Starch hydrolysis test
Objective:
- to test whether the organism produces the starch hydrolyzing enzyme amylase or not
Introduction:
The main aim of this test is to detect the ability of organism to produce enzyme amylase. Starch is a polysaccharide composed of monomers unit α-D-glucose. These monomers units are arranged in repeated fashion giving two types of polymers, ie. Amylose and amylopectin.
Amylose is the linear polymer of α-D-glucose linked by α (1,4) glycosidic bond. Amylopectin is linear as well as branched polymer of α-D-glucose linked by α (1,4) glycosidic bond at linear point and α(1,6) glycosidic bond at branch point.
The enzyme amylase is an extra cellular enzyme produced by microorganism which hydrolyses the starch breaking α (1,4) glycosidic bond to produce dextrin, maltose and glucose molecules.
Starch when treated with iodine solution, it gives blue color complex. The iodine intercalates in the helical portion of starch molecule and produces blue color complex. When starch is hydrolyzed by amylase produce by microorganism, adding iodine does not change color of the media as there is no starch available for reaction. This indicates positive hydrolysis test. Usually soil bacteria gives positive hydrolysis test whereas members of Enterobacteriaceae gives negative starch hydrolysis test.
Requirements:
- Media: 1% starch agar media
- Gram’s iodine solution
- Culture: 24 hours culture of Bacillus spp and E. coli
Procedures:
- prepared agar media with 1% starch.
- Inoculate the starch agar plate with the given organism ( E. coli and Bacillus in separate plate) using inoculating loop making small central line or streak in the plate.
- Incubate the plates at 37 °C for 24 hours.
- Flood the plates with gram’s iodine solution
- Observe the plates after few minutes. A clear zone around the inoculated area indicates starch hydrolysis; positive test
