Objectives of Starch Hydrolysis Test
- To determine the ability of the organism capable of hydrolyzing starch.
- To differentiate organism based on their ability to hydrolyze starch with the enzyme, α- amylase.
- It aids in the differentiation of species from genera Corynebacterium, Clostridium, Bacillus, Bacteroides, Fusobacterium, and members of Enterococcus.
Principle of Starch Hydrolysis Test
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 semipermeable membrane and thereby used by the endoenzymes.
Amylase production is known 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. In the laboratory setting, it is tested by performing starch hydrolysis test or starch test to determine the presence or absence of amylase enzyme. The test utilizes iodine as an indicator. Starch in the presence of iodine produces dark blue colouration of the medium as iodine is trapped in the helical structure of starch and a yellow zone or clear zone around a colony in a blue medium indicates amylolytic activity. The clear zone indicates hydrolysis of starch into monosaccharides which cannot bind the iodine molecule and appear as clear zone around the bacterial growth. Size of the clear zone is directly proportional to the starch hydrolyzing activity of the strain under study.
Procedure of Starch Hydrolysis Test
- Using a sterile technique, make a single streak inoculation of organism to be tested into the centre of labelled plate.
- Incubate the bacterial inoculated plates for 48 hours at 37°C.
- Following incubation, flood the surface of the plates with iodine solution with a dropper for 30 seconds.
- Pour off the excess iodine.
- Examine for the clear zone around the line of bacterial growth .
Result interpretation of Starch Hydrolysis Test
Positive test: a clear zone around the line of growth after addition of iodine solution.
Negative test: dark blue colouration of the medium
Quality control of Starch Hydrolysis Test
Escherichia coli ATCC25922- Negative reaction, no clearing.
Bacillus subtilis ATCC6633- Positive reaction clearing colony around.
- Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen edition. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
- Sigmon J. 2008. The starch hydrolysis test. http://www.asmscience.org/content/education/imagegallery/image.3172
- Aneja K.R. 2003. Experiments in Microbiology, Plant Pathology and Biotechnology, fourth revised edition, New Age International (P) limited, Ansari road, Daryaganj, New Delhi-110002.