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Soil is a complex medium for life. It consists of numerous microorganisms, such as bacteria, fungi, protists, and insects (Campbell and Reece 2008). These microorganisms aid in decomposing plant and animal detritus into a more useful substances, producing nutrient rich soil (Smith and Smith 2001). Out of these microorganisms, bacteria are one of the most diverse and extensive that are involved in decomposition. If these microbes did not carry out these processes, all nutrients would be lost as inaccessible organic matter. Bacteria are involved in almost all biological processes as well as many industrial ones. Distributions of these bacteria in the soil depend on temperature, acidity, and salinity, of the surrounding environment.
Many plants, fungi, and animals live in symbiosis with bacteria; however, many bacteria can cause infections and disease in these organisms (Smith and Smith 2001). Isolating bacteria helps to identify it, which can lead to understanding its limitations. It can then be determined what the bacteria is useful for, or if it is harmful to other organisms. By isolating these bacteria, studies can be conducted to potentially find cures for known diseases. However, a pure culture has to be isolated first before any further tests are preformed on the bacteria. By isolating a culture, physiological characteristics, biological tests and environmental responses can be observed to help determine the identification of the bacterium.
All procedures are based on the Winter 10A Biology 203 lab manual (Egger and Robertson 2010). Dilutions of 10-2 to 10-7 of the agricultural and forest soil were prepared with water. Then, 2 streak plates were made using the 10-2 dilutions for both soil samples. Using melted TSA medium, 8 pour plates were prepared for dilutions of 10-4 to 10-7 for each sample. Using 0.1mL of the 10-2 dilutions of both soils, 2 spread plates were assembled.
One week later, all the plates were observed and 4 different bacterial cultures were chosen to be sub-cultured from the agricultural and forest soil dilutions of 10-2 (spread plates). These were cultured on to 4 new TSA streak plates. Gram staining was performed on all 4 of the sub-cultured bacteria. Their cellular morphology was observed under a microscope.
The next week, the 4 sub-cultures' morphology was observed. Then, 2 starch agar plates were produced using the 4 bacterial colonies, 2 on each plate; as well, 2 SIM deeps were inoculated with each bacterium. These were then incubated for 48 hours at 25 ÌŠ C. The 4 bacterium were also inoculated in 4 peptone, 4 ammonium sulphate, and 4 nitrite broth tubes. These were then incubated for 7 days at 25 ÌŠ C. Each bacterium was then placed into 4 nitrate broth tubes and 4 thioglycollate medium tubes and was incubated for 7 days at 22 ÌŠ C.
The following week, the bacteria were tested for starch hydrolysis, H2S production, motility, ammonification, nitrification, denitrification, and catalase activity. As well, the bacteria were made on TSA plates to be incubated at 4, 22, 37, and 50 ÌŠ C. They were also placed on TSA plates of concentrations of 0, 0.5, 2, and 5% NaCl, and to broth tubes to be tested at pH 3, 5, 7, and 9. In the last week, the bacteria's growth was noted at each temperature, pH and NaCl concentration. One bacterium was picked for identification.
The isolated bacterium was found to be raised in elevation, shiny and white in appearance, and smooth in texture. The colony also appeared viscous and was 15mm in diameter. All of the sub-cultures did not change in appearance from the parent colony. As shown in Table 1, the bacterium responded positively to majority of the biological tests. Under 400 times magnification, bacillus shaped cells (dimensions 2.0µm x 0.5 µm) were observed and were found to be in a strepto cell arrangement.
Table 1. Environmental responses and physical properties of K. terrigena.
22 ÌŠ C
Optimal NaCl concentration
Based on the environmental responses and morphology of the isolated bacteria culture, it was found to belong to genus Klebsiella and the most likely species was K. terrigena. The morphology and viscosity of the bacterium helped to place it in the genus Klebsiella; the gram staining, starch hydrolysis, and catalase activity helped to narrow the identification to the species K. terrigena (Egger 2010). K. terrigena bacteria are also found in unpolluted water and soils, as well as drinking water and vegetation (Krieg and Holt 1984). This is consistent with the experiment as the bacteria were isolated from forest soil.
This bacterium is found in a wide range of habitats, and is an opportunistic pathogen for humans and other animals, and is commonly found in hospital environments. Usually when this genus is found in nature it is often due to human and animal contamination, but in other cases it is not directly linked to either (Bagley 1985). When found in soil and vegetative environments it may be in an early life stage waiting to be consumed or picked up by an animal so it can mature and reproduce in a more suitable environment (Willey et al. 2008).
The identification of the bacteria might have been more confidently identified if a test for indole production was conducted, as K. terrigena is positive for this (Krieg and Holt 1984). DNA sequencing could have also helped to confirm the species of the isolate. The resulting DNA sequence could then be compared to sequences of known bacteria to find a match. Some limitations to this experiment would be that it is easy to identify the incorrect bacteria due to insufficient testing. The life stage of the bacterium was also unknown and therefore is a limiting factor because the bacterium could appear or behave differently depending on what stage it is in of its life cycle, leading to inadequate or misleading results. If the tests in the identification manual were conducted during different life stages of the bacteria, then the results may not be comparable. Also, many bacterial colonies can have similar results to the same tests; therefore, if more tests were performed then a better identification could have been made.
A high amount of error in this experiment is due to lack of repetition. If the tests were repeated numerous times then the results of each test could have been compared and a statistical analysis could be conducted. The results could have also been altered by human contamination as it is inevitable. The inoculating loop could have been too hot when making sub-cultures; this could have resulted in little or no bacterial growth. Although there was a lot of possible error, the isolated bacterium can was confidently found to be of genus Klebsiella and the most likely species being K. terrigena. Further studies should be conducted to support this identification.
Bagley, S.T. 1985. Habitat Association of Klebsiella species. Infection Control. 6 (2): 52-58.
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Egger, K.N. 2010. Common soil bacteria Key. UNBC.
Egger, K.N. and Robertson, S. 2010. Microbiology Laboratory Manual. Prince George, British
Columbia: UNBC. pp. 1-32.
Krieg, N.R. and Holt, J.G. 1984. Bergeys Manual of Systematic Bacteriology, Vol. 1. Baltimore:
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Smith, R.L. and Smith, T.M. 2001. Ecology and Field Biology, 6th ed. New York: Benjamin Cummings. pp. 64.
Willey, J.M., Sherwood. L.M., and Woolverton, C. 2008. Prescott, Harle & Klein's
Microbiology, 7th ed. New York: McGraw- Hill.