Starch Hydrolysis Test: Principle, Procedure and Results

Starch, the most important source of carbohydrate for human, is a polysaccharide mixture of two polymers, amylose and amylopectin, the latter being predominant. Amylose is a linear polysaccharide of several thousand α-D-glucose linked by 1,4-α-glycosidic bonds. Amylopectin is a branched-chain polysaccharide composed of glucose units linked primarily by α-1,4-glycosidic bonds but with occasional α-1,6-glycosidic bonds, which are responsible for the branching.  

Principle            

Starch molecules are too large to enter the bacterial cell, so only bacteria that secrete exoenzymes (α -amylase and oligo-1,6-glucosidase) are able to hydrolyze starch into subunits (dextrin, maltose, or glucose).  These molecules are readily transported into the bacterial cell to be used in metabolism.

In starch hydrolysis test (also known as amylase test), we use starch agar, which is a differential nutritive medium with starch added.  The test organisms are inoculated onto a starch plate and incubated at 30°C until growth is seen (i.e. up to 48 hours).  The petri plate is then flooded with an iodine solution. Depending on the concentration of the iodine used, iodine turns blue, purple or black in the presence of starch. When bacteria capable of producing α-amylase and oligo-1,6-glucosidase are grown on starch agar, they secrete these enzymes into the surrounding areas. Clearing around the bacterial growth indicates that the organism has hydrolyzed starch.

Test Objective: To determine if the organism is capable of breaking down starch into maltose through the activity of the extra-cellular α-amylase enzyme.

Uses: Starch hydrolysis test is used to differentiate members of various genera including Bacillus, Clostridium, Corynebacterium, Fusobacterium, Enterococcus, Pseudomonas, and Streptococcus. These genera have both amylase-positive and amylase-negative species.

Test procedure

1. Pick a few colonies of test organism using a sterile swab or loop.
2. Streak a starch plate in the form of a line across the width of the plate. Several cultures can be tested on a single agar plate, each represented by a line or the plate may be divided into four quadrants for this purpose.
3. Incubate plate at 37 °C for 48 hours.
4. Add 2-3 drops of 10% iodine solution directly onto the edge of colonies. Wait 10-15 minutes and record the results.

Interpretation:

1. Positive test (“+”): Characteristic purple-black color will appear in the medium. However, a clear halo will appear around the colonies of amylase positive species.
2. Negative test (“-“): Characteristic purple-black color will appear in the medium, right up to the edge of isolated colonies of amylase negative species.

Starch hydrolysis test results of selected organisms

References and further reading

1. Archana Lal, Naowarat Cheeptham. 2012. Starch agar protocol.
2. Madigan MT, Martinko JM, Stahl DA, Clark DP. 2012. Brock biology of microorganisms, 13th ed. Benjamin Cummings, San Francisco, CA.