Coagulase Test: Principle, Procedure, Results

The coagulase test is a crucial biochemical assay used to identify Staphylococcus aureus, which uniquely produces the enzyme coagulase, differentiating it from coagulase-negative staphylococci (CONS). Coagulase is also the virulence factor ofS. 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 aureusproduces two forms of coagulase, bound and free;

1. A slide coagulase test is done to detect bound coagulase or clumping factor.
2. 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. lugdunensisand S. schleiferimay give positive slide coagulase test results.

Note:Colonies from amannitol salt agar (MSA)culture are not suitable for coagulase testing. The organism must first be cultured on nutrient agar or blood agar.

Slide Coagulase Test Procedure

1. 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).*
2. Make similar suspensions of control positive and negative strains to confirm the proper reactivity of the plasma.
3. 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.
4. 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 thetube coagulase test.

Observation

1. Coagulase positive: Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping in saline or water drop.
2. Coagulase-negative: No clumping in either drop

Interpretation

****Slide coagulase test is the main method used to identify S. aureus in clinical laboratories but it has some limitations.

1. About 15% of ordinary strains of S. aureus and many more of MRSAgive negative reactions.
2. 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 forS. aureus.

Tube Coagulase Test

Tube coagulase test detects free coagulase (staphylocoagulase) which reacts with coagulase-reacting factor (CRF). CRF is a plasma protein, often described as a thrombin-like molecule, that serves as an activator. Staphylocoagulase forms a complex with CRF, and this complex then indirectly converts soluble fibrinogen in plasma to insoluble fibrin, resulting in a clot.

When 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 .Staphylokinase is more active at 35°C than at room temperature (around 25°C). Therefore, if a clot forms early and is then lysed by staphylokinase at 37°C, you might miss it. This is why tubes must be examined periodically, and negative tubes are held overnight at room temperature, where staphylokinase activity is reduced, allowing any delayed or transient clot to persist and be observed.

Tube Coagulase Test Procedure

1. 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.
2. Emulsify several isolated colonies of test organism in 1 ml of diluted rabbit plasma* to give a milky suspension.
3. Incubate the tube at 35°C in ambient air or in a water bath for 4 hours.
4. Examine at 1, 2, and 4 hours for clot formation by tilting the tube through 90**°.**
5. Leave negative tubes at room temperature overnight and re-examine.(This step is essential, for some strains of S. aureus, including many MRSA, whichproduce a delayed clot thatis rapidly lysed at 37°C by the organism’s staphylokinase.)

Observation

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.

1. Coagulase Positive: Clot of any size eg.Staphylococcus aureus
2. 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 faecaliscan destroy the anticoagulant and cause clotting. False-positive or false-negative results can occur if the plasma is not sterile.

Reporting

• 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. lugdunensisand S. schleiferi.
• A negative slide coagulase test is not valid and should be followed with a tube test for confirmation.

Quality Control

• 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.
• Organisms

S. aureus ATCC 25923—coagulase positive Staphylococcus epidermidisATCC 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.

Limitationsof Coagulase Test

1. **Do not use citrated blood, as false-positive results can occur.**Sodium citrate acts as an anticoagulant by binding calcium ions. Some bacteria, like certain species of Enterococcus faecalis, possess citrate lyase enzymes that can metabolize citrate, releasing calcium ions. This re-released calcium can then allow the plasma’s own clotting factors to initiate coagulation, leading to a false-positive clot unrelated to bacterial coagulase.
2. Do not perform coagulase testing from colonies grown on mannitol salt agar. High salt concentrations in MSA can interfere with the enzyme activity or cause autoagglutination of some Staphylococci, leading to false-positive or unclear results.
3. Methicillin-resistant S. aureus (MRSA) can be deficient in bound coagulase, which results in a negative slide test.
4. S. intermediusand 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
5. S. lugdunensisand 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. intermediusby a negative tube coagulase test.
6. 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