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.

principles of coagulase test

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

slide coagulase testPicture 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

glass slide coagulase test

(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

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.)

positive coagulase test

(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

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