The coagulase test helps to identify Staphylococcus aureus which produces the enzyme coagulase (coagulates) plasma from coagulase-negative staphylococci (CONS). Coagulase is also the virulence factor of S. aureus, It converts soluble fibrinogen in plasma to insoluble fibrin, and the fibrin coat the bacterial cells making protecting them from opsonization and phagocytosis. Staphylococcus aureus produces two forms of coagulase, bound and free;
- A slide coagulase test is done to detect bound coagulase or clumping factor.
- A tube coagulase test is done to detect free coagulase.
Slide Coagulase Test
Slide coagulase test is done to detect “bound coagulase” or “clumping factor”. The clumping factor is a fibrinogen binding cell surface receptor present in the cell walls of most, but not all, S. aureus. The clumping factor acts directly on the fibrinogen in the plasma resulting in clumping. Slide coagulase test is rapid, but it requires several colonies and lacks sensitivity.
As the clumping factors are not present in all S. aureus, some strains will give a negative slide coagulase test. In addition, clumping factors can be masked by cell surface capsular polysaccharides. So, the negative slide tests must be confirmed by tube coagulase test. Other species of Staphylococcus, namely, S. lugdunensis and S. schleiferi may give positive slide coagulase test results.
Slide Coagulase Test Procedure
- Emulsify a staphylococcal colony in a drop of water on a clean and grease-free glass slide with a minimum of spreading (If the isolate does not form a smooth, milky suspension, do not proceed with the test).
- Make similar suspensions of control positive and negative strains to confirm the proper reactivity of the plasma.
- Dip a flamed and cooled straight inoculating wire into the undiluted plasma at room temperature, withdraw, and stir the adhering traces of plasma (not a loopful) into the staphylococcal suspension on the slide. Flame the wire and repeat for the control suspensions.
- Read as positive a coarse clumping of cocci visible to the naked eye within 10 seconds. Read as negative the absence of clumping or any reaction taking more than 10 seconds to develop, but re-examine any slow reacting strains by the tube coagulase test.
- Coagulase positive: Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping in saline or water drop.
- Coagulase-negative: No clumping in either drop
Slide coagulase test is the main method used to identify S. aureus in clinical laboratories but it has some limitations.
- About 15% of ordinary strains of S. aureus and many more of MRSA give negative reactions.
- Few species of coagulase-negative staphylococci give positive reactions.
Note: All coagulase-negative slides must be confirmed using a tube coagulase test as the definitive test for S. aureus.
Tube Coagulase Test
Tube coagulase test detects free coagulase (staphylocoagulase) which reacts with coagulase-reacting factor (CRF). CRF is a thrombin-like molecule. Staphylocoagulase and CRF combine to indirectly convert fibrinogen to fibrin. A suspension of the organism is suspended and incubated with plasma at 37°C. Clot formation within 4 hours indicates a positive test.
The coagulase clot thus formed can be destroyed by S. aureus fibrinolysin or staphylokinase, a plasmid-carried enzyme that is more active at 35°C than at 25°C. So the tube coagulase tubes must be examined periodically (1, 2, and 4 hours) for clot formation. Negative tubes should be held overnight at room temperature.
Tube Coagulase Test Procedure
- Prepare a 1-in-6 dilution of the plasma in saline (0.85% NaCl) and place 1 ml volumes of the diluted plasma in small tubes.
- Emulsify several isolated colonies of test organism in 1 ml of diluted rabbit plasma* to give a milky suspension.
- Incubate the tube at 35°C in ambient air or in a water bath for 4 hours.
- Examine at 1, 2, and 4 hours for clot formation by tilting the tube through 90°.
- Leave negative tubes at room temperature overnight and re-examine.
(This step is essential, for some strains of S. aureus, including many MRSA, which produce a delayed clot that is rapidly lysed at 37°C by the organism’s staphylokinase.)
Read as positive any degree of clot formation. Often the plasma is converted into a stiff gel that remains in place when the tube is tilted or inverted, but sometimes clots are seen floating in the fluid.
- Coagulase Positive: Clot of any size eg. Staphylococcus aureus
- Coagulase Negative: No clot (plasma remains wholly liquid or shows only a flocculent or ropy precipitate). eg. Staphylococcus epidermidis
Note: Rabbit plasma is preferable, as it gives better clotting, is free from inhibitors, and is safe. Human plasma contains sodium citrate as an anticoagulant, and some citrate utilizing bacteria such as Enterococcus faecalis can destroy the anticoagulant and cause clotting. False-positive or false-negative results can occur if the plasma is not sterile.
- Report as Staphylococcus aureus if the tube coagulase test is positive and the organism is catalase-positive and a Gram-positive coccus in clusters.
- For a negative tube coagulase test from catalase-positive, Gram-positive cocci in clusters that have creamy white colonies, report as “coagulase-negative staphylococci.”
- A positive slide coagulase test can be reported as S. aureus; however, the test should be confirmed with a tube test from nonhemolytic or only slightly hemolytic colonies from sterile sites, such as blood, to separate S. aureus from S. lugdunensis and S. schleiferi.
- A negative slide coagulase test is not valid and should be followed with a tube test for confirmation.
- Do not use plasma that has not been stored refrigerated or frozen or that appears turbid.
- Perform quality control of coagulase plasma on new lots prior to using them.
- S. aureus ATCC 25923—coagulase positive
- Staphylococcus epidermidis ATCC 12228 or ATCC 14990—coagulase-negative
Where I can get the plasma?
Rabbit plasma may be obtained by centrifuging blood to which 0.1% EDTA has been added as anticoagulant. Alternatively, it may be obtained lyophilized from a commercial supplier and reconstituted by the addition of water.
Citrated human plasma may be obtained from a blood bank, but the blood must have been screened and found free from viral (Hepatitis B, HIV) antigens and antibodies. It must be handled with the precautions appropriate for all human body fluids.
Store the plasma in small portions at -20°C and keep a stock of in-used plasma at 4°C bringing it to room temperature before use.
Limitations of Coagulase Test
- Do not use citrated blood, as false-positive results can occur.
- Do not perform coagulase testing from colonies grown on mannitol salt agar.
- Methicillin-resistant S. aureus (MRSA) can be deficient in bound coagulase, which results in a negative slide test.
- S. intermedius and S. hyicus may be positive in the tube test; these species are generally found only in dogs and pigs, respectively, but are as infectious as S.aureus when they infect humans. Both form nonhemolytic colonies on fresh blood agar plates and are Voges-Proskauer negative, which separates them from S.aureus. S. intermedius is also pyrrolidonyl-β-naphthylamide (PYR) positive
- S. lugdunensis and S. schleiferi give positive slide test but negative tube test. They can be separated from S. aureus by their strongly positive PYR reaction and from S. intermedius by a negative tube coagulase test.
- Staphylococcus argenteus is a novel staphylococcal species, which is also tube coagulase-positive and PYR negative.
References and further readings
- Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology. https://doi.org/10.1128/9781555818814
- Katz, D. S. (2010, November 11). Coagulase Test Protocol. https://www.asmscience.org/content/education/protocol/protocol.3220
- Staphylococcus Argenteus: Another Coagulase Positive Staphylococcus. ASM.Org. Retrieved June 4, 2021, from https://asm.org/Podcasts/Editors-in-Conversation/Episodes/Staphylococcus-Argenteus-Another-Coagulase-Positiv