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Microbes have developed many different strategies to inhabit most, if not all of the different environments present on earth. They are found in the soils of the earth in great abundance, ranging from 4.8x109 cells in the forest soil, to 1.8x1010 in pasture soil (Wiley et al. 2008). In the soil different microbial communities are found on the surface of the soil and throughout its depth (Wiley et al. 2009). There is such a large diversity that less than 1% of all soil microbes have been cultured (Wiley et al. 2009). The variability of microbes found in different soils depends on the nutrients found in the soil, and the decomposition rates and litter accumulation (Wiley et al. 2009). The temperate region soils are usually rich in resources, as there decomposition rates are slower than that of the primary production rates, and in temperate coniferous soils, the excessive accumulation of organic matter causes the soil to suffer and the lack of decomposition results in organic acids being produced and solubilize aluminum and other soil components resulting in a bleached zone (Wiley et al. 2009). In temperate coniferous soils the most prominent way to maintain nutrient cycling is by fire, usually in the form of controlled burns (Wiley et al. 2009). With this information in mind this study set out to isolate and identify a bacterium from the forest soil through a variety of different tests.
1 gram of forest soil was added to 99 ml of distilled water. Further dilution of this sample was done up to 10-7. From the original dilution a streak plate was made using an inoculating loop and aseptic technique, which includes sterilizing the inoculating loop in 70% ethanol, and passing it through the flame of a Bunsen burner. Sub-culturing was done from this original plate by preparing a new streak plate with the isolated bacterial colony. The original colony was also examined under microscope to determine the features of the individual bacterium. Additionally gram staining was done on the colony from the original streak plate. Further tests were done from the sub-cultured streak plate that included starch hydrolysis prepared on a starch agar plate, H2S and motility that were done in SIM deeps, ammonification done in a peptone broth, nitrification done in ammonium sulfate and nitrite broths, denitrification done in a nitrate broth, catalase done by adding hydrogen peroxide to the colony on a second streak plate, and oxygen tolerance investigated in thioglycollate medium. At a later date additional tests that were done are temperature, on TSA agar plates, pH, done in TSB tubes at pH 3, 5, 7 and 9, and osmotic pressure, done on TSA plates of 0, 0.5, 2 and 5% NaCl concentrations. (adapted from Egger, 2009)
The original colony that was isolated from the original streak plate was orange, convex with a smooth texture and an opaque/translucent appearance. When the colony was observed under the microscope at 1000x magnification, it had a rod shape, and was arranged in clusters. The dimension of the bacterium was 3-4 µm x 0.5-1 µm. After this was done the colony underwent gram staining which resulted in a positive gram stain result, as the bacteria appeared purple under the microscope. The bacterium appeared to hydrolyze starch because when iodine was added, a clear zone formed around the bacteria growth. The next test examined was the SIM deep. The SIM deep did not have any black participate which indicated that there was no hydrogen sulfide production, but there was movement away from the original stab line which showed that the bacterium had motility. Following the SIM deep, all of the tests that were done to determine the bacterium's reaction with nitrogen in any form were examined and all came back negative as shown in Table 1. When inspecting the test tubes contain thioglycollate medium, it was seen that the bacterium grew throughout the tube, but with a higher concentration near the top of the medium. This shows that the bacterium is a facultative anaerobe. When the bacteria on the TSA plate had hydrogen peroxide added to it, it began to foam, which indicated that it was positive for the catalase test. The final three tests were done to determine the optimum conditions under which this bacterium grew. The results for the optimal tests are summarized in Table 1. The optimal pH test revealed that it was able to grow under all pH conditions it was put into, even though its optimal pH was 7. For both the optimal temperature test it did grow at 4°C and 22deg;C, but not as well as it grew at 37deg;C. The salt concentration test revealed that it had similar growth at 0 and 0.5 % NaCl, with a small amount also growing at 2%.
One distinction that focused the bacterium search was the color of the colonies. The colonies of bacterium that were isolated on the sub-culture streak plate were orange (Table 1) in appearance. The colour of the colony is the same as what is stated in the National Standard Method (2007) that was put out in conjunction by many microbiology groups of the United Kingdom for Cellulomonas sp. Bergey's Manual of Systematic Bacteriology (Sneath et al. 1986) has the colours of the Cellulomonas genus as ranging from lemon yellow to canary yellow. The colour for Cellulomonas sp. appears to be quite variable as a study done by Rahman et al. (2009) has the Cellulomonas sp. as light brown and brown. The rest of the colony identification aspects associated with Cellulomonas sp. are quite constant throughout the different books and journals. The colonies can be round in shape, opaque, convex and smooth (Sneath et al 1986; Rahman et al 2006).
The unknown bacteria had many characteristics that were similar to many other bacteria. However, there were a number of characteristics that made it easier to identify. Having a gram positive bacterium greatly reduced the amount of bacteria that it could be, as the majority of bacteria are gram negative. The second factor that helped narrow down the search for the bacterium was that is a rod bacterium. When looking at the gram positive, rod bacteria, it was easiest to narrow the search down by the size of the bacteria. The unknown bacterium ranged in size from 0.5-1 µm x 3-4 µm. This was comparable to Cellulomonas sp. as they range in size from 0.5-0.8 µm x 2-4 µm (Sneath et al. 1986; Washington et al. 2006). Also the shape of the rods, which varied (Table 1) from straight to V shaped also is constant with what is stated for Cellulomonas sp. in Sneath et al. (1986), and Washington et al. (2006). The remaining bacterium identification tests shown in Table 1 had comparable results for Cellulomonas sp. in Sneath et al. (1986), Washington et al. (2006) and Rahman et al. (2009). They all show motility, starch hydrolysis, NO3 reduction and catalase as positive test results. They also all show negative results for the H2S test, ammonification, indole production, and nitrification. As well as the previously mentioned tests Sneath et al. (1986), Washington et al. (2006) and Rahman et al. (2009) all have the bacterium Cellulomonas sp. as facultative anaerobes, mesophiles, and neutrophiles. The commonality of all these tests with the unknown bacterium that was isolated led to the belief that the bacterium isolated was indeed Cellulomonas sp. Further tests would need to be conducted to determine which species of Cellulomonas that it was.
Though the unknown bacterium was positively identified, there are many other tests that could be done that would insure that indeed it was Cellulomonas sp. Some of the other tests that could have been done are tests for what source of sugars they utilize in production, as results for these tests are listed in both Sneath et al. (1986) and Washington et al. (2006), what was found in their cell walls, hydrolysis of gelatin (Sneath et al. 1986; Washington et al. 2006) and whether or not the bacterium needs biotin and thiamine for growth (Sneath et al. 1986). These tests would help concrete or discourage the results of the tests shown in Table 1. The tests that were performed for this experiment are limited because they are general tests, that can have variable results based on techniques used in the lab, and not all tests are entirely conclusive. Some tests need to have other tests done to solidify the results found, especially in the nitrifying and denitrifying tests as the results were not always decisive. One of the other bacterium that was isolated in this experiment, though it was not identified in this report, had a positive test result for nitrifying, but then within a few minutes changed to a negative result. The results for this test then came into question for the results for the other bacterium.
The unknown bacterium, Cellulomonas sp., was found in the agricultural soil. The possible reasons for it being found in agricultural soil is that it is most likely one of the bacteria that helps to break down organic material in and on the soil. One of its major roles is the break down of cellulose, and the principal enzymes found in Cellulomonas sp. have been widely study (Sneath et al. 1986). Cellulomonas sp. can also be found in waste materials with high cellulose content (Rahman et al. 2009; Sneath et al. 1986). In one study done by Riis et al. (2003) it was found to help break down diesel fuel that had been leached into the soil. Cellulomonas sp. major role appears to be that of soil waste reduction.
The results of the tests summarized in Table 1 led to the identification of the unknown isolated bacterium. The many tests corresponded to similar results posted in Sneath et al. (1986), Washington et al. (2006), and Rahman et al. (2009) for the Cellulomonas genus. The identification of the bacterium that started with colony and then bacterium characteristics narrowed the search for the identity of the unknown bacterium to Cellulomonas sp. The ability to identify the bacterium is a considered a success for this experiment.
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