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Coagulase Test Principle and Procedure

In this coagulase test principle and procedure post we have briefly explained about  Coagulase test in microbiology, principle, objectives, requirements, procedure, uses and limitations.

Coagulase test in microbiology used coagulase protein produced by Staphylococcus aureus that may coagulate plasma into a gel in a tube or agglutinate cocci on a slide. S.aureus can be distinguished from other coagulase-negative staphylococci with this Coagulase test in microbiology. Free coagulase and bound coagulase are produced by the majority of S.aureus strains.

Bound coagulase is a cell wall associated protein, whereas free coagulase is an extracellularly released enzyme. In a tube Coagulase test in microbiology, free coagulase is found, while a slide coagulase test detects bound coagulase. S.aureus isolates can be screened with a slide Coagulase test in microbiology, and tube coagulase can be employed for confirmation.

Only one antigenic type of bound coagulase exists, although there are seven antigenic forms of free coagulase. Coagulase that is free is heat labile, whereas coagulase that is bound is heat stable.

Coagulase Test in Microbiology

Slide Coagulase Test

Principle

Clumping factor is another name for bound coagulase. It forms a fibrin clot on the cell wall by crosslinking the and chain of fibrinogen in plasma. As a result, individual coccus adheres to one another, causing clumping.

Coagulase Test

Procedure

1. With a grease pencil, divide the slide into two halves. One should be labelled “test,” while the other should be labelled “control.

2. Apply a drop of distilled water to each surrounding region. To make a smooth suspension, emulsify one or two colonies of Staphylococcus on a blood agar plate in each drop.

3. A drop of citrated plasma is added to the test suspension, which is then well stirred with a needle. Put nothing in the other drop that will be used as a control.

4. The control suspension is used to rule out false positives due to auto agglutination; cocci clumping within 5-10 seconds are considered positive.

5. Some S.aureus strains do not produce bound coagulase and must be detected using a tube coagulase test.

Tube Coagulase Test

Principle

The free coagulase secreted by S.aureus reacts with coagulase reacting factor (CRF) in plasma to form a complex, which is thrombin. This converts fibrinogen to fibrin resulting in clotting of plasma.

Coagulase Test

Coagulase test in microbiology, Tube Coagulase Test

Procedure

1. Three test tubes are taken and labeled “test”, “negative control” and “positive control”. Each tube is filled with 1 ml of 1 in 10 diluted rabbit plasma. To the tube labeled test, 0.2 ml of overnight broth culture of test bacteria is added.

2. To the tube labeled positive control, 0.2 ml of overnight broth culture of known S.aureus is added to the tube labeled negative control, 0.2ml of sterile broth is added.

3. All the tubes are incubated at 37C and observe the suspensions at half hourly intervals for a period of four hours.

4. Positive result is indicated by gelling of the plasma, which remains in place even after inverting the tube.

5. If the test remains negative until four hours at 37C, the tube is kept at room temperature for overnight incubation.

Results and Interpretation

Result

Slide Coagulase Test: A positive Coagulase test in microbiology result is the production of clumps within 10-15 seconds. There should be no clumping in the saline and plasma mixture.

Tube Coagulase Test: Any degree of clotting, from a loose clot suspended in plasma to a solid clot, indicates a positive coagulase test.  If the Coagulase test in microbiology is negative, the plasma remains liquid.

Coagulase test

Coagulase test in microbiology, Image Source: http://laboratorytests.org/coagulase-test/

Controls

Positive is shown by: Staphylococcus aureus, S. pseudintermedius, S. intermedius, S. schleiferi, S. delphini, S. hyicus, S. lutrae etc.

Negative is shown by: Staphylococcus epidermidis, S. saprophyticus, S. warneri, S. hominis, S. caprae etc.

Applications

1. Coagulase test in microbiology is used to identify and differentiate S.aureus from coagulase negative staphylococci.

2. While slide Coagulase test in microbiology is useful in screening, tube coagulase test is useful in confirmation of coagulase test.

3. Not all S.aureus strains produce coagulase; such rare strains are identified by thermonuclease test.

4. Some Coagulase test in microbiology negative staphylococci such as S.lugdensis and S.schleiferi are known to give positive slide Coagulase test in microbiology while S.hyicus and S.intermedius are known to give positive tube coagulase test.

Limitation

1. The slide Coagulase test in microbiology should be read very quickly, as false positives can occur. Auto agglutination may occur.

2. The slide Coagulase test in microbiology should not be performed with organisms taken from high-salt media such as Mannitol Salt Agar, as the salt content can create false positives.

3. Over mixing may cause the clot to break down. The tube test is more reliable than the slide test.

4. Samples must be observed for clotting within 24 hours. This is because some strains that produce coagulase also produce an enzyme called fibrinolysin, which can dissolve the clot.

5. Therefore, the absence of a clot after 24 hours is no guarantee that a clot never formed. The formation of a clot by 12 hours and the subsequent disappearance of the clot by 24 hours could produce a so-called false negative if the Coagulase test in microbiology were only observed at the 24-hour time.

Further Readings

Reference