Coagulase test: Principle, Procedure and Interpretation

· Tests for identification of bacteria
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    Prasil Pradhan
Coagulase test is done to distinguish Staphylococcus aureus from other Coagulase negative Staphylococci (CONS) like Staphylococcus epidermidis, Staphylococcus saprophyticus etc. Staphylococcus  aureus produces two types of coagulase, i.e. free coagulase and bound coagulase. Free coagulase is an extracellular enzyme that can be detected in tube coagulase test while bound coagulase is a cell wall associated protein that can be detected in slide coagulase test.
Principle of Slide coagulase test:
Bound coagulase is also known as clumping factor. It cross-links α and β chain of fibrinogen in plasma to form fibrin clot that gets deposited on the cell wall of the cocci. As a result, individual coccus sticks to each other and clumping can be observed.

Procedure for Slide Coagulase test:
    1. Emulsify few colonies of Staphylococci from culture in a drop of normal saline on two ends of clean glass slide.
    2. Label one as “test” and the other as “control”. The control suspension serves to rule out false positives due to autoagglutination.
    3. Mix a drop of rabbit or human plasma with the test suspension.
    4. Observe agglutination or clumping of cocci. Agglutination within 5-10 seconds is considered as positive. Some strains of S. aureus may not produce bound coagulase, and such strains  must be identified by tube coagulase test.

 

 
Principle of Tube coagulase test:
The free coagulase secreted by S. aureusreacts with coagulase reacting factor (CRF) present in plasma to form a complex, thrombin. This converts fibrinogen to fibrin resulting in clotting of plasma.
Procedure for Tube coagulase test:

  1. Take three test tubes and label them as “test”, “negative control” and “positive control”.
  2. Fill each test tube with 1 ml of 1:6 dilution of rabbit or human plasma in normal saline.
  3. Add 0.1 ml of overnight broth culture to the tube labeled test. Also add 0.1 ml of overnight broth culture of known S. aureus to the tube labeled positive control and 0.1 ml of sterile broth to the tube labeled negative control.
  4. Incubate all the tubes at 37oC and observe up to four hours.

 

Positive result is indicated by clotting of the plasma, which remains in place even after inverting the tube. If the test remains negative until four hours at 37oC, leave the tube at room temperature for overnight incubation. Some strains on continued incubation produce fibrinolysin that lyses the clot.

Slide coagulase test is useful as a screening test while tube coagulase test is useful in confirmation of coagulase test . Not all strains of S. aureus produce coagulase, such rare strains are identified by thermonuclease test. Some species of Staphylococci such as S. lugdunensis and S. schleiferi  are slide coagulase positive while S. hyicus and S. intermedius are tube coagulase positive.

Note: Citrated plasma should not be used as some citrate utilizing bacteria (Pseudomonas) may utilize citrate to give false positive result.

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