Understanding the identity of microorganisms is crucial for several purposes. These include identifying the causative agent of a disease in a patient, which is essential for determining the appropriate treatment. Additionally, knowing the correct microorganism is necessary for producing specific foods or antibiotics. In this study, I employed all the methods I had learned in the microbiology laboratory class to identify an unknown bacterium.
The goal of this lab was to distinguish two unknown bacteria cultures through different tests. These tests aimed to identify specific biochemical characteristics of the bacteria, such as enzymatic reactions or metabolic pathways. By using these tests, the unique features of each bacteria species were highlighted, allowing for their identification. Additionally, each person in the class received a mixture of 2 different microbes in their broth.
The mixture was streaked onto different plates: TSA, BAP, MAC, and CNA. These plates can selectively support the growth of either gram-positive or gram-negative bacteria, while others do not have any specific selectivity. On the TSA plate, there were large hazy white colonies observed, along with a smaller number of orange colonies. Streaking the mixture onto a BAP plate allowed for the isolation of a pure culture of gram-positive bacteria, which would be used in the subsequent differential tests. On the other hand, streaking onto TSA/MAC plates allowed for the growth of gram-negative bacteria for further testing. The procedure is as follows:
The catalase test was conducted exclusively on gram (+) bacteria since it does not aid in distinguishing gram (-) bacteria. This is because all unidentified gram (-) bacteria could potentially exhibit catalase positivity. The purpose of this test is to identify the existence of catalase, an enzyme that aids in breaking down toxic hydrogen peroxide generated during the direct transfer of high-energy electrons to oxygen. To perform the catalase test, hydrogen peroxide is introduced into the culture and the observation of gas bubbles indicates a positive outcome for catalase.
If no bubbles are observed during the catalase test, it indicates that the culture is negative. The results were positive. The citrate test, conducted only on gram (-) bacteria, determines whether an organism can use citrate as its sole carbon source. Bacteria with citrate-permease possess this ability. The test medium contains bromthymol blue dye, which is green at a neutral pH but turns blue in a basic pH. Bacteria that can survive and utilize citrate convert ammonium phosphate to ammonia and ammonium hydroxide, which makes the agar more alkaline and causes it to turn blue.
The blue color conversion indicates a positive result in the citrate test, while no color change signifies a negative result. However, I believe that the citrate test was unsuccessful and should have yielded a positive result. The tube showed no bacterial growth and did not align with the results obtained from the previous test. The urease test was conducted on both gram-positive and gram-negative bacteria to examine urea decomposition. Urease-positive organisms hydrolyze urea into ammonia, which overcomes the buffer in the medium and causes it to change from orange to fuchsia. Rapid urease-positive bacteria will cause the broth to turn pink within 24 hours.
Both Urease-negative bacteria and the unknown organism produced a yellow color change in the broth, indicating a negative reaction. To test for resistance and sensitivity to the antibiotic bacitracin, a bacitracin test was performed on the gram (-) bacteria. If a zone of inhibition is present around the disc, it means the bacteria is sensitive to bacitracin. If there is no clear zone around the disc, the bacteria is resistant to the antibiotic. The unknown S was streaked onto BAP plate 1, and then a bacitracin disc was placed on the plate and incubated for 48 hours.
After 48 hrs of incubation, a zone of inhibition appeared around the bacitracin disc, indicating that the bacterium has sensitivity to bacitracin. To test for nitrate reduction, the unknown gram (-) culture was inoculated into a nitrate broth and allowed to grow. The first test involved adding 2 drops of sulfanilic acid and napththylamine to the medium to check for the presence of nitrite. If nitrite is present, the medium turns red, indicating a positive result.
If the medium remains unchanged, a second test is conducted to determine if nitrite was further reduced. During this second test, zinc powder is introduced into the broth to facilitate the reduction of any nitrate present to nitrite. If nitrate is present when the zinc is added, the reduction of this compound will cause the medium to turn red, indicating a negative result due to the previously added reagents. Conversely, if the medium does not change color following the addition of zinc, it signifies a positive result for nitrate reduction, as the just reduced nitrite remains undetectable. The outcome obtained from this step was positive.
The Triple Sugar Iron agar slant (TSI) was utilized for the purpose of testing glucose and lactose fermentation, as well as H2S production. Gas production was observed to detect fissures resulting from gas production during fermentation. The color change of the agar’s initially red slant and butt to yellow confirms glucose fermentation. The presence of H2S could be attributed to the reduction of thiosulfate within the media. The production of this compound triggers a reaction with ferrous sulfate, resulting in the formation of a black precipitate.
Thus, the culture is considered positive for H2S production if a black precipitate is observed and negative if no black precipitate is seen. After incubation, the results were A/A for gram (-) bacteria and K/K for gram (+) bacteria, with no black precipitate present in either tubes. The flow chart for Unknown 126 shows that it is a Gram-positive coccus, with positive catalase and negative tests for Staph. aureus, Strep. pyogenes, Staph. epidermidis, Strep. aglactea, Staph. saprophyticus, Enterococcus faecalis, Micrococcus oseus, and Strep pneumonia. On BAP Gamma testing, the results were negative for TSI (k/k), positive for Micrococcus roseus, Staph. Saprophyticus, and Staph. Aureus. Further testing on Micrococcus roseus was conducted using Urease (Negative)/Bacitracin (S) tests. Based on these multiple differential and biochemical tests on Unknown 126, it was determined that the bacteria in question is Micrococcus roseus.
Based on the test results and analysis, it has been determined that M. roseus is the unknown 126. The flow chart reveals that the unknown is Gram stain-negative, urease-negative, and positive for various bacteria including Escherichia coli, Citrobacter freundii, Enterobacter cloacae, Proteus vulgaris, Enterobacter aergenes, Protues mirabilis, Pseudomonas aeruginosa, Serratia marcescens, Salmonella enteriditis, and Klebsiella pneumonia. Additionally, according to the TSI results: Escherichia coli, Salmonella enteriditis,and Klebsiella pneumonia show an acid-acid (A/A) reaction; Proteus vulgaris shows an acid-alkaline (A/K) reaction; while Enterobacter cloacae shows an alkaline-alkaline (K/K) reaction.
The text presents the findings of tests carried out on different types of bacteria, including Enterobacter aerogenes, Escherichia coli, Enterobacter cloacae, and Klebsiella pneumonia. The test for Hydrogen Sulfide (H2S) indicated that Enterobacter aerogenes did not produce it. Moreover, a test was conducted to measure Nitrate levels in these bacteria, revealing positive results for both Escherichia coli and one sample of Klebsiella pneumonia. Another set of tests aimed at determining Urease activity yielded negative outcomes. It is important to note that there is no information provided about the MR-VP test tube. Nonetheless, by utilizing available tests, an unidentified sample labeled as “Unknown S” was identified as Enterobacter cloacae.
Conclusion
After conducting multiple tests, I was able to narrow down my choices but encountered an issue with the gram-negative bacteria during the MR-VP test. The specific test tube needed for this test was missing, resulting in two different microbes. To come to a conclusion, I had to rely on available results and make an educated guess. In the end, it appears that the unknown S (G-) could potentially be Enterobacter cloacae. This particular microbe is characterized as being gram-negative, rod-shaped, and a facultative anaerobe. E. cloacae is recognized as an opportunistic pathogen and is accountable for causing various infections such as lower respiratory tract infections, skin and soft-tissue infections, urinary tract infections (UTIs), and endocarditis.
