An experiment like this one allows students to apply their existing knowledge of organisms and laboratory procedures to a challenging task. By conducting familiar experiments on unknown bacteria and incorporating information about specific bacteria, it becomes possible to identify a mixed culture. This process gradually eliminates bacteria that do not match the results obtained.
Tube number twelve was acquired for conducting a variety of tests on different microorganisms, encompassing both Gram positive and Gram negative types. The Gram positive organisms consisted of Bacillus cereus, Clostridium perfringens, Corynebacterium xerosis, Enterococcus faecalis, Streptococcus agalactaie, Staphylococcus aureus, and Staphylococcus epidermidis. Conversely, the Gram negative organisms included Enterobacter aerogenes, Escherichia coli, Moraxella catarrhalis, Neisseria flavescens, Psuedomonas aeruginosa Proteus vulgaris,and Salmonella typhimurium.
I started my investigation by performing a Gram stain on my bacteria. To create a smear, I added a drop of water to the middle of a glass slide and then transferred a loopful of bacteria from my tube into the droplet. I followed aseptic technique by flaming the neck of the test tube and inoculating loop before and after transferring the bacteria. After that, I allowed the smear to dry fully on the glass slide.
After my smear had dried, I placed it in my staining rack, which I had taken from my lab drawer. I started the Gram stain by flooding the slide with crystal violet, and then waited for thirty seconds before washing the slide with water for five seconds. I then flooded the slide with Gram’s iodine and waited for sixty seconds before washing the slide again with water for five seconds. To decolorize my slide, I washed it with 95% ethanol until no more crystal violet washed off, and once again, I washed the slide with water for five more seconds.
After flooding the slide with safranin and waiting for sixty seconds, I washed it with water for five seconds. I then used bibulous paper to blot the slide dry until it was completely dry. Next, I placed the slide under the microscope to examine the morphology of my bacteria for exercise 7. Moving on to the next test, I used tryptic soy agar(TSA) plates as my medium. I started by dividing the medium into four quadrants using a wax penicil to perform the streak plate technique.
First, I employed the aseptic technique to move a small quantity of bacteria using my inoculating loop into the initial quadrant. While continuing to use the loop, I spread the bacteria across the quadrant while ensuring to stay within it. Subsequently, I sterilized the loop by subjecting it to flame and utilized it to streak some bacteria from the first quadrant into the second quadrant. I repeated this procedure until all four quadrants were streaked (exercise 8).
On another identical TSA plate, I performed the same procedure, incubating one under aerobic conditions at 37oC and the other under anaerobic conditions at 37oC. To continue the experiment, I utilized Columbia colistin nalidixic acid agar (Columbia CNA) with 5% sheep blood as my medium. On this medium, I employed the identical streak plate technique as the TSA plates and incubated it at 37oC. Additionally, I employed MacConkey agar as a medium to identify lactose fermentation, applying the same streak plate technique with my bacteria and incubating the plate at 37oC.
In my next experiment, I aimed to identify the existence of nitrate reductase in my microorganisms. To achieve this, I cultivated the microorganisms in a nitrate broth which contains potassium nitrate as the nitrate source. This particular medium is equipped with Durham tubes that serve to capture any nitrogen gas generated during denitrification (as shown in exercise 29). I proceeded by transferring bacteria from my initial tube to the nitrate broth tube aseptically. Subsequently, I incubated the tube at a temperature of 37oC.
I conducted multiple tests in addition to the ones mentioned earlier before I narrowed down my bacteria based on morphology. However, this was a mistake as I could have identified my unknown bacteria by solely running the aforementioned tests. The additional tests I performed include the Oxidase test, Indole test, Methyl Red test, Voges Proskaer test, Citrate Permease test, Nitrate Reductase test, urease test, coagulase test, triple sugar iron (TSI) test, CAMP factor test, and DNase test. Unfortunately, these tests proved to be irrelevant in discovering my unknown microorganisms since the class’s identification table did not provide results for these specific bacteria.
Hence, I should not have proceeded with them. I obtained numerous satisfactory outcomes from the experiments conducted in the laboratory. Upon preparing the smear and applying stain to the glass slide containing my organisms, I examined them under the microscope. I identified purple, Gram positive rods and numerous reddish Gram negative cocci. Furthermore, I observed growth on both the anaerobic and aerobic TSA plates.
I was motivated to perform two separate Gram stains by transferring bacteria from each of the plates onto different glass slides. With each slide, I repeated the Gram stain procedure and examined them under the microscope. I discovered that the Gram positive rods only thrived on the anaerobic TSA plate, while the Gram negative cocci grew in both anaerobic and aerobic environments. Additionally, I noticed substantial growth on both the Columbia CNA and MacConkey plates.
After incubating the bacteria in the nitrate broth, I proceeded with the experiment by introducing nitrate reagent A (sulfanilic acid) and nitrate reagent B (a-napthylamine) into the broth. There was no change in color, so I decided to include zinc dust in the mixture. However, even after this addition, there was still no color change. Conducting the initial Gram stain was essential in determining the bacteria’s morphology.
While excluding all Gram negative rods like E. aerogenes, E. coli, P. aeruginosa, I successfully identified Gram positive rods and Gram negative cocci during this process.
I successfully eradicated various types of bacteria, including Gram-negative bacilli such as E. coli, K. pneumoniae, P. vulgaris, and S. typhimurium, as well as Gram-positive cocci like E. faecalis.
agalactiae, S. aureus, and S. epidermidis were growing as Gram positive rods in TSA anaerobic conditions, therefore C could be eliminated.
Xerosis cannot be found in anaerobic conditions. It was not possible to eliminate any Gram negative cocci as they can grow in both aerobic and anaerobic conditions. By observing the growth on the Columbia CNA medium, I determined that one of the bacteria had g-hemolysis as there was no change observed in the medium around the colonies.
The purpose of the MacConkey plate is to identify organisms that ferment lactose. The presence of the neutral red indicator allows for differentiation between lactose-fermenting and lactose-nonfermenting organisms. When an organism ferments lactose, it releases acidic by-products, which lowers the pH of the medium surrounding the colony. As a result, the colony absorbs the red indicator, causing it to exhibit a pink to brick red color.
My colonies showed clear absorption of the indicator as they displayed a distinct red color instead of being translucent. This observation indicates that the lactose in my bacteria is being fermented, enabling me to rule out B. cereus as a potential candidate. Consequently, I identified my Gram positive bacterial unknown as C.
Erfringens is the Gram-negative bacterium that I have identified. The nitrate broth test indicated a negative result, as there was no color change after the addition of the reagents and zinc dust. This indicates that the microorganism further reduced the nitrites through denitrification to produce nitric oxide, nitrous oxide, or nitrogen gas. From the remaining two bacteria, N. flavescens displayed a negative nitrate reductase reaction, confirming it as my Gram-negative bacterium. To summarize, I have identified my two unknown bacteria as a Gram-positive C.
Utilizing html tags, there is a presence of both a Gram positive C. perfringens and a Gram negative N. flavescens.
