Bacteria that are gram negative, aerobic or facultatively anaerobic, non-endospore forming, rod-shaped bacteria that produce gas from lactose at 35 degrees celcius (coliforms) can be differentiated by the production or lack of production of tryptophanase. This microbial enzyme hydrolyzes tryptophan into ammonia, pyruvic acid, and indole. Once an unknown bacterium is isolated and diluted in liquified agar medium, indole production can be tested. Using aseptic technique, an inocculating loop is used to transfer a loop full of bacteria to a test tube of 1% tryptone, a medium with high amounts of tryptophan. After incubation at 35 degrees celcius for 48 hours, a dropperful of Kovacs' reagent is added to the tryptone culture. This acid solution of para-dimethylaminobenzaldehyde in alcohol, in response to indole produced from the hydrolysis of tryptophan, creates a red layer that floats on the surface of the medium. This indicates an indole (+) test. If a red layer is not formed, the bacteria do not produce the enzyme tryptophanase and are thus unable to hydrolyze tryptophan into indole.