An experiment such as this one serves the purpose of allowing us, the students, to apply what we already know about any organism and any laboratory procedure to the difficult task at hand. It is possible to identify a mixed culture by running familiar experiments on the unknown bacteria and taking information already known about specific bacteria and applying it to the results. This helps to slowly eliminate any bacteria that do not correspond with the results. I obtained tube number twelve to run various tests on. There are only two microorganisms, one is Gram positive and one is Gram negative, inside the test tube. The Gram positive possibilities are to be narrowed down from Bacillus cereus, Clostridium perfringens, Corynebacterium xerosis, Enterococcus faecalis, Streptococcus agalactaie, Staphylococcus aureus, and Staphylococcus epidermidis. The Gram negative possibilities are to be narrowed down from Enterobacter aerogenes, Escherichia coli, Moraxella catarrhalis, Neisseria flavescens, Psuedomonas aeruginosa, Proteus vulgaris, and Salmonella typhimurium.
I began my quest by conducting a Gram stain on my bacteria. I prepared a smear by placing a drop of water onto the center of a glass slide and then removed a loopful of bacteria from my tube to place within the water droplet. I used the aseptic technique while doing this making sure to flame the neck of the test tube and inoculating loop before and after transferring bacteria. Then, I waited a while in order to let the smear dry completely on the glass slide. Once it was dry, I placed my smear in my staining rack that I obtained from my lab drawer. I began the Gram stain by flooding the slide with crystal violet and waited for thirty seconds before I washed the slide with water for five seconds. Next, I flooded the slide with Gram's iodine and waited for sixty seconds before I again washed the slide with water for five seconds. Then, I decolorized my slide by washing it with 95% ethanol until no more crystal violet washed off the slide and yet again, washed the slide with water for five more seconds. Finally, I flooded the slide with safranin and waited for sixty seconds before washing it with water for five seconds. I blotted the slide dry with bibulous paper until it was dry and placed it under the microscope for viewing to figure the morphology of my bacteria(exercise 7).
The next test I ran using tryptic soy agar(TSA) plates as my medium. I began using the streak plate technique by diving the medium into four quadrants with a wax penicil. Then I used the aseptic technique to transfer a small amount of bacteria with my inoculating loop to the first quadrant. Still using the loop, I streaked the bacteria across the quadrant making sure to remain in that quadrant. Then, I flamed the loop and used it to streak some bacteria from quadrant one to quadrant two. I kept on repeating this process until all four quadrants were streaked (exercise 8). I did the same thing on another identical TSA plate and incubated one under aerobic conditions at 37°C and the other under anaerobic conditions at 37°C. Next, I used Columbia colistin nalidixic acid agar (Columbia CNA) with 5% sheep blood as my medium. I used the same streak plate technique on this medium as I used on the TSA plates and incubated it at 37°C as well. Then, I used MacConkey agar as a medium to detect lactose fermentation, used the same streak plate technique with my bacteria, and incubated the plate at 37°C.
My next experiment was done to detect the presence of nitrate reductase by growing my microorganisms in a nitrate broth. This broth contains potassium nitrate as the source of nitrate. This medium also contains Durham tubes which trap any nitrogen gas produced during denitrification (exercise 29). I aseptically transferred bacteria from my tube to the tube of nitrate broth and then incubated the tube at 37°C. I ran numerous tests as well as the ones...