The slide was allowed to cool to room temperature. The first reagent to go on the slide was crystal violet, which was allowed to sit on the heat fixed bacteria for one minute. The crystal violet was gently rinsed off the slide with DI water and the slide was covered lightly with Gram's iodine, which was allowed to stand on the slide for one minute. After one minute the Gram's iodine was gently rinsed off with DI water and 95% EtOH was used to decolorize the bacteria. Enough 95% EtOH was used so that the run-off reagent became clear. The slide was then gently rinsed with DI water. The last reagent to be put on the slide was 0.25% safranin, which was allowed to stand for one minute, and was rinsed off gently with DI water. The slide was gently dried by pressing it lightly between bibulous papers. Viewing the slide under 100X, Unknown 9 was determined to be comprised of two bacteria; a gram-positive cocci and a gram-negative rod. These were temporarily named Bacteria A and Bacteria B, respectively. Gram stains and Quadrant Streaks were done until the bacteria were successfully isolated, at which time a slant was made for each isolated bacteria to be kept frozen in storage for reference (Leboffe & Pierce, 2010). .
After Bacteria A and Bacteria B were isolated, more specific testing could be done to determine their identities. Some of the tests performed were done on both bacteria to collect more general data, while other tests were more specific to each bacteria. One test that was done on both bacteria was a Fluid Thioglycollate Medium test. This test was used to determine the bacteria's aerotolerance. Results of this test were used to narrow down the many types of Gram-Positive Cocci and Gram-Negative Rods. An inoculation loop was first sterilized and allowed to cool. The cap on the Fluid Thioglycollate Medium tube was removed and the top of the tube was gently swiped over the flame at an angle to destroy any contaminants around the top.