Methods Several tests were conducted in order to isolate the unknown bacteria.

All tests were done using prior techniques from Bensons Microbiological Lab Manual, and methods from Brown1. To conduct tests on the unknown culture, two slants inoculated with the unknown and incubated at 25 C and 37 C to determine optimal growing temperature. An agar was prepared using Method B plate streaking technique featured in Brown1. Afterwards, three agar plates were separated into four separate quadrants for each lab mate. One plate inhibited growth of gram positive cells, the other inhibited growth of gram negative, and the third was the control. Using the normal plate culture, a gram stain test was done using Brown1 techniques. Next, an FTM tube, a nutrient broth tube, a gelatin tube, and a control slant were prepared using the plate culture. Oxidation and fermentation tests were conducted to determine the capabilities of the unknown. Durham tubes were used to determine if the unknown ferments sugars. Three sugar tubes consisting of glucose, lactose and mannitol were tested for fermentation. Next a citrate test was conducted by inoculating the citrate slant with the unknown. This was done to see if the unknown could use citrate as a sole carbon source. Following this test a nitrate broth was used to determine if the unknown could reduce nitrate, indicating nitrate respiration. First vile A+B was added to the Durham tube, and then vile C was added. A red color indicates ammonia or hydroxylamine production. Vile A consisted of sulfanilic acid, vile B consisted of dimethylalpha-napthylamine, and vile C consisted of zinc powder. Next an oxidation test was done to determine if the unknown carries out respiratory metabolism. A plate was divided into four quadrants. One quadrant was for each of the two lab partners and the other two quadrants were inoculated with bacterias SA and AF. The unknown was

swabbed off the plate and a drop of oxidase reagent was placed on it. Next, a catalase test was done to check for production of catalase. A small amount of the bacteria was smeared on a slide then a drop of 3% H2O2 added. Next a Voges Proskauer test (VP) test was done by pipetting the entire culture out of the tube, then using 18 drops of Barrits reagent A and B. Then the tube was shook vigorously and the presence of a red color was observed. Following this test a Methyl red (MR) test was done to test fermentation of glucose. Three drops of pH indicator was added to the MR tube and observed. The next period hydrolytic reactions were used to test for hydrolytic and degradative enzymes. A plate was divided into four quadrants for each lab mate. Each was inoculated with the unknown bacteria, and a ring of Bacillus subtillis was placed in the middle as a control. Four tubes each had a separate test for hydrolysis. One tube tested for hydrolysis of casein, another for urea hydrolysis. The other tubes tested for degradation of tryptophan, and deamination of phenylalanine. Each tube was inoculated with the unknown and observed the following week. The final set of tests followed the Multiple Test Media section of the lab manual. A test tube containing litmus milk was inoculated with the unknown bacteria. Litmus milk is purplish blue before inoculation. The tubes were left to inoculate for as long as possible. Next a SIM medium test was done to test for production of hydrogen sulfide and motility. The SIM medium contains .7% agar, hydrolyzed casein and ferrous salt. The medium was inoculated by stabbing. The final test was the Kiglers Iron agar, which tests for fermentation of glucose, lactose and production of hydrogen sulfide. Kligers Iron agar contains .1% glucose, 1% lactose, peptone, ferrous salts, and phenol red as a pH indicator. The slant was inoculated using the loop technique. All observations were recorded in the lab notebook.