One of the biochemical tests that microbiology students may use to identify a bacterial species is the starch hydrolysis test. In this test, starch agar is inoculated with the species in question. After incubation at an appropriate temperature, iodine is added to the surface of the agar. Iodine turns blue-black in the presence of starch. Absence of the blue-black color indicates that starch is no longer present in the medium. Bacteria which show a clear zone around the growth produce the exoenzyme amylase which cleaves the starch into di- and monosaccharides (1). These simpler sugars can then be transported into the cell to be catabolized. Bacillus species are known to produce the exoenzyme, amylase (2).
In this demonstration, the action of two bacterial species, Bacillus subtilis and Escherichia coli, is compared on starch agar. After inoculation in the shape of the corresponding bacterial name initials, EC for E. coli and BS for B. subtilis, the plates were incubated for 24 hours at 37°C. Iodine, which changes color from a yellow-brown to blue-black in the presence of starch, was applied to the agar surface and allowed to stand for 10 minutes. The E. coli starch agar plate turned completely blue-black which indicated that all the starch was still present (Fig. 2.). This is a negative reaction for the starch hydrolysis test. The B. subtilis produced a clear zone around the growth which is a positive reaction (Fig. 1.) and indicates that the starch has been removed in the area around the bacterial inoculum . B. subtilis produced the enzyme amylase which hydrolyzed starch in the agar. If the species produces and releases amylase, starch hydrolysis in the agar should occur.