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Systematic Methodology Used in the Identification and

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Systematic Methodology used in the Identification and
Isolation of Escherichia coli and Bacillus cereus from an Unknown Culture.

By: Richard Martinez

MCB-2010L Microbiology Lab
Dr. James Rogers

Unknown bacteria were determined to be Escherichia coli and Bacillus cereus due to their morphological, physiological and metabolic properties.

I. Abstract In a laboratory setting, it often becomes necessary to identify an unknown organism. In this experiment, researchers classified some unidentified bacteria based on their physical structure, colony morphology, optimal conditions and metabolic properties. A Gram stain using crystal violet, iodine, and safranin and a simple stain using methylene blue characterized the organism’s cell walls. Cultural behaviors were classified by inoculating the organisms onto nutrient agar and incubating them at 30° and 37° C for 48 hours, and observing their behaviors, as well as using Mannitol and phenol red media for fermentation and acid production. Optimal growth temperatures were determined by incubating nutrient agar plates of the organisms at 30° C and 37° C. The metabolic profile was created by inoculating the bacterium into broths containing lactose, mannitol, and citrate and incubating the tubes at 30° and 37° C for 48 hours, then observing them for color change. The stains revealed that the bacteria were both Gram-negative and Gram-positive bacilli.
Organism “A” was shown to not grow on MacConkey agar and to have limited growth on EMB agar. It was also a non-lactose, non mannitol fermenting and grew best at a temperature of 30° C, making it a mesophilic non-coliform. The oxidation and fermentation tests showed that the organism is a facultative aerobe that does not ferment sugars, produces acid below a pH of 4.4 and is able to metabolize citrate.
Organism “B” was shown to be a lactose fermenter that...

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