Ensuring that a clean glass slide was being used, the slide was carefully labeled with a circle drawn on the underside, center to help direct the position of the bacteria sample smeared. After a loop was sterilized, a bacteria contained in the Eppendorf sample tube was grabbed with an inoculating tube. The loop of bacteria was placed onto the center of the slide over the opposite side of the circle drawn. The slide was placed inside of an incubator and carefully monitored until the bacteria smear on the slide turned a white/clear color. After the Bunsen burner was started, the slide was passed through the flame 2-3 times, ensuring to not hold the slide over the flame too long. Holding the slide over the flame to closely and too long would result in no bacteria present on the slide during stain testing. After the slide had completely dried, the student carefully held the glass flat in the air over a staining tray and applied crystal violet for 1 minute, ensuring to cover the entire slide. After 1 minute, the slide was titled downward at a 45 º angle and rinsed with DI water until the water running off was clear. Using the same technique, the slide was held flat again and Gram’s Iodine was then applied for 1 minute and then rinsed off with DI water. Decolorizer was then added for 3 seconds and rinsed off with DI water. Lastly, Safranin was applied for 1 minute and then rinsed off with DI water. The wet slide was placed carefully in between bibulous paper until dry. Using proper microscope and oil technique, the student examined the slide to view the bacteria morphology. Stock Culture: 2 TSB broth tubes were labeled with student name, date, teacher assistant, bacteria sample, class section, and TSB. Using a sterilized loop, a portion of an isolated colony on the MAC plate was grabbed and placed into the broths. The student then used the vortex shaker and incubated the tubes at 37 ºC. Kesmarki 5
IMVC Tests: A SIM broth media, Methyl Red Vogues-Proskauer broth, and Simmon’s Citrate Medium was supplied to each student by a TA. Both needles and loops were sterilized to perform the IMVC tests. The Indole and Citrate tests used needles, where as the Methyl and Vogues tests used a loop. To perform the Indole test, the SIM broth tube cap was removed and the tip of the tube was passed through the flame, followed by an agar deep stab inoculation with a sterilized needle. After inoculation, the tube was passed through the flame again and the cap was replaced. Following the same aseptic techniques, the citrate test was performed by a light streak slant inoculation of the Simmon’s Citrate Medium tube with a sterilized needle. The Methyl Red and Vogues-Proskauer tests were performed by inoculating a loop of bacteria into the MRVP broth with the same and proper aseptic technique done during the Indole and citrate tests. All tests were incubated for at least 24 hours at 37 ºC. IMVC Reagent’s: After the IMVC tests were incubated for at least 24 hours at 37 ºC, reagents were added to each test. Reagents were passed out by the TA. The Indole test was started first and the student
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- Fall '16