There may be no sign of a single living organism in a sample of soil, water or another type of sample taken from the natural environment that is viewed by the unaided eye. However, if the sample is viewed through a microscope, hundreds to even millions of microorganisms may be observed in a field of view only 50 µm long. A handful of soil may contain more than 10 billion bacteria of thousands of different species, making the soil the most complex ecosystem on earth (Torsvik and Ovreas 2002).
For their growth and survival, microorganisms depend on the nutrients and conditions of their surrounding environment. Since there is a vast range of different environments in the world, bacteria can be classified into different groups based on the amount of oxygen, salt, or other environmental condition that they are exposed to.
The majority of bacteria that is cultivated from soil are aerobic, requiring oxygen to survive. These bacteria break down complex organic substances into carbon dioxide, water and ammonia (Clark 2005). Since every other organism on the planet depends on the breakdown of these complex organic substances, either directly or indirectly, soil bacteria are of huge importance to sustaining life on earth.
A single bacterial colony was isolated from a sample of agricultural soil and a series of different tests were used in order to classify and identify the bacterium. Using the data collected, the ecological role of the bacterium in its environment was also considered.
METHODS
Serial dilutions of 1 gram of agricultural soil sample were made in sterile deionized water (Robertson and Egger 2010). Various pour broths, slants, deeps and streak plates were inoculated with samples from the 10-2 dilution. The rest of the dilutions, 10-3 to 10-7, were used for the pour plates. These broths and plates were used to culture bacterial colonies that were present in the soil sample.
One of the colonies was chosen to be cultured in one of the mediums, the streak plate for our experiment, and we used a sub-culturing technique to isolate the bacterium. A loopful of bacteria from the chosen colony was inoculated and prepared on a streak plate similar to the one used for the culturing technique. This process of sub-culturing allowed for the isolation of the single bacterial colony.
The appearance, size, texture, and other physical properties of the colony of the isolated bacterium was observed and recorded. The bacteria was also observed under the microscope for the size, shape and arrangement of a single bacterium from the colony. A Gram-staining procedure was used on the colony to determine if the bacteria were Gram-positive or Gram-negative.
Different environmental factors on the bacteria were tested. Bacteria were inoculated onto four plates that were incubated at temperatures 4, 22, 37 and 50°C. Bacteria were also inoculated onto four TSB tubes of different pH, 3, 5, 7 and 9. The bacteria's tolerance for osmotic pressure was also examined by inoculating the bacteria onto four different TSA plates at different salt concentrations of 0, 0.5, 2, and 5% NaCl.
Since bacteria are capable of producing or breaking down different chemicals, we tested these biochemical characteristics of the bacteria. A series of differential media were inoculated and after incubation each medium was examined for the presence of specific end products. The bacteria were tested for starch hydrolysis, the production of H2S and indole, ammonification, nitrification, denitrification, and nitrogen fixation (Robertson and Egger 2010).
RESULTS
The following tables summarize the results from the different tests performed on the unknown bacteria isolate.
Table 1.1 Morphological Test Results for the Identification of the Unknown Isolated Bacterium Found in a Sample of Agricultural Soil
Test Results
Colony morphology
White/creamy, filamentous, dull appearance, opaque, medium texture, diameter of 8 mm
Cell morphology (1000x magnification)
Rod shaped, cells in bunches, 2-4µm x 1µm
Gram Stain
Positive
As noted in Table 1.1, the isolated colony was non-pigmented and had filamentous form, almost looking like small feathers. Under a magnification of 1000x, the bacteria were rod-shaped with dimensions 2-4µm x 1µm and found in bunches. During the Gram stain procedure, the bacteria stained purple indicating that they were Gram-positive.
Table 1.2 Effects of Environmental Conditions for the Growth of the Unknown Isolated Bacterium Found in a Sample of Agricultural Soil
Test Results
Oxygen Tolerance
Obligate aerobe
Optimal Temperature
22°C
Optimal pH
7
Optimal salt concentration
0
Fluctuating environmental conditions seemed to have a large effect on the isolated bacteria. The bacteria grew only on the medium incubated at 22°C, and there was no sign of growth on the other plates incubated at the other temperatures. As shown in table 1.2, the optimal growth was found on the plate containing a pH of 7. However, there were some bacteria present on the plate of pH 9, but there was no growth present on the plates smaller than the pH of 7. The greatest amount of growth was observed at 0% NaCl concentration. There was some growth present on the 0.5 and 2% NaCl concentrations but there was no sign of survival on the 5% NaCl plate.
Table 1.3 Biochemical Test Results for the Identification of the Unknown Isolated Bacterium Found in a Sample of Agricultural Soil
Test Results
Starch Hydrolysis
-
H2S reduction
-
Motility
+
Ammonification
+, medium yellow color
Indole production
-
Denitrification (N03-to NO2-)
+
Denitrification (N03-to NH4+ or N2)
-
Nitrification (NH4+ to NO2-)
-
Nitrification (NO2-to N03-)
+
Catalase
+
The results from the biochemical tests are listed in table 1.3. When treated with iodine, there was no color change indicating that starch hydrolysis was not present. There was no black precipitate present in the SIM deep, and no color change after the application of Kovac's reagent indicating that there was no H2S or indole produced, respectively. There was growth in areas away from the stab line indicating that the bacterium was positive for motility. A medium yellow color was observed in the peptone broth with Nessler's reagent indicating that the bacteria were positive for ammonification. The unknown bacteria isolate was able to reduce nitrate to nitrite which was confirmed in the color change when sulfanilic acid and N,N-dimethyl-1-1-naphthylamine were added to the culture. The growth of the bacteria was present only near the top of the tube indicating that the microorganism was an obligate aerobe. Catalase activity was present indicated by the formation of bubbles after adding hydrogen peroxide to the colony.
DISCUSSION
The results of the many tests that were used to identify our unknown bacterial isolate to be of the genus Nocardia. Although not unique to this group of bacteria, rudimentary to extensively branched vegetative hyphae, 0.5-1.2 mm in diameter, are found growing on the surface of agar of these bacteria (Williams et al. 1989).This string-like, filamentous form was observed on our agar plate. One of the colonies was measured to be 0.7 mm in diameter. Nocardia are also documented to be aerobic, rod-shaped to coccoid, and catalase positive. The experimental results support the data. The growth of our unknown bacteria occurred only at the top of the broth of the test tube where most oxygen was available and there was no growth observed in the other parts of the test-tube. Only rod-shaped organisms were seen under the microscope and bubbles were observed after added hydrogen peroxide to test for catalase.
However, not all of our experimental results match the literature data. Nocardia almost always have aerial hyphae, that can be viewed microscopically, and long chains of well to poorly formed conidia may occasionally be found on the hyphae (Williams et al. 1989). Our bacterium isolate had a medium texture observed by the unaided eye and aerial hyphae were not observed under the microscope. There were no conidia present when the specimen was looked at under the microscope, however, more parts of the bacterial colony should have been considered. Also, Nocordia are documented to be non-motile organisms. Motility turned out to be positive for our bacterial isolate. The results may have been an error during the experiment. The stab line may have taken a horizontal route into the tube causing bacteria to grow and giving a false result of motility.
Other tests should be considered to further confirm our bacteria isolate to be of the genus Nocardia. Bacteria that belong to this genus are largely defined on the basis of cell envelope lipid and peptidoglycan composition (Williams et al. 1989). Another test that can be performed to confirm the genus of our isolate bacteria is to test the susceptibility to the antibiotics bleomycin and mitomycin which can help determine if our bacteria belongs to the genus nocardia or rhodococci (Williams et al. 1989).
Our microorganism is likely to have en ecological importance to its environment. It is not surprising to see that the bacteria isolate grew best at conditions similar to those found in the soil, summarized in table 1.2. Many soil microorganisms act as controllers of atmospheric trace gases including H2, CO, CH4, and OCS (Conrad 1996). Almost all species of Nocardia are positive for the production of nitrate reductase (Williams et al. 1989). Nitrate reductases are enzymes that reduce nitrate (NO3) to nitrite (NO3). Our isolated bacteria tested positive for denitrification. Bacteria that are capable of denitrification prevent excess nitrate from accumulating in the soil and leaching into water. They reduce the nitrate into nitrite which is ultimately reduced by other microorganisms into N2, a component of the atmosphere.
The isolation and identification of our bacteria from agricultural soil was successful. The majority of the results from our test support that the bacterial isolate belongs to the filamentous, Gram-positive, aerobic genus Nocardia. However, further tests should be considered before confirming this genus because other groups of bacteria may share all of these common characteristics.
