Unknown Experiment BI 303 Danion Terauds and Brian Turek 3/31/05 Structure: The layout of this lab report will be in chronological order to show test and deductive patterns. Our lab report will also include photographs, where possible, and other graphical representations of our results. Procedure: Given the choice of 13 possible bacteria, we created a spreadsheet (Table 1.1) with the bacteria and their reactions to particular tests, according to Bergey’s Manual of Determinative Bacteriology, 9th Edition. We then compared our unknown bacteria test results with the spreadsheet in order to eliminate the possible bacteria from the list. This was done only when it did not meet two separate tests or characteristics of our unknown. We chose that possible bacteria had to fail two criteria before being eliminated because of the possibility a test failure or inconclusive results. As a procedural constant, in the beginning of every lab period we made it a point to inoculate fresh nutrient broth and/or inoculate a nutrient agar slant with our unknown. A fresh culture is imperative for the Gram’s stain, and redundancy is important incase of contamination. Tests/Results Unknown Sample: Tube #7 Streak Plate (BI 303 lab manual pg. 64-67) o Date Inoculated: 3/15/05 Results Recorded: 3/17/05 o Sample Age: < 24 hours (original stock) o Media: Nutrient Agar o Notes: We ran this procedure first to isolate colonies of the bacteria, to determine if the culture was pure. Also, we can determine visible characteristics of the colonies, to serve as a preliminary identifier of the bacteria. o Results: Yellow colonies and uniform colonies. (Fig. 1.1) o Discussion: With our results, we can determine that the culture is pure. Also, the yellow colonies point toward M. luteus, M. phlei, S. epidermidis, and S. aureus. Fig. 1.1 – Streak plate results. (Note: Thinning yellow colonies.) Simple Stain (BI 303 lab manual pg. 7-8) o Date: 3/15/05 o Sample Age: < 24 hours (original stock) o Stain: Methylene Blue o Notes: We decided to run this test because of its fast results and we can use the physical attributes, size, and grouping to help identify the bacteria. Also, we can compare the physical attributes to the future Gram’s stain for consistency. o Results: Blue spherical clusters (grape like) throughout the slide. Cells are cocci and ~1.0µm in size. (Fig. 1.2) o Discussion: Our results indicate a defining structure of the Staphylococcus species. Grape-like cluster Fig. 1.2 – Simple stain results. (Note: Image transcribed onto paper from the slide sample.) Gram’s Stain (BI 303 lab manual pg. 13-16) o Date: 3/15/05 o Sample Age: < 24 hours (original stock) o Notes: The Gram’s stain is a definitive test for determining and classifying bacteria. o Results: Cells are stained purple, cocci shaped, tetrads and clusters, and cells are ~ 1.0µm in size. o Discussion: The purple stained cells are indicative of Gram positive bacteria. The physical attributes and groupings were similar to the simple stain, but revealed more of a tetrad arrangement. Hanging Drop Method (BI 303 lab manual pg. 23-26) o Date: 3/15/05 o Sample Age: < 24 hours (original stock) o Notes: Due to the instant results and the inexpensiveness to perform, we decided that this would be an efficient way of gathering more data. o Results: No motility was detected. o Discussion: Because of no motility, we decided that further tests needed to be performed to validate non-motility. Motility Stab (BI 303 lab manual pg. 23-26) o Inoculation Date: 3/15/05 Results Recorded: 3/17/05 o Sample Age: < 24 hours (original stock) o Media: Soft agar (~0.4% agarose) o Notes: Because of the hanging drop test results, we thought that it would be necessary to perform a more definitive test to determine motility. o Results: No motility. No growth within the agar, but growth was abundant on a surface streak. (Fig. 1.3) o Discussion: These results confirm the assumption that our unknown is in fact non-motile, and is a possible aerobe. Fig 1.3 – Motility stab. (Note: brown spot on picture is a glass stain, not motility or growth. 3/15/05 Results Synopsis The key test results taken on 3/15/05 were from the Gram’s stain and simple stain. The clustered pattern of bacterial arrangement and the positive Gram’s test eliminates many of the possible bacteria (Table. 1.2), and pointing our tests in the direction of M. luteus, S. epidermidis, and S. aureus. Mannitol Salt Agar Test (BI 303 lab manual pg. 66-67) o Inoculation Date: 3/17/05 Results Recorded: 3/22/05 o Sample Age: 48 hours (cultured slant – 3/15/05) o Notes: The MSA test was performed to differentiate S. aureus from S. epidermidis and M. Luteus. o Results: Low growth of bacteria and no color change to agar. o Discussion: Due to the lack of Mannitol fermentation (positive fermentation would yield a yellow hue); we can determine that the unknown bacterium is not S. aureus. The low growth on the MSA, points at the possiblility of the bacteria being halotolerant. Fig. 1.4 – MSA test. (Note: No yellow color change.) 3/17/05 Results Synopsis On 3/17/05 we were able to observe the results from the streak plate and the motility stab, both inoculated on 3/15/05. The streak plate results were indicative of M. luteus (bright yellow colonies). The motility stab (negative result) does not differentiate between the possible bacteria that are left (refer to table 1.2), but only validates them. Oxidase Test (BI 303 lab manual pg. 101-103) o Date: 3/22/05 o Sample Age: 5 days (cultured slant – 3/17/05) o Notes: Due to the unknown bacteria’s inability to ferment mannitol, it left us to differentiate between the possibility of M. luteus and S. epidermidis. The oxidase test differentiates M. luteus, which produces a positive result, and the Staphylococcus species which is considered to be an oxidase negative species. o Results: The oxidase test strip turned purple in the presence of the unknown sample. o Discussion: A purple result in indicative of an oxidase positive bacterium, Microccocus luteus. Catalase Test (BI 303 lab manual pg. 97-98) o Date: 3/22/05 o Sample Age: 5 days (cultured slant – 3/17/05) o Notes: We decided to run the Catalase test, because of its inexpensiveness, quick results, and its further criteria for validation. o Results: An effervescent reaction occurred. (Fig. 1.5) o Discussion: Bubbles are indicative to a Catalase positive reaction. Fig. 1.5- Catalase test. (Note: bubbles forming on the slide.) 3/17/05 Results Synopsis On this day of results, we found that the incubated MSA did not ferment, indicating that the unknown bacteria is not S. aureus. From the oxidase test, we were able to rule out the Staphyloccous species. Thus we conclude that our unknown species is Micrococcus luteus. Conclusion We conclude that our unknown species (#7) is Micrococcus luteus. From our results and discussions, that we have already stated, reasonably justify our assumption. Post Experiment Disscussion Reviewing our tests and results we found that some tests were unnecessary or not performed in the correct chronological order. If we were to run the experiment again, we would: 1.) Create a streak plate. 2.) Perform a Gram’s stain, 3.) Perform an oxidase test. Using this pattern we could reasonably identify M. luteus. We feel that this experiment helped hone our laboratory skills, and improved our deductive laboratory techniques. Resources Benson, Brown, et al. (2005). BI 303 General Microbiology. United States: McGrawHill Holt, J. (Ed.), et al. (1994). Bergey's Manual of Determinative Bacteriology. 9th ed. Baltimore: Williams & Wilkins.