Decarboxylase Reactions

Identifying members of the Enterobacteriaceae, a group that contains many medically important bacteria, is often difficult. It is especially difficult to separate members of the genera Klebsiella, Enterobacter and Citrobacter. Measuring the ability of these bacteria to decarboxylate certain amino acids has proven a useful taxonomic tool. The amino acids that have proven to be most useful are lysine, arginine and ornithine.

Under anaerobic conditions, specific decarboxylase enzymes are able to remove the carboxyl group from these amino acids and thus change the pH of the media to make it more alkaline. A pH indicator in the media, such as brom cresol purple, can detect this shift and provide an easy way to measure these reactions. To create suitable anaerobic environments the bacteria are grown in broth tubes of the appropriate media, which have been capped after inoculation with sterile mineral oil.

Materials:

1. Overnight cultures of Enterobacter aerogenes, E. coli and Klebsiella pneumoniae.

2. Three tubes each of lysine, arginine and ornithine decarboxylase broth.

3. Sterile mineral oil.

Procedure: Inoculate each culture into each of the three types of decarboxylase media. Add a layer (0.5 – 1 cm) of sterile mineral oil and incubate for 4 days at 37 C. Check daily for a change in color of the medium, yellow is a negative result while various shades of purple indicate a positive result.