3 Tests To Identify Escherichia Coli

1443 words - 6 pages

In order to identify an unknown bacterium a variety of tests can be performed. The unknown bacterium that underwent a few of these tests was determined to be Escherichia coli. A Gram stain, citrate utilization test using Simmons citrate agar, and a urease detection test with phenol red were performed on the assigned bacterium. The unknown bacterium was determined to be E. coli because the tests concluded that the specimen was Gram-negative, bacilli, citrate utilization negative, and urease production negative. It is concluded that E. coli cannot utilize citrate as its sole carbon source and it cannot convert urea into ammonia and carbon dioxide.

It is important in microbiology to be able to identify an unknown microorganism. Many tests that characterize functions of bacteria have been developed to accomplish this task. The unknown bacterium that was tested was discovered to be Escherichia coli. E. coli is a Gram-negative, bacillus bacterium. Gram-negative means there is an outer membrane surrounding the thin peptidoglycan layer of the cell. Bacillus means that the bacterium is rod-shaped. The E. coli was identified as Gram-negative and bacilli from performing a Gram stain. A Gram stain uses crystal violet for the primary dye and safranin as a counter stain (1). Gram-positive bacteria have a very thick peptidoglycan layer in their cell wall. This thick cell wall will stay stained purple by the crystal violet-iodine complex, and Gram-negative bacteria will be decolorized and stained pink from the safranin counter stain (1). The peptidoglycan layer’s thickness determines whether it is stained pink or purple.
To test an organism’s ability to use citrate as a source of carbon, a citrate utilization test is. This test is done by the use of Simmons citrate agar. A transport protein enzyme, citrate permease, is required to utilize citrate; an organism that does not produce citrate permease cannot utilize citrate as a carbon source. Sodium citrate is the only carbon source the medium contains, therefore if the organism cannot utilize citrate as a carbon source, it cannot convert the nitrogen source, ammonium dihydrogen phosphate, to ammonium and ammonium hydroxide (4). If the organism can use citrate as a carbon source, the ammonium compounds produced change the pH of the agar from neutral to alkaline, thus changing the color of the medium from green to deep blue. The deep blue color is produced by the pH indicator bromthymol blue included in the medium. Note that growth on the agar alone not accompanied by the change in color, does not indicate a positive test for citrate utilization because there is no evidence of the conversion of the nitrogen source to ammonium compounds (4).
Urease is an enzyme produced by some organisms that degrades urea to ammonia and carbon dioxide. To test an organism’s ability to produce the enzyme urease, a urease detection test is performed (2). The urease broth is a differential medium that contains urea,...

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