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The intended purpose of the study was to identify a bacterial-based organism which was extracted from either agricultural soil or forest soil. There were other key components of the study, such as the selection of different methods used to identify bacteria in their forms of growth and their sustainability in different environments. The commonly used test of inoculating and monitoring the growth of organisms in test tubes and on plates was introduced.
Soil diversity among microbes is enormous; a thousand species may be found in one gram of soil (Meier et al.2007). There are many factors that are relevant to what types and how abundant a microbe may be in a given soil (Mummey et al.2006). Both biotic and abiotic factors can influence the types of microbes, and it remains a challenge to determine soil microbe diversity (Mummey et al.2006). Such factors as light, water, heat and nutrient availability (carbon, nitrogen, etc.) may factor into a microbe's longevity.
Along with the already described factors can be predation from other microbes and other environmental factors that can affect all forms of life. Even stratification of the microbe in the scale of being closer to the surface or further down in the soil, where less oxygen is available and less light may shine, can greatly influence its metabolism and its functionality in being aerobic or anaerobic (Mummey et al.2006).
Material and Methods:
A sample of agricultural soil was taken and placed in distilled water. It was then diluted to the sixth power by taking 1ml of the original sample and progressively diluting it five more times. Then an inoculating loop and aseptic technique was used to culture four different types of bacteria from the soil. From there, a series of tests were used to determine the type of cultured bacteria. As an example, to determine whether the bacteria was gram positive or negative, a crystal violet stain, Gram's iodine, 95% ethanol and Sarafin stain were all used in a procedure detailed in our lab manual (Lab Manual.2010).
Next after being viewed in culture with just general observations, the cell shape and physiology were determined using a microscope. Under the microscope, the cell shape, arrangement and dimensions, as well as reassurance of the gram staining identification and a comparison to prepared slides of gram positive and negative bacteria per guidelines in the lab manual (Lab Manual.2010), were used to further identify each of the cultured bacteria.
A second series of tests were run to determine if carbon, nitrogen and/or sulfur cycling were part of the bacteria's metabolism. A-amylase and iodine were used to indicate the presence of starch (Lab Manual.2010). Kovacs reagent and Nessler's reagent were used to determine the presence of H2S and ammonia, respectively (Lab Manual.2010). To test for nitrification, there were many steps to determine how the nitrification was occurring; the reagents used were Nessler's reagent, Trommodorf's reagent combined with sulfuric acid, and diphenylamine reagent and sulfuric acid to determine which cycle of nitrification was being used by the respective bacteria (Lab Manual.2010). Another test for denitrification involved reagents A, B, and C if needed (Lab Manual.2010). Oxidase and hydrogen peroxide were used to test for aerobic respiration (Lab Manual.2010). The use of thioglycollate medium allowed for the determination of oxygen tolerance by locating the position of optimal growth in the test tube (Lab Manual, 20 10).
Other environmental factors were assessed as well, such as temperature, pH, and osmotic pressure. All tests were performed as detailed in the lab manual. Using TSA plates and varying the temperature and osmotic pressure, optimal, minimum and maximum temperatures and osmotic pressures for growth were established for each bacterial culture. TSB tubes and a spectrophotometer were used to determine optimal, maximum and minimum pH levels for growth (Egger, 2010). After performing the tests, one bacterium was chosen for identification using resources from the library and supplied sheets.
Table 1.1 Bacterial identification basic characteristics
Single = Rod
Length = 4um
Width = 1um
Preferred Growth in Thioglycollate
Top = Obligate Aerobe
A preliminary look at the bacteria, after isolation and culturing from the agricultural soil, showed a slightly shiny; circular bacteria, with slight convective shape and optical opaqueness when examined closely with the naked eye. The results of the preliminary examination are summarized in Table 1.1. It should be noted that the bacteria did seem filamentous in nature as there seemed to vein like connections within the bacterial growth as a whole. Upon further inspection under a microscope, the bacteria were determined to be rod shaped with a tendency to cluster in arrangement. It had a dimensional length of 4um and a width of 1um, was gram positive and showed positive for the presence of peptidoglycan as recorded in Table 2.2. Also the bacteria tended to grow optimally on the top of the thioglycollate test tube indicating that it is an obligate aerobe.
Table 2.2 Bacterial respiration forms, and determinate reaction results
(+) for (yellow/brown colour)
The results of the H2S test and motility for the bacteria were negative. However, the bacteria tested positive for nitrification by producing a variety of colors in the described tests previous detailed in the above "methods" section of this report. It also showed positive signs for denitrification by producing a pink to red color in the first stage of adding reagents A and B. The bacteria also produced a positive reaction towards the catalase and oxidase reactions as it bubbled and turned purple in each of the respective test procedures as summarized in Table 2.2. The optimum temperature obtained for growth of the bacteria was 37 oC; a maximum temperature for growth of 50 oC and minimum of 22 oC indicated that the bacterium was mesophilic. The optimum pH of 6 for growth is shown in Table 2.2; the maximum and minimum pH values found for growth demonstrate that the bacteria is a neutrophile at its optimal pH. The osmotic pressure test produced results shown in Table 2.2 that demonstrate a range between 0 and 2% being the optimal range for growth in an osmotic environment.
Based on the above findings and comparison of the information to that found in the Bergey Manual of Systematic Bacteriology, it has been determined that the bacteria inoculated and cultured is of the streptomyces genus. It tested positive for gram staining, hydrolysis, ammonification, and denitrification (NO3 to NO2) as described by the manual (Bryant et al,1989). The bacteria also had the following characteristics of the streptomyces genus: aerobic respiration, mesophilic, a neutrophile for pH, and halotolerant to osmotic pressure changes. It also reacted positively in both the oxidase and catalase tests as does the streptomyces genus. The bacteria was also distinct in that it had filamentous looking veins and seemed to be cotton like in appearance, which is also a common characteristic of streptomyces (Bryant et al. 1989). It should be noted that in the manual it states that streptomyces may have pigmentation but it can also pick this pigment up from its surrounding environment, which may have been the reason why the cultured lab bacteria contained no pigment (Williams et al.1989).
A resistance test to certain antibiotics, and maybe a look at whether the bacteria could produce spores or endospores, may have helped to further ensure the classification of the bacteria. Spore chain morphology and a spore surface ornamentation examination may have helped to better classify the species of streptomyces. There are some incongruencies between the cultured bacteria and streptomyces as the cultured bacteria has a slight diameter difference from the given book value. Also streptomyces and streptoverticullum are quite close in characteristics except for areas pertaining to spores and mycelium branching (Bryant et al. 1989). This again may have been resolved by looking at the spores closer under an electron microscope.
Streptomyces is abundantly found in natural soil environments and areas where there is organic decay. Very few species of it have been found to be pathogenic to humans and animals. It has lately been found to be better to use than E. coli for production of antibiotics, as it is commonly a nonpathogenic filamentous bacteria that has a high capacity for secreting protein. In particular, Streptomyces lividans has the ability to secrete human proteins at a commercially viable level (Binnie et al. 1997).
The determination of the bacteria streptomyces and culturing of it was interesting in all aspects as so many tests were used to narrow down the identification of the bacteria to just its genus. It seems hard to believe that so many bacteria may co-habitate in such a small area of soil. However, the importance of bacteria and their influence on the environment from decomposition to antibiotic production is immeasurable. Streptomyces seems to be very useful and highly functioning in both its natural environment and for use of in antibiotic and medicinal purposes.
Binnie, C., D. Cossar, and D.I.H. Stewart. Heterologous biopharmaceutical protein expression in streptomyces. Trends in biotechnology 15. 315-320.
Biology 203 Laboratory Manual. 2010. Aseptic technique and culturing of soil microorganism. pp 4-11. University of Northern British Columbia, Prince George, B.C.
Biology 203 Laboratory Manual. 2010. Microscopes and morphology of bacteria and fungi. pp.12-20. University of Northern British Columbia, Prince George, B.C.
Biology 203 Laboratory Manual. 2010. Biochemical testing and nutrient cycling. pp. 21-28. University of Northern British Columbia, Prince George, B.C.
Biology 203 Laboratory Manual. 2010. Effect of environmental factors on bacterial growth. pp. 29-33. University of Northern British Columbia, Prince George, B.C.
Bryant, M.P., N.Pfennig, and J.G. Holt.1989. Bergey manual of systematic bacteriology volume 3. Lippincott, Williams &Wilkins. New York. pp. 2452-2471.
Mummey, D., W. Holben, J. Six, and P. Stahl. 2006. Spatial stratification of soil bacterial populations in aggregates of diverse soils. Microbial Ecology 51: 404-411.
Meier, C., B. Wehrli, and J.R. van der Meer. 2007. Seasonal fluctuations of bacterial community diversity in agricultural soil and experimental validation by laboratory disturbance experiments. Microbial Ecology 56: 210-222.
Williams, S.T., M.E. Sharpe, and J.G. Holt. 1989. Bergey manual of systematic bacteriology volume 4. Lippincott, Williams &Wilkins. New York. pp. 1962.