Coagulase Test: Introduction, Principle, Types, Test Requirements, Procedure, Result Interpretation, List of Coagulase Positive and Negative Bacteria, Limitations, and Keynotes

Introduction of Coagulase Test

The coagulase test is an enzymatic assay that is applied to differentiate Staphylococcus aureus (positive) from coagulase-negative staphylococci (CoNS). Coagulase is an enzyme formed by S. aureus which changes soluble fibrinogen of plasma to insoluble fibrin. S. aureus produces two forms of coagulase. One is bound coagulase while the other is free coagulase.

1. A slide coagulase test is performed to detect bound coagulase or clumping factors.
2. A tube coagulase test is used to detect free coagulase.

Principle of Coagulase Test for Bacteria

A clinically significant and pathogenic species of staphylococci is Staphylococcus aureus. It is different from other species by the presence of coagulase. This enzyme is a thermostable thrombin-like substance that activates fibrinogen to form fibrin, resulting in a fibrin clot. This is demonstrated in the test tube by the formation of a clot when plasma is inoculated with Staphylococcus. The substance is known as free coagulase and it is detectable in tube coagulase test since it is liberated by the cell. In most, but not all, S. aureus, fibrinogen binding cell surface receptor is also present in the cell wall, called bound coagulase’ or clumping factor and it is detectable on slide coagulase test.

The clumping factor is illustrated by the ability of the organism to act directly on the fibrinogen of the plasma to clump it in a slide coagulase test. The test for clumping factor is rapid but requires several colonies and this factor is not present in all Staphylococcus aureus. In addition, the clumping factors can be covered by cell surface capsular polysaccharides and can be present in other species, namely, Staphylococcus lugdunensis and Staphylococcus schleiferi and the reason, negative slide tests must be followed with a confirmatory tube test.

Requirements for Coagulase Test

• Test organisms: Colonies of gram-positive cocci in clusters that are catalase-positive, as part of the identification of S. aureus.

• Plasma: Note: Rabbit plasma is preferred  rather than human plasma due to the following reasons:

1. Human plasma for the test is less sensitive.
2. It is potentially infectious with human pathogenic viruses.

• Other accessories-

1. Loops or sterile sticks
2. Glass or plastic tubes
3. clean and grease-free glass slides or black-coated cards
4. Physiological saline

• Quality control strains

1. Positive Control (PC):  Staphylococcus aureus ATCC 25923
2. Negative Control (NC): Staphylococcus epidermidis  ATCC 12228

Procedure for slide Coagulase Test Method

It proceeds for the detection of bound coagulase.

1.  Put a drop of physiological saline on each end of a slide or you can also put it on two separate slides.
2. Emulsify a colony of the test bacteria (To avoid misidentifications, only perform this test on classic-looking white to yellow, creamy, opaque, hemolytic colonies of gram-positive cocci in clusters that are catalase-positive. Hemolysis should only be observed on a fresh blood agar plate at equal and less than 18 hours) in each of the drops to make two thick suspensions. Note: Avoid taking colonies from mannitol salt agar (MSA) culture because of not suitable for coagulase testing. The organism must first be cultured on nutrient agar or blood agar.
3. Place just a loopful of plasma into one of the suspensions, and mix gently.
4. Look for clumping of the organisms within 10 seconds, no plasma is added to the second suspension.
5. This is used to differentiate any granular appearance of the organism from true coagulase clumping or agglutination.
6. Follow the above steps for control strains too for validation of the test methodology.

Procedure for Tube Coagulase Test Method

1. Take 0.5 ml of plasma which is diluted 1 in 5 with saline. If there is stored plasma, bring it to room temperature (25°C).
2. Inoculate one colony of Staphylococcus growing on a non-inhibitory medium.
3. Incubate the tube at 37°C  without CO₂ for up to 4 hours and observe for clot formation hourly. Do not agitate the tube during observations; rather, gently tilt it to observe the clot.
4. If the clot is not observed at that time, reincubate the tube at room temperature and read after  20 hours.
5. Follow the above steps for control strains too for validation of the test methodology.

Observations

In a positive slide test, evoke clumping of the organism shows the presence of the bound coagulase whereas in a positive tube test, the plasma in the tube clot and does not flow when the tube is inverted. One thing we should know; on continued incubation, the clot may be lysed by fibrinogen secreted by some strains.

Result and Interpretation

For the slide coagulase test

Positive: Clumping or agglutination within 10 secs

Negative: No clumping within 10 secs

For the tube coagulase test

Positive: Clot formation

Negative: No clot formation

All strains producing negative slide tests must be tested with the tube coagulase test. If there is any degree of the clot, the test should consider positive.

List of Coagulase Positive and Negative Bacteria

Coagulase-positive staphylococci

Staphylococcus aureus

Staphylococcus schleriferi

Staphylococcus felis

Staphylococcus intermedius

Staphylococcus lutrae

Staphylococcus hyicus

Staphylococcus lodgunensis

Only slide positive organisms

Staphylococcus lodgunensis

Staphylococcus schleriferi

only tube coagulase tests positive

Staphylococcus hyicus

Coagulase Negative Organisms

Staphylococcus epidermidis

Staphylococcus epidermidis

Staphylococcus epidermidis

Limitations of Coagulase Test

1. Coagulase assay cannot be performed from growth on mannitol salt agar.
2. Methicillin-resistant S. aureus (MRSA) can be deficient in bound coagulase, which results in a negative slide test.
3. S. intermedius and S. hyicus may be positive in the tube coagulase test.
4. Do not use citrated blood, as false-positive results may occur.

Keynotes

1. Do not practice leaving the test at 37°C for more than 4 hours, since S. aureus fibrinolysin can lyse the clot.
2. If 4 hours of incubation at 37°C is inconvenient for staffing, the test is most sensitive when incubated at room temperature(25°C) for the entire time, but the clot may take longer to form.
3. Alternatively, if the tube is left at 37ºC, add a drop or two of 5% calcium chloride (CaCl2) at 24 hours. If a clot forms, the isolate is coagulase-negative; if a clot does not form, fibrinolysin has lysed a previously formed clot and this result confirms the isolate as coagulase positive but the reference for this article is unpublished data of H. D. Isenberg, editor in chief Clinical Microbiology Procedures Handbook, original and second editions.
4. The coagulase clot can be destroyed by S. aureus fibrinolysin or staphylokinase, a plasmid-carried enzyme that is more active at 35°C than at 25°C.

Further Readings

1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
2. Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
3. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
4. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
5. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
6. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.