Starch Hydrolysis Test Introduction

Biochemical tests are generally used to determine the ability of the microorganism to be stable in the enzymatic field.

Starch hydrolysis test is one such test that helps us to identify the species of bacteria that has ability to hydrolyze starch which is present in the form of amylose and amylopectin, with the help of the enzyme a-amylase and the oligo-1,6-glucosidase.

Starch Hydrolysis Test is often performed to differentiate the species from the specific genera’s like Clostridium and the Bacillus. As the amylose and the amylopectin molecules are large, they cannot be passed through the cell wall of the bacterium easily.

As the bacteria use these enzymes as a source of carbon, which is very important for them, so these bacteria produce secretes a a-amylase and the oligo – 1, 6 glucosidases in the extracellular spaces, which helps to break the starch molecules into the smaller subunits of glucose which can enter directly into the glycolytic pathway.

Thus, during hydrolysis of the starch test, iodine reacts with the starch and results in the formation the brown color. And it also results in the clear zone around the growth of bacteria. Bacillus subtills is positive starch hydrolysis.

Starch Hydrolysis Test

Starch is one of the complex polysaccharides which can be seen, found abundantly in plants and it is commonly deposited in the form of large granules in the cytoplasm of the cell.

Starch consists of two major components namely amylose and amylopectin which are discussed above. In that amylose contain the units of D-glucose which is linked in a linear manner by the alpha – 1, 4 linkages, having two non-reducing ends and rhe reducing ends.

Amylopectin is one of the branched polysaccharides. These type of molecules contains a shorter glucose chain unit that are linked by an alpha-1, 4 and are joined together each other by an alpha-1, 6 linkages.

Here, the major component of the starch is hydrolyzed by an a- amylase, that is present in some kinds of bacteria as in fungi. This contains an ability to degrade the starch and it is used as a criterion for determining the amylase for producing a microbe.

Starch Hydrolysis Test Objective

 The main aim of the test is to determine the ability of an organism to hydrolyze the starch.

 This test also helps to differentiate the organisms based on their alpha-amylase enzyme activity.

Starch Hydrolysis Test Principle

Many kinds of bacteria produce an extracellular enzyme which are used to catalyze the chemical reactions that takes place outside the cell. At this time, nutrient sources like starch which are too large for the cell membrane to absorb, are breakdown into smaller molecules and they are transported into the cell through the process of diffusion.

However, in starch hydrolysis test, the test bacteria are grown on the agar plates which contains starch. Here, if the bacteria have its own ability to hydrolyze starch, in such way the rest of the medium that does not contains starch are kept non-hydrolyzed.

If there is no color change in the medium, when the hydrolysis process takes place iodine solution is being added here as an indicator to the agar plate after incubation. Whereas, on the other hand the non-hydrolyzed starch forms a dark blue color on adding iodine.

At the same time, a transparent clear zone can be seen, that is formed around the colonies which hydrolyses the starch, where the rest of the plate remains dark blue in color due to the reaction of iodine with starch.

Starch Hydrolysis Test Reagents

Media: The significant media used here for starch hydrolysis test is starch agar, which is one of the simple nutritive medium. Whereas the beef extract and the pancreatic digest of the gelatin provides the medium a rich source of nitrogen, vitamins, carbon, and amino acids. Agar acts as a solidifying agent, where as starch acts as the carbohydrate.

Composition of Media: Here about 5 grams of the peptic digest of the animal tissue is added per liter, sodium chloride of about 5 grams and yeast extract of 1.5-gram, Beef extract of about 1.5 gram, all each per liter are added along with the soluble 2 gram of agar in a pH of about 7.4.

Starch Hydrolysis Test Procedure

Initially, sterile technique is used to make a single line of streak in the inoculated organism that is present in the center of the labelled plate.

Then the inoculated plate is incubated at a temperature of about 37ºC for about 48 hours.

After incubation, the surface of the plates is flooded with the iodine solution using a dropper for about 30 seconds.

Then the excess iodine is poured off, then the clear zone is seen around the line of the bacterial growth.

Starch Hydrolysis Test Results

Positive Starch Hydrolysis Test: In positive results, a clear zone is formed around the area of the growth of the inoculated organisms after addition of iodine solution.

Negative Starch Hydrolysis Test: In case of negative result, a blue black or purple color of the medium is observed in the medium.