Biochemical tests

COAGULASE TEST

Written by MicroDok

Coagulase test test is used to distinguish pathogenic Staphylococcus aureus (which is coagulase positive) from nonpathogenic strains of S. aureus (which is coagulase negative). Coagulase is an enzyme that 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 any of the following two ways:

Slide method

  1. Use pure cultures from solid culture media (preferably BA) 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.
  8. The other portion of emulsified culture without plasma remains the same and serves as the negative control of the test.

Tube method

  1. Wash three small test tubes thoroughly.
  2. Label each of the tubes as tube A (test culture tube), tube B (positive control), and tube C (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 aureus culture to tube B (positive control tube).
  6. Add 0.5 ml of sterile broth to tube C (negative control tube).
  7. Incubate all tubes at 37oC after proper mixing.
  8. Observe the tubes hourly for the presence of clotting. It is vital to tilt or slant the tubes when looking out for clotting.
  9. Presence of coagulation within 1-4 hrs indicates a coagulase positive test.

Slide method of coagulase test

Tube method of coagulase test

REFERENCES

Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. 2nd Cambridge University Press, UK. Pp. 178-187.

Willey J.M, Sherwood L.M and Woolverton C.J (2008). Harley and Klein’s Microbiology. 7th ed. McGraw-Hill Higher Education, 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.

World Health Organization (1993). Laboratory Biosafety Manual, 2nd edn. Geneva: WHO.

World Health Organization (2003). Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens.  WHO/EMC/97.3. Geneva: WHO.

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MicroDok

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