Ensuring that a clean glass slide was being used the

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