COAGULASE TEST

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Coagulase test is used to identify bacteria that produce the enzyme coagulase, and are thus coagulase-positive. It is used to distinguish pathogenic Staphylococcus aureus (which is coagulase positive) from nonpathogenic strains of S. aureus (which is coagulase negative). This test is used to differentiate Staphylococcus aureus (positive) from Coagulase Negative Staphylococcus (CONS). Coagulase is an enzyme produced by S. aureus that converts (soluble) fibrinogen in plasma to (insoluble) fibrin. Staphylococcus aureus produces two forms of coagulase, viz: bound and free coagulase. Coagulase causes blood plasma to clot. The enzyme is a key virulence factor in the pathogenicity of pathogenic S. aureus because the clot formed helps to shield the bacterium from phagocytosis. Free coagulase reacts with a plasma cofactor called coagulase reacting factor (CRF) to produce coagulase-thrombin (a thrombin-like particle) which promotes the change from fibrinogen to fibrin forming a clot called coagulum. Free coagulase (detected by tube coagulase test) and bound coagulase (detected by slide coagulase test) are the two types of coagulase enzymes produced by pathogenic strains of S. aureus. Coagulase test can be performed in two ways viz: slide method (using a clean glass slide) and tube method (using test tubes).

SLIDE METHOD OF COAGULASE TEST

  1. Use pure cultures from solid culture media (preferably blood agar) to perform this test.
  2. Divide a clean glass slide into two parts using a grease pencil.
  3. Place a drop of normal saline or distilled water on each side of the slide.
  4. Pick a colony or speck of the overnight culture using a sterilized inoculating loop.
  5. Emulsify the culture with each of the drops of normal saline to form a homogeneous suspension. This must be separately done for each side of the slide.
  6. Add a drop of plasma (from human or animal origin) to only one of the suspensions on the slide and stir for about 5 secs.
  7. Lookout for clumping which does not re-emulsify. Presence of clumping indicates a positive test (Figure 1).
  8. The other portion of emulsified culture without plasma remains the same and serves as the negative control of the test.

  TUBE METHOD OF COAGULASE TEST

  1. Wash two small test tubes thoroughly.
  2. Label each of the tubes as tube A (test culture tube) and tube B (negative control).
  3. Aseptically dispense 0.5 ml of plasma into each of the three tubes.
  4. Add 0.5 ml of the test broth culture to tube A.
  5. Add 0.5 ml of sterile broth to tube B (negative control tube).
  6. Incubate all tubes at 37oC after proper mixing.
  7. Observe the tubes hourly for the presence of clotting. It is vital to tilt or slant the tubes when looking out for clotting (Figure 1).
  8. Presence of coagulation within 1- 4 hrs indicates a coagulase positive test.
Figure 1. Slide and tube method of coagulase test. A = Slide method of coagulase test. B = Tube method of coagulase test. Photo courtesy: https://www.microbiologyclass.com

Further reading

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23rd edition. McGraw Hill Publishers. USA.

Goldman E and Green L.H (2008). Practical Handbook of Microbiology, Second Edition. CRC Press, Taylor and Francis Group, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.

Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.

Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

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