Introduction Identifying an unknown microorganism is useful to many aspects of life.

Knowing the identity of various organisms is valuable in determining and treating diseases, making food, and antibiotics. The purpose of this laboratory experiment is to identify an unknown bacterium through performing a series of biochemical tests. A variety of methods and tests that have been learned and done in the microbiology laboratory were applied to the unknown microorganism in order to identify it. Materials and Methods The laboratory instructor handed out an unknown bacterium labeled #4. Many procedures that were learned in the laboratory have been applied to this unknown in order to identify it. Tests from the laboratory manual, Microbiology: Laboratory Theory and Application, were utilized in the identification of the unknown. The unknown mixed culture was first inoculated onto a nutrient agar plate through use of the quadrant streak method. The plate was then incubated for 24 hours at a temperature of 37C. After incubation, isolated colonies were present in the nutrient agar plate. The Gram stain test was performed on the isolated colonies, in which pink rods were observed under a microscope. Therefore, it was concluded that the bacteria was gram-negative. The gram-negative bacteria was then subcultured onto a new nutrient agar plate with a sterile loop, in which the plate was then incubated overnight again at 37C. The growth on the subculture plate was then used to perform a total of 5 biochemical tests in order to identify the unknown. Not all were needed, but were performed for accuracy. The first test performed was the Phenol Red Broth test, which is a differential test medium used to determine if an organism is capable of acid production from fermentation (Leboffe). Three different mediums of glucose,

sucrose, and lactose were inoculated with the unknown test organism and incubated at 37C. Two days later, the broths were observed to be yellow and there were bubbles in the tubes of each medium. As a result, the unknown bacterium was capable of fermentation with acid and gas end products. The next test performed was the Methyl Red test to detect whether or not the organism was capable of performing mixed acid fermentation (Leboffe). Following incubation of a sample of the pure culture in the broth, 3 drops of Methyl Red reagent were added. The broth turned red, resulting in mixed acid fermentation. The Citrate test was performed next to determine whether the organism was able to use citrate as its main source of carbon (Leboffe). The agar slant was streaked with the unknown test organism with a sterile loop and incubated for 48 hours. No color change was observed, which showed that the unknown did not utilize citrate. By then, the unknown could have been identified; however more tests were performed for better accuracy. The SulfurIndole-Motility (SIM) test was then performed next to determine whether the unknown was able to produce indole, reduce sulfur, or whether or not it was motile (Leboffe). The SIM tube was stab-inoculated with a sterile loop and incubated at 37C. It was observed that the tube contained growth radiating from the stab line, which confirmed motility. There was no sulfur reduction because there was no black in the medium. Kovac's reagent was then added to the medium, in which a red alcohol layer could be observed. This showed that tryptophan was broken down into indole and pyruvate, a positive test for indole production. The final test performed was the Urea Hydrolysis test to determine urease activity of the unknown. The broth was inoculated with the test organism and incubated for 24 hours. It was confirmed that the organism tested negative for urea hydrolysis when the broth was observed to be orange.

Results Table 1 includes all the biochemical tests performed on the unknown, their purpose, observations, and test results. Although six tests were used in order to determine the identity of the unknown bacterium, four are shown in the flowchart. This is because not all of the tests were needed, but were used for better accuracy in determining the identity of the unknown organism. Table 1: Biochemical Test Results Test Purpose Gram Stain To determine gram reaction Phenol Red To determine Glucose/Sucrose/Lactose fermentation Broth Methyl Red To determine mixed acid fermentation Citrate To determine citrate utilization Urea To determine urea hydrolysis Sulfide-Indole-Motility (SIM) To determine motility, sulfur or indole production Observations Pink rods Yellow broth, bubble in tube Red No color change Orange broth Results Gram negative Fermentation with acid/gas end products (+) Mixed acid fermentation (-) No growth (-) No urea hydrolysis, organism does not produce urease or cannot live in broth (+) Motile. (-) Sulfur is not reduced. (+) Tryptophan is broken down into indole & pyruvate.

Radiating growth. No black in medium. Red in alcohol layer of Kovacs reagent

Flowchart Unknown #4 Gram Stain Test

Gram negative Rod Phenol Red glucose/lactose/sucrose Test (+) Citrobacter fruendii Enterobacter aerogenes Escherichia coli Klebsiella pneumoniae (-) Proteus mirabilis Proteus vulgaris

Methyl Red Test (-) Enterobacter aerogenes Klebsiella pneumoniae (+) Citrobacter fruendii Escherichia coli

Citrate Test (+) Citrobacter fruendii (-) Escherichia coli

Discussion/Conclusion After performing all the various biochemical tests on the unknown test organism, unknown #4 was found to be Escherichia coli. This gram-negative organism is capable of fermentation with acid and gas end products, as it tested positive in both the glucose and sucrose broth as well as the lactose broth. E. coli is also capable of mixed acid fermentation,

which was proven when it tested positive during the Methyl Red test. When the Citrate test was performed, the bacterium tested negative as citrate is not utilized by E. coli. The SIM test proved that the unknown organism was surely E. coli when it confirmed that sulfur was not reduced, indole was produced, and the organism was motile. Lastly, the Urea Hydrolysis test confirmed that no urea hydrolysis occurred because the organism does not produce urease or cannot live in the broth. All of these results matched the characteristics of this bacterium and were consistent with the data, which is why it is safe to assume that unknown #4 is E. coli. E. coli is a gram-negative, rod-shaped bacterium member of the Enterobacteriaceac family and can generally be found in the human and animal gut intestines. It is typically harmless, but can also cause disease and serious food poisoning in its host. E. coli is extensively studied and serves as a model organism for prokaryotes. References Leboffe, Michael J., and Burton E. Pierce. Microbiology: Laboratory Theory and Application. Englewood, CO: Morton Pub. 2008. Print.