Isolation of amylase producing microorganism

Many bacteria produce extracellular enzymes used to catalyse chemical reactions outside of the cell. In this manner, nutrient sources, such as starch, that are too large to be absorbed through the cell membrane can be broken down into smaller molecules and transported into the cell via diffusion. In the starch hydrolysis test, the test bacteria are grown on agar plates containing starch. If the bacteria have the ability to hydrolyse starch, it does so in the medium, particularly in the areas surrounding their growth while the rest of the area of the plate still contain non hydrolysed starch. Since no colour change occurs in the medium when organisms hydrolyse starch, iodine solution is added as an indicator to the plate after incubation. While the non hydrolysed starch forms dark blue colour with iodine, its hydrolysed end products do not acquire such dark blue colour  with iodine. 

Screening for Amylase Activity (Starch Iodine Test):

 Starch is a complex polysaccharide found abundantly in plants and usually deposited in the form of large granules in the cytoplasm of the cell. 

 Starch consists of 2 components—amylose and amylopectin, which are present in various amounts.

 The amylose consists of D-glucose units linked in a linear fashion by α-1,4 linkages. It has 2 non-reducing ends and a reducing end.

 Amylopectin is a branched polysaccharide. In these molecules, shorter chains of glucose units linked by α-1,4 are also joined to each other by α-1,6 linkages.

 The major component of starch can be hydrolysed by a-amylase, which is present in some bacteria while well known in case of fungi.

 The ability to degrade starch is used as a criterion for the determination of amylase production by a microbe. 

 Isolated colonies were picked up from each plate containing pure culture and streaked in straight lines in starch agar plates with starch as the only carbon source.

 After incubation at 37ºC for 24-48 hrs., individual plates were flooded with Gram’s iodine (Gram’s iodine- 250 mg iodine crystals added to 2.5gm potassium iodide solution, and 125ml of water, stored at room temperature) to produce a deep blue coloured starch-iodine complex.

 In the zone of degradation no blue colour forms, which is the basis of the detection and screening of an amylolytic strain. 

 The colonies which were showing zone of clearance in starch agar plates were maintained on to nutrient agar slants 

 Consequently, transparent clear zones are formed around the colonies that hydrolyse starch while the rest of the plate show a dark blue coloration as iodine forms the coloured complex with starch.