I. Introduction
The ability to accurately identify bacteria is important, especially in a healthcare setting. Correctly identifying bacteria and their metabolic characteristics provides information that is useful in prescribing treatments for patients. The purpose of this lab is to use various tests to separate and identify two different species of bacteria.
II. Materials and Methods
The instructor provided a test tube labeled #112 that contained a cloudy mixture of two strains of bacteria, one Gram positive and one Gram negative. Protocols for the various tests were followed from the course’s Lab Manual (1). All procedures were carefully completed using a sterile technique to prevent contamination. All culture plates and tubes were grown in an incubator at 37oC. The unknown bacteria mixture was streaked onto a nutrient agar plate in an attempt to isolate a single colony of each species. Pure cultures were then obtained for each bacterial species. Gram staining was used as a starting point to identify the bacteria and to confirm the isolation of a pure culture of each bacterial species. Gram-stained slides were viewed on a compound light microscope at 1000X using oil immersion. Following Gram staining various tests were done to further narrow down the identity of the two bacteria. The specific media and reagents used for each test are listed in Table 1. All tests included a non-inoculated control tube or media plate for comparison.
| Table 1. Reagents and Media Used For Each Test- Protocol details in lab manual (1) | ||
|
Test |
Media |
Reagents |
| Gram Stain | Nutrient Agar | Crystal Violet, Gram Iodine, Gram Decolorizer, Safranin Red |
| Simmons Citrate | Simmons Citrate Agar Slant | |
| Methyl Red | Nutrient Broth | Methyl Red |
| Voges Proskauer | Nutrient Broth | VP Reagent A and VP Reagent B |
| Indole | SIM tube | Indole Reagent |
| Glycerol Fermentation | Glycerol, Peptone, and Phenol Red | |
| Maltose Fermintation | Maltose, Peptone, and Phenol Red | |
| Nitrate Reduction | Nutrient Broth | Nitrate Reagent A and Reagent B, powdered Zinc |
| Oxidase | Filter Paper | Oxidase Reagent |
| Urea | Urea Broth and Phenol Red | |
III. Results
| Table 2. Test Results for Strain A | |||
|
Test |
Observation |
Results |
|
| Gram Stain | Red rods | Gram Negative | |
| Simmons | Media remained green | Cannot use citrate as sole source of carbon | |
| Urea | Broth remained yellow | Cannot hydrolyze ammonia | |
| Indole | Media immediately turned red | Has the enzyme tryptophanase | |
| Methyl Red | Broth turned red | Produces acid from glucose metabolism | |
| Voges Proskauer (V-P) | Yellow, no color change | Does not produce acetyl methyl carbinol | |
| Nitrate Reduction | No color change after Reagent A & B. Turned red after adding zinc | Cannot reduce nitrates | |
| Table 3. Test Results for Strain B | |||
|
Test |
Observation |
Results |
|
| Gram Stain | Purple rods | Gram Positive | |
| Simmons | Media became blue | Can use citrate as sole source of carbon | |
| Oxidase | Colorless | Lacks the enzyme cytochrome oxidase | |
| Methyl Red | Broth remained yellow | Does not produce acid from glucose metabolism | |
| Glycerol Fermentation | Red, no color change | Does not ferment glycerol | |
| Maltose Fermentation | Red, no color change | Does not ferment maltose | |
Following the initial streaking of the unknown, colonies from two distinct strains were identified. Strain A was white, while strain B was yellow. Gram staining was then done and strain A was identified as a gram-negative rod and strain B was identified as being a Gram-positive rod. One colony was taken from each strain to isolate a pure culture. A pure culture for strain A was easily obtained, as verified by gram staining, but the pure culture for strain B was contaminated with some of strain A and therefore had to be re-isolated. Once each strain was isolated various tests were completed. The results of the tests are summarized in Tables 1 and 2 for strain A and strain B respectively. Flow charts depicting the order in which the tests were completed are shown in Figures 1 and 2. Although the identity of unknown A as E. coli was quickly ascertained, it was important to do a methyl red and an indole test because E. coli produces a positive result for these two tests, compared as negative for the others. The identity of unknown B was quickly narrowed down to two candidates because of its shape. Although seemingly superfluous, Simmon’s Citrate test served to validate the two potential candidates for the identity of unknown B. All of the tests to differentiate B. cereus and B. subtilis produced negative results. Therefore, it was important to complete multiple tests to validate the identity of unknown B as Bacillus subtilis.
IV. Discussion/Conclusion
The first test completed was Gram Staining. This allowed the two species of bacteria to be separated into Gram -, Unknown A, and Gram +, unknown B. The first test completed to identify Unknown A was Simmon’s Citrate. This test produced a negative result, which narrowed down the potential candidates to E. coli and P. vulgaris. The negative result obtained from the Urea test narrowed down the identity of unknown A to E. coli. Positive results for Indole and Methyl Red tests confirmed the identity of Unknown A as E. coli. This was further confirmed by the negative results of the V-P and Nitrate tests.
Unknown B was determined to be Gram + rods, which narrowed the candidates to B. cereus and B. subtilis. This was confirmed with a positive Simmon’s Citrate test. A negative Methyl Red test identified Unknown B as B. subtilis. This was further confirmed by negative results for Glucose Fermentation, Maltose Fermentation, and Oxidase tests.
Bacillus subtilis is one of the most studied bacteria with very well defined characteristics as its entire genome has been sequenced (2). B. subtilis bacteria are rod-shaped, Gram-positive bacteria that are generally found within the soil and plants. Recent research has found that B. subtilis is also found within the gastrointestinal tract of humans and animals (3). In response to stress such as nutrient depletion of carbon, nitrogen, or phosphate in the immediate environment B. subtilis can undergo the process of sporulation to produce metabolically inactive endospores (4). The advantage of entering a dormant state is that the endospore is highly resistant to stresses such as heat, radiation, and nutrient deprivation (4). When favorable conditions are reached the endospore can then undergo germination to become a growing vegetative cell again. B. subtilis can secrete large concentrations of protein into the media, which makes it useful in the industrial production of enzymes such as a-amylase and alkaline phosphatase (5, 6). Additionally B. subtilis produces over twenty-four different antibiotics including subtilin, surfactin, and bacillomycin (6).
V. Reference List
1. McDonald, V., Thoele, M., Salsgiver, B., and Gero, S. 2011. Lab Manual for General Microbiology Bio 203. St. Louis: St. Louis Community College at Meramec.
2. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A.M., Alloni, G., Azevedo, V., Bertero, M.G., Bessieres, P., Bolotin, A., Borchert, S., et al. 1997. The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390:249-256.
3. Hong, H.A., Khaneja, R., Tam, N.M., Cazzato, A., Tan, S., Urdaci, M., Brisson, A., Gasbarrini, A., Barnes, I., and Cutting, S.M. 2009. Bacillus subtilis isolated from the human gastrointestinal tract. Research in microbiology 160:134-143.
4. Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J., and Setlow, P. 2000. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiology and molecular biology reviews : MMBR 64:548-572.
5. Harwood, C.R. 1992. Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends in biotechnology 10:247-256.
6. Stein, T. 2005. Bacillus subtilis antibiotics: structures, syntheses and specific functions. Molecular microbiology 56:845-857.
