There are millions of different microorganisms in the soil, each with a different purpose. The different levels of microorganisms include earthworms, nematodes, arthropods, eukaryotic fungi, protozoa, algae, prokaryotic archaea, and bacteria (Robertson and Egger 2010). Each microorganism serves a different purpose. Some assist in decomposition, mineralization, nutrient cycling, storage, and release of nutrients (O'Gara 2010). By determining the identities of different microorganisms, we can begin to identify their spatial distribution and functions within the soil (Chenu 2006). In the study we performed, we wanted to take soil samples, from forest and agricultural soil, and be able to identify a single bacterial isolate to a genus level through the process of different biochemical test, testing environmental factors, and identifying different physical features.
Two different soil samples were used. Soil #1 was agriculture soil, and soil #2 was forest soil. We added each soil to two separate 10-2 deionized water beakers. Each soil sample was cultured into deionized water with dilutions from 10-3 through to 10-7. We then sub-cultured both 10-2 soil dilutions into three different culture tubes: broths, slants, and stabs. Streak plates and spread plates were made using the 10-2 dilution soil solutions. A pour plate was made for both soils using 10-4 through to 10-7 dilutions. Each culture was incubated. All steps were followed using the laboratory manual (Robertson and Egger).
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From our aerobic cultures, we produced pure cultures by sub culturing four different bacterial colonies. The colonies chosen were based on which colonies had the most difference in physical appearance, and the most distance from other colonies. Using the same colonies that were used to produce our sub-cultures, and following the steps using the reagents mentioned in the manual, we made a gram stain for each colony.
Our pure cultures helped to add detail to the unknown bacteria. We observed the colony and cell morphology (under 1000x oil immersion magnification). We preformed a number of biochemical test and tested environmental factors that may have affected the bacteria. The biochemical tests included testing for starch hydrolysis, H2S production, motility, the ability for ammonification, nitrification, and denitrification, as well as oxygen tolerance and catalase. The environmental factors that were tested were different pH levels (3, 5, 7, 9) different temperatures (4, 22, 37, and 50oC), and different osmotic pressures (0, 0.5, 2, and 5% NaCl). The incubation periods varied for each test (Robertson and Egger).
The isolate I chose to identify was from soil #2 (forest soil), on a pour plate with 10-4 dilution. From the pure culture the colony morphology was a circular, shiny, smooth, and convex. Its optical property was opaque and had pale yellow pigmentation. When we looked under the microscope at 1000x magnification, the cells were coccus-shaped and were formed in clusters of two or more. Each cell was approximately 2.5 to 3Âµm. Most importantly, the bacteria were a positive gram stain (Table.1).
Form: circular, Elevation: convex, Margin: entire, Appearance: shiny, Optical property: opaque, Texture: smooth, Colour: yellow pigmentation
Size: 2.5 - 3 ÂµM, coccus-shaped, cluster or cells
Denitrification (NO3- to NO2-)
Denitrification (NO3- to NH4+ or N2)
Nitrification (NO2- to NO3-)
Optimal Salt Concentration
Table 1. A summarization of the results of the physical appearance, biochemical tests, and tests on environmental factors to help identify bacterial isolate.
The biochemical tests showed important results (Refer to Table 1.). In the starch hydrolysis, after adding the iodine to the culture, the area around the bacteria streak was clear, so therefore the bacteria were positive, and could hydrolyze starch. In the H2S production test, there was no sign of a black precipitate (ferric sulphide) in the test tube, which indicated that there was no production of hydrogen sulphide (Robertson and Egger). However, in the same SIM deep off of the stab line, we could see some bacterial growth, which meant slight motility. We added the Kovac's reagent to see if the enzyme tryptophanase was present; it was not. When we tested for ammonification, we added the Nessler's reagent to a small amount of isolate. The mixture turned yellow, which indicated that the bacteria had a small to moderate amount of ammonia present.
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To test for nitrification we added the Nessler's reagent to the ammonium sulphate broth, we got a strong orange-red colour. By adding the Trommdorf's reagent and H2SO4 to the ammonium sulphate broth, the broth remained clear. By adding Trommdorf's reagent and H2SO4 to the nitrite broth, we got a red-brown colour. When we added the diphenylamine reagent and H2SO4 to the nitrite broth we got a blue colour. All of the above, other than the ammonium sulphate added to Trommdorf's reagent and H2SO4, indicated that the bacteria were positive for nitrification, and that the bacteria were positive for ammonification.
In the denitrification test, after adding both reagents A and B, the broth remained yellow, indicating that nitrate was not reduced or that nitrite was reduced all the way to ammonium ions or N2 (Robertson and Egger). Due to the results, we added reagent C, and the broth turned red, thus nitrate was not reduced to nitrite.
To test for oxygen tolerance we noted the area of growth in thioglycollate medium. There was growth throughout the tube, but more near the top, indicating that the bacteria were facultative anaerobes. However, the bacteria were observed a few days to late, and had already started to die and began to sink to the bottom, so the bacteria may have been obligate aerobes. The final biochemical test we did was to test for catalase. After adding H2O2, to the bacteria, bubbles formed, indicating that there was catalase activity.
Finally we tested for the optimal levels of environmental factors. The different temperatures indicated the most growth at 37oC, with little growth at 4oC and 22oC. However there was zero growth at 50oC. The bacteria were categorized as mesophiles. By measuring the wavelengths of the TSB tubes, the greatest wavelength, which is the optimal pH, was 0.287Î», which was at a pH of 7. The lowest to the third highest pH were 5, 9, and 3, respectively. The bacteria fell into the category of neutrophiles. Finally the amount of isolate growth at different levels of osmotic pressure showed that the optimal percent of NaCl was 0.5%, which had a large amount of growth. The minimum percent of NaCl was at 0%, and the maximum was at 5%. The bacteria were known as halotolerant.
From the results of the tests completed the identification of my bacterial isolate is most likely Sporosarcina, a spore-forming package (Holt et al. 1986). Sporosarcina have been located in garden and field soils, ones that have been occupied by man and dog, "- the base of trees where dogs have urinated.", and salt marsh soils (Holt et al.; Chomarat et at. 1990). Most species of Sporosarcina are not considered pathogenetic to humans, except Sporosarcina pulmonum, which may be related to respiratory tract and lung infections (Holt et al.; Chomarat et al. 1990).
Sporosarcina were identified by a number of different characteristics. As Table 1. summarizes, the bacteria were obligate aerobic, coccus-shaped, ranged in size from 2.0 - 3.0Âµm, and were gram positive. They had the ability to be motile with a randomly spaced flagella, in which they moved by tumbling (Holt et al.). Sporosarcina were identified by the formation of an endospore (Doi and McGloughlin 1992), and a capsule indicated by the smooth texture of the colony. Different species of Sporosarcina were distinguished by colony pigmentation, optimal temperatures, and the main location they were found at.
The possible and known roles of Sporosarcina were not well known. Bergey's ManualÂ® suggests that they play an active part in the decomposition of urea.
In order to verify that the isolate I identified was actually Sporosarcina, there are a number of other tests I could do to support my observations. A common two-step screening method is used to identify the different surface layers of gram positive bacteria which would help to quickly verify the bacteria that you are studying (Wahl et al. 2001). Another test that could be used to identify a species of Sporosarcina would be to use acetate of glutamate as a carbon and energy source on strains of Sporosarcina ureae (Holt et al.). We could have studied the nucleic acid sequencing, which would have given us a definite genus level.
In this study, there were some limitations to the tests that we performed. The maximum magnification of the microscopes was 1000x oil immersion magnification. If we had a more powerful microscope we would be able to see more bacterial detail. Also by having the limitation of only being able to observe our results once a week, we missed out on important indications that may have helped us to identify our isolate. Such as testing the oxygen tolerance; the bacteria were starting to die and falling to the bottom of the tube, giving us false results of the oxygen tolerance of our bacteria.
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Some major sources of error that could have led to false results include our gram staining techniques. We could have over-decolourized or over-washed our slides. These could have led to incorrect staining, giving us false positive, or false negative stains (Robertson and Egger). Also, working in teams of two, and different people's opinions of observations and techniques could have led to different interpretation of results.
From the results of the characteristics, biochemical tests, and tests on environmental factors, I can conclude that the identification of my isolate belongs to the genus Sporosarcina. We could, in future studies, attempt to identify the bacteria to a species level, or find more information about the roles they play in nature.
Chenu, C. The spatial distribution of microorganisms and their activities in soil structure. [Internet]. 2006 July 11 [cited 2010 Feb 23]. International Union of Soil Sciences and WCSS. Available from <http://a-c-s.confex.com/crops/wc2006/ tech program/P15502.HTM>
Chomarat, M., de Montclos, M., Flandrois, J.P., Breysse, F. 1990. Isolation of Sporosarcina ureae from a bronchial biopsy in a child with cystic fibrosis. European Journal of Clinical Microbiology and Infectious Diseases. 9:302-303.
Doi, R. H., and M. McGloughlin. 1992. Biology of Bacilli, Applications to Industry. Stoneham, Massachusettes, USA. pp 2-3.
Holt, J. G. Sneath, P. H. A., Mair, N. S. 1986. Bergey's ManualÂ® of Sustematic Bacteriology, vol. 2. Lippincott Williams and Wilkins, Baltimore, Maryland, USA. Pp 1202-1206.
O'Gara, F. Microbiology of Agricultural Soils. [Internet]. [cited 2010 Feb 22]. The IMPACT Project. Available from <http://www.ucc.ie/impact/homef.html>
Robertson, S and Egger, K. 2010. BIOL 203 Microbiology Laboratory Manual. UNBC.
Wahl, R., Raff, J., Selenska-Pobell, A., Mertig, M., Pompe, W. 2001. A fast screening method for surface layers on gram-positive bacteria. Biotechnology Letters. 23:1485-1490.