxuU8ZwqdLGraZVXdm2X5Gtc1bZK2wQ_cT0Br_W87squLX24M92DnImKkAaAlUUEALw_wcB
8. http://www.asmscience.org/content/education/imagegallery/image.3207
9. https://www.life.umd.edu/classroom/bsci424/LabMaterialsMethods/CoagulaseTest.htm
10. http://www.vumicro.com/vumie/help/VUMICRO/Coagulase_Test.htm