Various microbes can be found in the soil, on the ground. In soil, there are communities that can be composed of archaic, bacterial or prokaryotic colonies (Blair and Egger 2010). Different bacterial colonies differ from one another based on their morphological appearances and shape. The bacterial colonies can be assessed by their form, elevation, margin, appearance, optical property, texture and color.
Organisms in the soil require certain amounts of oxygen in order to survive harsh conditions. With oxygen in mind, the growth and survival of the organisms depends on the location of nutrients in the soil. For example, agricultural soil can be either rich or deficient in nutrients, but this depends on where the soil is located and how it is being taken care of (Blair and Egger 2010). This can also be the case for forest soil, however, in more heavily forested areas, nutrition richness would be greater as less wooded areas lose their nutrition level due to soil erosion.
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Not only can soil nutrition lead to growth of microbes, but it can help play a role in the pH, soil salinity and temperature of the microbe (Blair and Egger 2010). These factors can help with the determination of what type of microbe is present in a certain soil type. To determine what microbe is present in a soil type, tests of concentration, pH, soil salinity and temperature can be done to identify a bacteria. Therefore, the purpose of this study is to be able to isolate a bacterial colony from agricultural or forest soil and perform tests to identify the bacteria present.
Agricultural and forest soils were diluted to a 10-2 dilution and with that, dilutions of 10-3, 10-4, 10-5, 10-6 and 10-7 were created. The soil that was isolated for our study was agricultural soil. The 10-2 dilution was used for culturing in a broth, a deep, a slant and a streak plate, while using aseptic techniques (Blair and Egger 2010). Both soils were treated with the dilutions of 10-2 to 10-7. This was done in pour plates, with four of them being TSA plates (Tryptic Soy Agar) and the other four containing SDA (Saboraud Dextrose Agar). The agriculture and forest soils in the 10-2 dilution were cultured into spread plates, with one plate being aerobic and the other anaerobic. These cultures were left for a period of time so the bacteria could grow. From the grown bacteria, four samples of single bacteria were taken. These samples were then sub-cultured onto streak plates, using the technique of streaking (Blair and Egger 2010). The bacteria from the pour plates were determined by using the method of enumeration (plate counting method for the soil samples). After using the enumeration method, the selected bacteria were subjected to various testing to see what type of bacterium was present. One method done was gram-staining and from this, the bacterium was looked under a microscope to detect morphology, cell type, shape and size of the bacterium. On the sub-cultured bacteria, testing for starch hydrolysis was conducted. The first bacterium was tested with iodine for any starch hydrolysis, where the second was to look at the production of H2S in the SIM plates (Sulfide, Indole and Motility medium). Ammonification, nitrification, denitrification and nitrogen fixation were tested on the bacteria using the reagents of diphenylamine reagents, Nessler`s reagent, sulfanilic acid, sulphuric acid, N, N-dimethyl-1-1-naphthylamine with zinc and Trommsdorf`s reagent. A test was done for catalase by using 3% of hydrogen peroxide. A final set of tests done were involving temperature, pH and osmotic pressure. The sub-culture for temperature was done by streaking each of the four bacteria onto a streak plate and placing under the temperatures of 4°C, 22°C, 37°C and 50°C. The pH was tested by using TSB tubes and placing the bacteria into four different pH conditions (3, 5, 7, and 9) and taking an absorbance at a wavelength of 580 nm. By using the TSA medium containing different percents of NaCl concentrations (0%, 0.5%, 2% and 5%), the osmotic pressure was tested. From all the tests and isolations done, one bacterium was selected (bacterium 1) and identified (Blair and Egger 2010).
Table 1. Bacterial morphology for Bacterium 1
Always on Time
Marked to Standard
Circular, raised, filamentous, shiny, opaque, yellow in color, smooth
Cell shape and size
Circular, round shaped, dimensions of 1x1 μm
Table 2. Biochemical tests on Bacterium 1
Positive for NO2- but negative for NH3 or N2
Positive for NO2- but is negative for NO3-
Table 3. Environmental factors that can affect growth for Bacterium 1
22°C - 37°C
Optimal osmotic pressure (% NaCl)
Bacterium 1 was chosen for identification. It is a circular gram positive bacterium (Table 1). From the biochemical tests completed, bacterium 1 is able to hydrolyze starch but it has very minimal motility. The bacterium is also able to allow proteins to be broken down and form into many amino acids and let ammonification to be released. Not only that, bacterium 1 is able to oxidize ammonia into nitrate and reduce nitrate to nitrite. It can break down hydrogen peroxide as well (Table 2). The optimal environmental factors give an insight as to what environment the bacterium grows at its best (Table 3). Enumeration was also done to see how much of the bacterium was present, based on each condition in the tables above. By counting, it was determined that the bacterium was present in large numbers, mostly in the pour plates.
From all the tests completed and the results seen, bacterium 1 can be identified as it is a gram positive cocci bacterium. This and the bacterial morphology can help determine what genus the bacterium could belong to. The type of oxygen requirements for the bacterium can also help identify the identity. The bacterium is microaerophile, which is specific type of microorganism that requires oxygen to survive, but requires environments containing lower levels of oxygen than are present in the atmosphere (Be'er et al. 2009). With all of these assumptions, a genus can be determined, including the bacterial morphology and the catalase production levels.
From all the results, the genus of the bacterium could be Staphylococcus, as it is more evident in water and soil conditions (Bergey and Holt 1986). This genus is able to form in pairs and in irregular clusters. The colonies are also a pale orange in color, as seen in the study. Staphylococcus is able to tolerate a concentration of 5% NaCl, with a circular, smooth and spherical morphology. Nitrate is also able to be reduced into nitrite in this genus (Bergey and Holt 1986).
Based on results for the hydrolysis of starch, H2S reduction and osmotic pressure, a species can be narrowed in the genus of Staphylococcus. Even though all the results do not match a specific species, the bacterium is closest to the species S. Hyicus ssp. hyicus. It seems that this species is closest to the bacterium as it is spherical, with dimensions of 0.8x1.1 μm and is able to withstand concentration of 10% NaCl and a pH of 4.8-5.3, with the best temperature growth at 37°C. It is able to reduce nitrate to nitrite and it is able to produce ammonia from arginine (Bergey and Holt 1986). From the results, the optimal pH is 7, but the pH for S. Hyicus is between 4.8-5.3. This could be a result of the study being done in a controlled environment, and not in a forested area. With the results stating that nitrates can not be able to reduce lower than a nitrite, but S. Hyicus can, this could be due to the fact that the bacterium chosen was not able to be fully incubated on the streak plate, leading it to be unable to reduce nitrite further down (Kenyatta et al. 2003).
The genus and species can be mostly found in water and soil habitats, but it can also be found in humans and pig. However, S. Hyicus does not cause serious damage or harm to a human being. Since the sample isolated was the agricultural soil, and the species determined is S. Hyicus, the species is quite rich in nutrients and it can help in the growth process of trees and plants as nitrogen is present in it.
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Another way to possibly identify a bacterium is to isolate DNA from a soil sample and compare it to the DNA from other geneses'. Another method is to test for specific antigen antibody reactions, which is an example of serological tests (Ohba and Aizawa 1978). Since bacteria can serve as antigens, production of antibodies in a mammal and testing of antiserum can be done by agglutination testing. In this test, a drop of a bacterium is put onto a slide with the anti-serum of an infected mammal and looked at by the use of a microscope. If clumping happens, the bacterium is considered to be either the same or related to the bacterium used as the antigen (Ohba and Aizawa 1978).
A source of possible error that could have occurred in the duration of this study could have been that there was some sort of cross contamination that occurred during the incubation or sub-culturing process. The correct technique of aseptic inoculating could have not been done properly, therefore contamination could have resulted. Also, there could have been mis-labelling that occurred as the top of the plates used in the study were labelled instead of the bottom. This can result in the lid of a plate being put onto another plate that does not contain the specific bacterium as the label describes, thus leading to varied results.
With the isolation of a soil sample and various tests that have been done in order to determine what genus and species the sample could belong, it was found that the genus could most possibly be that of Staphylococcus. In this genus, the species could be, as the results are quite close to the characteristics of S. Hyicus. By being able to determine the genus and species, the study was a success.