Coagulase test

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

Coagulase Test Definition

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

1. To detect free coagulase and bound coagulase produced by different organisms.
2. To differentiate coagulase-positive Staphylococci from coagulase-negative Staphylococci.
3. 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; Is a 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 FC 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: Is another type of coagulase that is released by the organism.

• 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

1. 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.
2. 5% CaCl2 (optional)

Supplies

1. Loops or sterile sticks
2. Glass or plastic tubes
3. Glass slides

Procedure of Coagulase Test

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

Slide Test

1. About 10 µl of deionized water or physiological saline is added to a slide.
2. 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.
3. 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

1. The plasma is diluted with physiological saline. (Add 0.2 ml plasma in 1.8 ml saline)
2. 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.
3. The test tube is mixed and incubated at 37°C for an hour.
4. 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.

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

1. The coagulase test is used to determine the production of coagulase by different microorganisms.
2. This test can also be used to differentiate S. aureus from other Staphylococcal species.
3. 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.