STARCH HYDROLYSIS TEST

STARCH HYDROLYSIS TEST

AIM

            To differentiate bacteria based on their ability to hydrolyze starch with the enzyme α –amylase.

PRINCIPLE      

            Starch is a complex carbohydrate (polysaccharide), composed of two constituents –amylose, a straight-chain polymer of 200-300 glucose units, and amylopectin, a larger branched polymer groups. The α-D-glucose molecules in both amylose and amylopectin are bonded by 1,4-α-glycosidic (acetal) linkages. The two forms differ in that the amylopectin contains polysaccharide side chains connected to approximately every 30th glucose in the main chain. These side chains are identical to the main chain except that the number 1 carbon of the first glucose in the side chain is bonded to carbon number 6 of the main chain glucose. The bond is, therefore, a 1,6-α-glycosidic linkage. Starch is too large to pass through the bacterial cell membrane. Therefore, to be of metabolic value to the bacteria it must first be split into smaller fragments or individual glucose molecules. Organisms that produce and secrete the extracellular enzymes α-amylase and oligo-1,6-glucosidase are able to hydrolyze starch by breaking the glycosidic linkages between the sugar subunits into maltose, a disaccharide and some monosaccharides such as glucose.  These disaccharides and monosaccharides enter into the cytoplasm of the bacterial cell through a semi-permeable membrane and thereby used by the endoenzymes.

            The enzyme amylase is secreted out of the cells (an exoenzyme) into the surrounding media, catalyzing the breakdown of starch into smaller sugars which can then be absorbed by the cells for use.  Amylase production is known in some bacteria while well known in the case of fungi. Starch hydrolysis test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes a-amylase and oligo-1,6-glucosidase. In Starch hydrolysis test, Starch agar (Differential medium) was inoculated and incubated overnight at 37 °C. After incubation, Iodine was added to the surface of the Starch agar plate. Characteristic purple-black color will appear in the medium. However, a clear halo zone will appear around the colonies of amylase positive species. Starch hydrolysis test is used to differentiate members of various genera including Bacillus (Bacillus subtilis, Bacillus cereus and Bacillus megaterium are positive for Starch hydrolysis), Bacteroides, Clostridium, Corynebacterium, Fusobacterium and Enterococcus. These genera have both amylase-positive and amylase-negative species.

MATERIALS REQUIRED

1. Test bacteria
2. Starch agar plate
3. 10 % Iodine
4. Inoculation loop
5. Incubator

PROCEDURE

a)     Pick a few colonies of test organism using a sterile swab or Inoculation loop.

b)     Make a single line streak of the unknown bacterium across the Starch agar plate.

c)     Incubate the inoculated Starch agar plate overnight at 37 °C.

d)     After incubation, add 2-3 drops of 10 % Iodine solution directly onto the edge of colonies.

e)     Wait 10-15 minutes and record the results.

OBSERVATION AND RESULTS

·       Positive test - Characteristic purple-black color will appear in the medium. However, a clear halo zone will appear around the colonies of amylase positive species.

·    Negative test - Characteristic purple-black color will appear in the medium, right up to the edge of isolated colonies of amylase negative species.

(Left – Starch hydrolysis positive; Right – Starch hydrolysis negative)

Figure – 1: Starch hydrolysis test