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 37oC and the other under anaerobic conditions at 37oC. 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 37oC 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 37oC.
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 37oC.
I ran numerous tests as well as the ones described above before I narrowed my bacteria down due to morphology. This was a mistake in that my unknown bacteria could have been figured out only by running the tests listed above. The other tests I ran include the Oxidase test, the Indole test, the Methyl Red test, the Voges Proskaer test, the Citrate Permease test, the Nitrate Reductase test, the urease test, the coagulase test, the triple sugar iron(TSI) test, the CAMP factor test, and the DNase test.These tests ended up being a bit pointless to my discovery of my unknown microorganisms because the results of these on specific bacteria were not given in the table the class used for identification.
Therefore, I should not have followed through with them. There were many pleasing results to the tests I accumulated in lab. After preparing the smear and staining the glass slide of my organisms I observed them under the microscope and noticed that I had purple, Gram positive rods and many reddish colored, Gram negative cocci. After this I observed that there was growth on both the anaerobic and aerobic TSA plates.
This motivated me to do two separate Gram stains by inoculating bacteria from each of the plates onto separate glass slides. I repeated the Gram stain procedure with each slide and observed both under the microscope. I found that the Gram positive rods only grew on the anaerobic TSA plate while the Gram negative cocci grew in both anaerobic and aerobic conditions. I also observed vivid growth on both the Columbia CNA and MacConkey plates.
Following incubation of the bacteria growing in the nitrate broth, I continued the experiment by adding nitrate reagent A(sulfanilic acid) and nitrate reagent B(a-napthylamine) to the broth.No color change occurred so I then added a bit of zinc dust to the mix. Once again, no color change occurred. The purpose of conducting the initial Gram stain was to be able to figure out the morphology of the bacteria.
By doing this and discovering Gram positive rods and Gram negative cocci, I was able to eliminate all of the Gram negative rods: E. aerogenes, E. coli, P. aeruginosa, P.
vulgaris, and S. typhimurium. I was also able eliminate all of the Gram positive cocci which included E. faecalis, S.
agalactiae, S. aureus, and S. epidermidis.Then, by understanding that the Gram positive rods were growing in TSA anaerobic conditions, I could also eliminate C.
Xerosis from the possibilities. It could not be found in anaerobic conditions. I was unable to eliminate any Gram negative cocci because both of those bacteria are able to grow in both aerobic and anaerobic conditions. By observing growth on the Columbia CNA medium, I was able to understand that one of my bacteria had g-hemolysis because there was no change observed in the medium around the colonies.
The MacConkey plate was used to detect any lactose fermentation.The neutral red indicator serves to contrast lactose-fermenting from lactose-nonfermenting organisms. If an organism ferments lactose, acidic by-products are released and the pH of the medium around the colony drops. This causes the thriving colony to absorb the red indicator, causing it to appear pink to brick red in color.
My colonies clearly absorbed the indicator because they were a distinct red color and not translucent. This means that my bacteria ferments lactose and I could eliminate B. cereus from the possibility list. I then had discovered that my Gram positive bacterial unknown was C.
erfringens! Due to the fact that there was no color change in the nitrate broth after the addition of the reagents and zinc dust, there was clearly a negative result.This means that nitrites were further reduced by the microorganism in the process of denitrification to yield nitric oxide, nitrous oxide, or nitrogen gas. Between the two bacteria left to narrow down, N. flavescens was the one with a negative nitrate reductase reaction and is therefore my Gram negative bacteria! So, in conclusion, this means that my two unknown bacteria are a Gram positive C.
perfringens and a Gram negative N. flavescens.