Essay On Unknown Bacteria

Introduction:

Bacteria play a large role in our health, the environment, and most aspects of life. They can be used in beneficial ways, such as decomposing wastes, enhancing fertilizer for crops, and breaking down of substances that our bodies cannot. However, many bacteria can also be very harmful by causing disease. Understanding how to identify bacteria has numerous applications and is incredibly important for anyone planning to enter the medical field or begin a career in research. Having the background knowledge of identifying an unknown bacteria may one day aid healthcare professionals diagnose their patient with a particular bacterial infection or help researchers determine various clinical, agricultural, and numerous other uses for bacteria.

The purpose of this project was to identify unknown bacteria species from a mixed culture. The two unknown species were initially plated onto Tryptic Soy Agar (TSA), Eosin Methylene Blue (EMB), Mannitol Salt Agar (MSA), and blood agar plates to distinguish between the two different bacteria using colony size, color, shape, and growth characteristics. By identifying and inoculating the differing types of colonies, the two unknown bacteria were purified and able to be tested

separately. These tests were conducted using the techniques and procedures provided in the Eighth Edition of John P. Harley’s Laboratory Exercises in Microbiology. After performing various stains on each unknown, a series of tests provided by the Bergey’s Manual of Determinative Bacteriology were completed to limit the possible identities of the bacteria. Additional tests were also performed to confirm the identity of each unknown.
According to the results of the various laboratory tests, the Gram positive unknown #4 was identified as Streptococcus mutans and Gram negative unknown #4 was identified as Klebsiella pneumoniae.
Methods and Materials:
After receiving both unknowns inoculated in a single mixed broth, the bacteria were initially plated on TSA, EMB, MSA, and blood agar plates to distinguish between varying bacterial colonies. Observing the differing colony sizes, shapes, color, and growth pattern, the two differing species were plated on separate TSA plates to isolate each species. The purification of each species was critical to ensure that the tests were not contaminated with the other unknown bacteria. All tests used in the identification of the unknown bacteria are summarized in Tables 1 and 2. Within Table 1, the staining procedures are listed with their purpose and expected observations. Table 2 includes the laboratory tests used in identifying the unknown bacteria and descriptions of each test’s positive and negative results. The procedure used in each stain and test can be found in the Eighth Edition of John P. Harley’s Laboratory Exercises in Microbiology.

Results:
Gram Positive Unknown #4:
Using a Gram stain, the unknown bacteria was determined to be cocci shaped and linked in chains, suggesting that they are a streptococcus species. The two possibilities for gram positive streptococcus bacteria included Streptococcus mutans and Streptococcus pyogenes. Neither species was directly identified using Bergey’s Manual of Determinative Bacteriology due to the lack of information on Streptococcus mutans and lack of tests available for Streptococcus pyogenes. Initially, a catalase test was performed and the unknown had a positive result (Table 2). According to Bergey’s, neither Streptococcus species were expected to have a positive catalase test; therefore, several tests were performed in order to gain a better insight to the identity of the unknown bacteria. After comparing the test results summarized in Table 3 with the expected phenotypes of other bacteria presented by Bergey’s, many of the other possible bacteria species could be eliminated. Using the Biolog’s Microbial ID Sytem, the unknown was identified as Enterococcus faecalis. However, after comparing the test results of the unknown with the expected results of Enterococcus faecalis provided by Microbiology Info, Enterococcus was eliminated as a possible identity. E. faecalis had several differences in results, including the catalase test (-ve), methyl red (-ve), and Vogues-Proskaeur test (+ve) (MICROBIOLOGY INFO). After eliminating E. faecalis, the identity of unknown #4 was narrowed to S. mutans and compared with the test results of another peer who had S. mutans as one of their known bacteria. Both inoculates of bacteria shared similar results listed in Table 3, and these similarities in results confirmed the identity of unknown #4 as Streptococcus mutans.

Gram Negative Unknown #4:
After performing the stains listed in Table 1, the Gram negative bacteria was determined to be bacillus with a large capsule. Following the series provided in Bergey’s Manual of Determinative Bacteriology, an oxidase test was first performed and had a negative result, which led to the Enterobacteriaceae flowchart. Various tests were then performed following the expected results, including lactose fermentation (+ve), indole (-ve), MR-VP (+/-), and Triple Sugar Iron agar tests (-H2S Production). These results led directly to identifying Gram negative unknown #4 as Klebsiella pneumoniae. Identification using the Biolog’s Microbial ID System was attempted several times with ‘False Positive’ as the result each time. Therefore, in order to confirm this identity, several other tests were performed (Table 4). These results from the unknown matched those provided by Microbiology Info and the unknown also had a large capsule that K. pneumoniae is known for. Therefore, the confirmed the identity was Klebsiella pneumoniae.

Discussion:
After completing the various tests and comparisons, it was concluded that the two unknown bacteria were Streptococcus mutans and Klebsiella pneumoniae. We arrived at this conclusion by using Bergey’s Manual and Microbiology Info as sources to compare our results to. By using this information, we were able to limit our possible unknowns to just a few bacteria.
Arriving at the result of S.

mutans was problematic due to its difference with Bergey’s Manual result for the catalase test. However, after comparing it with a peers results, it seems very possible that the strain we are working with varies from the strain used in Bergey’s. Bacteria possess the ability to develop varying phenotypes within the same species due to frequent mutation and horizontal gene transfer. Therefore, it is possible that the results obtained in our lab may vary from those provided in Bergey’s Manual. Arriving to the conclusion that the Gram negative bacteria was Klebsiella pneumoniae was much more direct. Using Bergey’s Flowchart for identification, the bacteria shared the test results and had a similar shape and

capsule.
Additionally, the Biolog program was problematic due to its incorrect identification or lack thereof. For our Gram positive, it was identified as E. faecalis; however, the test results used to confirm the identity did not match the unknown’s. When using the Biolog system for the Gram negative bacteria, it repetitively reported false positives. With each new attempt to have a lower inoculate, the system still provided a false positive. In the future, I now know that the Biolog system may not be the most reliable source for bacterial identification.