Starch Hydrolysis Test Procedure

In this starch hydrolysis test procedure post we have briefly explained about starch hydrolysis test principle, objectives, requirements, procedure, uses and limitations.

Starch Hydrolysis Test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes α-amylase and oligo-1,6-glucosidase. Often used to differentiate species from the genera Clostridium and Bacillus.

Because of the large size of amylose and amylopectin molecules, these organisms cannot pass through the bacterial cell wall. In order to use these starches as a carbon source, bacteria must secrete α-amylase and oligo-1,6-glucosidase into the extracellular space.

These enzymes break the starch molecules into smaller glucose subunits which can then enter directly into the glycolytic pathway. In order to interpret the results of the starch hydrolysis test, iodine must be added to the agar. The iodine reacts with the starch to form a dark brown color. Thus, hydrolysis of the starch will create a clear zone around the bacterial growth.

Principle

Only bacteria that secrete exoenzymes (α-amylase and oligo-1,6-glucosidase) can hydrolyze starch into subunits because starch molecules are too big to enter the bacterial cell (dextrin, maltose, or glucose). These compounds are easily carried into the bacterial cell, where they are utilised in metabolism.

We employ starch agar, which is a differential nutritive media, in the starch hydrolysis test (also known as amylase test). The test organisms are injected onto a starch plate and kept at 30°C until they develop (i.e. up to 48 hours). The iodine solution is then poured into the Petri plate.

The amylose and iodine react together to generate a blue tint if there is no enzyme present, and hence no hydrolysis. In the presence of starch, iodine turns blue, purple, or black, depending on the proportion of iodine employed.

When bacteria that produce -amylase and oligo-1,6-glucosidase are cultured on starch agar, they secrete enzymes that hydrolyze the starch in the surrounding areas. Because there is no amylose in the medium around the bacterial colony, clearance occurs around it (there is no colour development).

Requirements

Materials

1. Inoculum Loop

2. Incubator at 37⁰C

Chemicals

1. Gram’s iodine

Composition

Starch agar is a basic nutritive media that has been supplemented with starch. Nitrogen, vitamins, carbon, and amino acids are all provided via beef extract and gelatin pancreatic digest. The solidifying agent is agar, while the carbohydrate is starch.

Ingredients gm/L: Meat Extract: 3.000, Peptic digest of animal tissue: 5.000, Starch soluble: 2.000, Agar: 15.000, Final pH (at 25°C) 7.2±0.1.

Preparation

Suspend 25 grams in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 10 minutes. Mix well and pour in sterile Petri plates.

Starch Hydrolysis Test Procedure

1. Using a sterile swab or loop, select a few colonies of the test organism. Make a line across the width of a starch plate with a paintbrush.

2. On a single agar plate, several cultures can be tested, each indicated by a line, or the plate can be divided into four quadrants for this purpose.

3. Incubate the plate for 48 hours at 37 degrees Celsius. Apply 2-3 drops of a 10% iodine solution immediately to the colony’s edge.

4. Wait 10-15 minutes before recording your findings. As the blue hue disappears after the addition of iodine, read the plates right away.

Results

Starch Hydrolysis Test

Starch hydrolysis Test: No clear zone around E-coli, Klebsiella pneumoniae and Staphylococcus aureus were observed indicating negative result.

Positive test: After adding iodine solution, a clear zone surrounding the line of growth indicates that the organism has hydrolyzed starch.

Negative test: The medium is blue, purple, or black in colour (depending on the concentration of iodine).

Applications

1. To distinguish between Bacillus, Clostridium, Corynebacterium, Fusobacterium, Enterococcus, Pseudomonas, and Streptococcus members of distinct genera. There are amylase-positive and amylase-negative species in these genera.

2. To separate Streptococcus bovis (positive) from other viridans group streptococci that are bile-esculin positive, 6.5% NaCl negative, and pyrrolidonyl-β-naphthylamide (PYR) negative.

Limitations

1. Do not use a glucose starch medium since glucose metabolism may interfere with the experiment.

2. The organisms become non-viable once the iodine is applied.

3. Partial hydrolysis results in a red-violet tint, and the test should be repeated after more incubation.

Further Readings

Reference