Starch Hydrolysis Test - Principle, Procedure, Result, Interpretation, Introduction
Introduction to Starch Hydrolysis Test
The biochemical test starch hydrolysis test is used to determine if an organism is able to hydrolyze starch by producing an exoenzyme called amylase.
The complex carbohydrate starch is made up of a mixture of amylopectin and amylose in the proportion of 1:4. The exoenzyme amylase hydrolyzes starch into maltose ( a disaccharide) and monosaccharides such as glucose. These sugars which are now broken down are transported into the bacterial/fungal cytoplasm through the semipermeable membranes where they are further cleaved by endoenzymes.
Principle of Starch Hydrolysis Test
The absence or presence of starch in the medium is detected by using an iodine solution as an indicator. If the medium contains starch the inclusion complex between iodine and amylose fraction of starch is formed. This is the principle of the Starch Hydrolysis Test.
This complex in turn changes the linear amylase molecule to become coiled into spirals with hydrogen bonds between the hydroxyl group of adjacent loops. The arrangement of iodine molecules in the center of the spinal allows interaction of the electron orbitals of many iodine atoms. As a result, the blue color is developed after changes in light absorption.
In the case of starch being hydrolyzed and no longer present in the medium, the addition of iodine solution does not change the color of the medium and stays clear.
Procedure of Starch Hydrolysis Test
The procedure of the Starch Hydrolysis Test includes:
Take a sterile starch agar and streak the test organism on it following aseptic conditions. (each plate can have a single streak of multiple test organisms)
Incubate the inoculated plate at 35-37°C for 18-24 hours (for bacteria) or at 28°C for 3-4 days (for fungi) in an inverted position.
Following incubation, flood the surface of the media with an iodine solution
Pour off the excess solution
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Fig: Bacillus subtilis before addition of iodine solution (A), after addition of iodine solution (B) - positive reaction as clear zone seen around colony in B (Source: ASM)
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Fig: E. coli before addition of iodine solution (A), after addition of iodine solution (B) - negative reaction as no clear zone seen around colony (Source: ASM)
Result, Interpretation of Starch Hydrolysis Test
The result of the Starch Hydrolysis Test can be interpreted as follows:
Positive
A transparent zone develops around the colonies after the addition of iodine solution while the area without colonies turns dark blue.
Negative
No transparent zone develops around the colonies and the entire media color changes to dark blue.
Starch hydrolyzing organism
A list of Starch hydrolyzing organism include:
Bacillus subtilis
Aspergillus niger
Non-starch hydrolyzing organism
A list of non-starch hydrolyzing organism include:
E.coli