Starch Hydrolysis Test: Principle, Reagent, Protocol, and Application

Starch Hydrolysis Test is a widely used test for the characterization of bacteria using the enzymes alpha-amylase and oligo-1,6-glucosidase, starch (amylose and amylopectin) which can be hydrolyzed by bacteria, and this ability is tested for using the starch hydrolysis test. Clostridium and Bacillus species are frequently used to distinguish one another.

starch hydrolysis

What actually starch hydrolysis is all about?

Starch Hydrolysis in literal meaning is defined as from glucose monomer units, starch is a condensation polymer. These substantial starch molecules decompose into more manageable sugar molecules when they interact with water. The starch has allegedly been hydrolyzed. In order to determine whether starch has been hydrolyzed, we look for the little sugar molecules that are created during the process.

Principle of starch hydrolysis

The test microorganisms are cultivated on agar plates containing starch for the starch hydrolysis test. If the bacteria can hydrolyze starch, they do so in the medium, especially where they are growing, while the remaining portions of the plate still contain non-hydrolyzed starch.

Which types of bacteria shows this test?

Starch Hydrolysis Test

Positive starch hydrolysis

After adding iodine solution, the area surrounding the line of growth becomes transparent, signifying that the organism has hydrolyzed starch.

Negative starch hydrolysis

Depending on the iodine concentration, the medium can be colored blue, purple, or black.

Protocol of starch hydrolysis

The procedure for the starch hydrolysis are discussed below:

  1. A few colonies are picked from the pure culture plate using a sterile brush or sterile loop. And is spread in a starch plate across the width of the plate in the shape of a line.
  2. A single agar plate can be used to test multiple cultures, with each culture represented by a line. Alternatively, the plate can be divided into four quadrants (like a pie plate) for this purpose.
  3. The plate is Incubate for 48 hours at 37 °C.
  4. 2-3 drops of a 10% iodine solution is directly applied to the colony edges. And Wait 10-15 minutes, then note the outcomes.
  5. The medium will get dark if the test positive (“+”). The amylase-hydrolyzed starch, however, will look clear in the vicinity of solitary colonies.
  6. A negative test result (“-“) will result in the medium being darkly colored all the way to the edge of any isolated colonies.

Example of Starch hydrolysis test

Starch hydrolysis test positive bacteriaStarch hydrolysis (-ve)
Bacillus subtilisStreptococcus agalactiae
Bacillus cereusStaphylococcus epidermidis
Bacillus megateriumEscherichia coli


http://Bird, R., and R. H. Hopkins. 1954

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