3 tests to identify Escherichia coli

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.

Introduction

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 ...

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...n dioxide. The ammonia would cause the broth’s pH to be alkaline, which would turn the broth bright pink (3). Since the broth stayed the same orange/yellow color, we know that the urea in the broth was not degraded to ammonia, meaning that the organism does not produce the enzyme urease.

Literature Cited

1) Bartholomew, J.W., and T. Mittwer. 1952. The Gram stain. Microbiology and molecular biology reviews. 16: 1-29.

2) Krajewska, B. 2009. Ureases I. Functional, catalytic and kinetic properties: A review. Journal of Molecular Catalysis B: Enzymatic. 59: 9-21.

3) Roberts, G.D., C.D. Horstmeier, G.A. Land and J.H. Foxworth. 1978. Rapid urea broth test for yeasts. Journal of Clinical Microbiology. 7: 584-588.

4) Vaughn, R.H., J.T. Osborne, G.T. Wedding and J. Tabachnick. 1950. The utilization of citrate by Escherichia coli. Journal of Bacteriology. 60: 119-127.