Biochemical tests

AMYLASE (STARCH HYDROLYSIS) TEST

Written by MicroDok

Amylase (Starch hydrolysis) test is used to identify bacteria that hydrolyze starch (including amylopectin and amylose) with the help of the enzyme amylase. Amylase is an enzyme that hydrolyzes starch into maltose, glucose, and dextrin’s.  Some bacterial isolates including Bacillus and Clostridium have the ability to produce α-amylase and other enzymes that degrade starch molecules into smaller and simple sugars (e.g. glucose), and this serves as the basis for the identification of such microorganisms in the laboratory.

Procedure

  1. Perform this test using pure cultures of the test isolate.
  2. Prepare nutrient agar plates according to manufacturer’s instructions.
  3. Pour thin layers of the prepared agar on Petri dish plate(s) and allow to set.
  4. Prepare another smaller quantity of nutrient agar base modified with 1 % soluble starch and sterilize by autoclaving. This is the starch agar medium.
  5. Pour the starch agar medium on the already poured thin layer of nutrient agar earlier prepared to make a very light upper layer. In some cases, iodine is incorporated in the agar medium during preparation.
  6. Allow plate(s) to set and cool.
  7. Inoculate the test bacteria by a single streak or inoculate isolate at the center of the Petri dish.
  8. Incubate plates at 37oC for 18-24 hrs.
  9. Flood plate(s) with iodine and drain off excess iodine leftovers. Iodine is known to react with starch to give a blue-black color. Observe the plate(s) for a change in colouration. Iodine reacts with the starch in the media to form a dark-brown or yellow-halo colour that results in a clear zone around the bacterial growth on the agar plate. This is indicative of a positive starch hydrolysis test result. Absence of this colouration shows a negative result.

Illustration of starch hydrolysis test. Plate A shows a positive starch hydrolysis test result, as indicated by a non-blue halo surrounding the bacterial growth. Plate shows a negative test result, as indicated by the absence of a non-blue halo surrounding.

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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