Coagulase Test- Principle, Procedure, Types, Result, Uses

A coagulase test is a biochemical test that is used to differentiate Staphylococcus aureus from other Staphylococci species like S. epidermidis and S. saprophyticus on the basis of the ability to produce the coagulase enzyme.

• The coagulase test is an important test that differentiates the species of the genus Staphylococci into two groups; Coagulase positive Staphylococci and Coagulase Negative Staphylococci.
• The coagulase enzyme acts as a virulence factor in some organisms as it interacts with the fibrinogen present on the host’s cell surface.
• Organisms with coagulase usually have a protective barrier around themselves, increasing their pathogenicity and resistance against the immune system.
• Coagulase is of two types; free coagulase and bound coagulase, each of which is detected by different methods.
• The bound coagulase is called the clumping factor and is detected rapidly by a slide test. The free coagulase, in turn, is detected in the test tube as a result of the formation of a clot.

Objectives of Coagulase Test

• To detect free coagulase and bound coagulase produced by different organisms.
• To differentiate coagulase-positive Staphylococci from coagulase-negative Staphylococci.
• To identify and differentiate S. aureus from other Staphylococcal species.

Principle of Coagulase Test

Coagulase is an enzymatic protein that is a thermostable thrombin-like substance, which converts fibrinogen into fibrin resulting in clotting or clumping. In S. aureus, two different forms of coagulase are found; free coagulase and bound coagulase.

Bound coagulase

• The clumping factor, termed bound coagulase, can be detected rapidly in the slide test, but this test requires several colonies and lacks sensitivity.
• S. aureus produces another substance in its cell wall, protein A, which binds to the F_C moiety of human immunoglobulin G (IgG) and thus acts as a coagulase-reacting factor.
• When latex or erythrocyte particles are coated with IgG and with human fibrinogen, a staphylococcus will agglutinate if either clumping factor or protein A is present in the bacterial cell wall.
• The presence of the clumping factor is demonstrated by the ability of the organism to act directly on the fibrinogen in the plasma to clump it in a slide assay.
• The test for the clumping factor is rapid but requires several colonies, and also the factor might not be present in all S. aureus organisms.

Free coagulase

• Another type of coagulase that is released by the organism is free coagulase.
• Free coagulase is different from bound coagulase in that the clotting mechanism of free coagulase requires the activation of a plasma coagulase-reacting factor (CRF), which is a modified or derived thrombin molecule, to form a coagulase-CRF complex.
• The plasma added to the tube is preferably rabbit plasma which acts as a binding factor.
• The complex then reacts with fibrinogen to form the fibrin clot in a test tube.

Microorganisms Tested

• Colonies of Gram-positive cocci in clusters that are catalase-positive, as part of the identification of S. aureus.
• Positive blood cultures containing Gram-positive cocci in clusters for rapid detection of S. aureus.

Reagents and Supplies Used

Reagent

• Frozen plasma (preferably rabbit plasma) with EDTA. Human plasma is commonly not used for the test, as it is less sensitive and potentially infectious with human pathogenic viruses.
• 5% CaCl₂ (optional)

Supplies

• Loops or sterile sticks
• Glass or plastic tubes
• Glass slides

Procedure of Coagulase Test

Coagulase can be detected by two different methods; tube test and slide test.

Slide Test

• About 10 µl of deionized water or physiological saline is added to a slide.
• Several colonies from a fresh culture are collected with an inoculating loop and are emulsified into the water to obtain a smooth milk-colored suspension.
• A drop of a rabbit or human plasma is added to the slide, and the clumping is observed immediately, not to exceed 10 seconds.

Tube Test

• The plasma is diluted with physiological saline. (Add 0.2 ml plasma in 1.8 ml saline)
• 5 ml of the diluted plasma is then added to a test tube. About 5 drops of the test organism culture are added to the test tube.
• The test tube is mixed and incubated at 37°C for an hour.
• The tube is finally observed for clot formation. If no clotting is observed, the tube should be examined at 30 minutes intervals of up to 6 hours.

Result Interpretation of Coagulase Test

Figure: Coagulase Test. Lower A- Slide coagulase test for clumping factor. The left side is positive; the right side is negative. Lower B- Tube coagulase test for free coagulase. The tube on the left is positive, exhibiting a clot. The tube on the right is negative. Above: Negative coagulase test in Staphylococcus epidermidis (left) and positive coagulase tests in Staphylococcus aureus (center and right). Lower Image Source A and B: Bailey and Scott’s Diagnostic Microbiology. Elsevier and Above Image Source: New HanseN (Wikipedia).

Slide Test

• A positive test is the demonstration of the agglutination of the bacterial cells after the plasma is added.
• A negative test is demonstrated by the lack of agglutination.

Test Tube

A positive test meets one of the following criteria:

1. Complete clot formation or any degree of clot formation before 24 hours.
2. No clot formation after the addition of 1 or 2 drops of 5% CaCl₂ to a tube without a clot at 24 hours.

A negative test meets one of the following criteria:

1. A lack of clot formation at 24 h at 25°C.
2. No clot after 24 hours at 35°C, but after the addition of 1 or 2 drops of 5% CaCl₂ to the tube, a clot forms.

Coagulase Positive organisms: Staphylococcus aureus and other animal host bacteria like  S. pseudintermedius, S. intermedius, S. schleiferi,  S. delphini, S. hyicus, S. lutrae, S. hyicus

Coagulase Negative organisms: Staphylococcus epidermidis, S. saprophyticus, S. warneri, S. hominis, S. caprae, etc.

Uses of Coagulase Test

• The coagulase test is used to determine the production of coagulase by different microorganisms.
• This test can also be used to differentiate S. aureus from other Staphylococcal species.
• Staphylococcal species are differentiated into coagulase-negative and coagulase-positive species on the basis of the production of the coagulase enzyme.

Limitations of Coagulase Test

• Methicillin-resistant S. aureus 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.
• S. lugdunensis and S. schleiferi produce slide coagulase, but the reaction is more efficient if the human plasma is used rather than rabbit plasma.
• Citrated blood should not be used as false-positive results can occur.
• Coagulase testing cannot be performed from growth on mannitol salt agar.

References and Sources

1. Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook, 3.17.1.1–3.17.48.3.DOI:10.1128/9781555818814.ch3.17.1
2. Berke, A., and R. C.   Tilton.  1986. Evaluation of rapid coagulase methods for the identification of Staphylococcus aureus. J Clin. Microbiol. 23 :916–919.
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