Starch hydrolysis on Mueller-Hinton agar. Light
halos around
Bacillus subtilis colonies after adding
Lugol solution
Starch Hydrolysis (Amylase Test)
DESCRIPTION
Some bacteria are able to hydrolyze starch from the medium by producing alpha-
amylase. The ability to degrade starch is used as a criterion for bacterial identification,
mostly Gram-positives like Corynebacterium, Clostridium or  Bacillus.

Medium composition: nutritive agar supplemented with soluble starch 2%.
Admin note: Mueller-Hinton agar can be used to determine starch hydrolysis as it
contains 1.5% starch in composition, but the reaction may be less evident than the
recommended medium.

PROCEDURE:
Inoculate a starch agar plate with fresh culture and incubate 24 hours at 37 ˚C, 24-48
hours. After incubation, flood the plate with Gram’s iodine / Lugol solution (iodine 20g
in 100 ml  50˚ethanol).
Wait for 30 seconds then remove the excess iodine.

When iodine is added to starch, it turns dark blue. If starch has been hydrolyzed, then
it is not available to react with the iodine and the area around the bacterial growth is
clear.
Positive result: clear zone around colonies.
Negative result: dark blue to purple medium.

NOTE
Adding iodine / Lugol solution to agar plate will result in killing the culture.

QUALITY CONTROL
Bacillus subtilis ATCC 6633 - positive control
Escherichia coli ATCC 8739 - negative control

REFERENCES:
1. H. Raducanescu, V.Bica-Popii,1986. Bacteriologie veterinara, Ed. Ceres, Bucuresti.
2. Murray, P.R., Baron, E. J., Jorgensen, J.J., Pfaller, M.A., and Yolken, R.H. Manual of
Clinical Microbiology, 8th ed. ASM Press: Washington, DC, 2003.
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