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In the natural world, a wide variety of microorganisms exist within the soils of the earth. The types of life forms vary from fungi to nematodes to protozoa to bacteria. Each serve their own purpose in the environment, and contribute to the overall structure of the community (Willey et al. 2008).
The focus of our experiments is on bacteria. Like other microorganisms, bacteria play a vital role in their respective communities, and have their own specific needs. Bacteria are very diverse, and are classified into groups depending on their metabolism, morphology, and other characteristics.
Depending on their oxygen tolerance, they are separated into the groups of obligate aerobes, microaerophiles, obligate anaerobes, aerotolerant anaerobes, or facultative anaerobes. Cellular shape and arrangements are also used to classify bacteria. The amount of peptidoglycan making up the cell wall of a bacterium can be used to determine if a bacterium is gram negative or gram positive. Bacteria are further classified by what nutrient and energy sources they use, as heterotrophs or autotrophs, as well as by their growth in varying environmental conditions, such as temperature and pH (Willey et al. 2008).
Bacteria have a fundamental role in the environment. They are very important in the cycling of nutrients, and many bacteria are a key factor in the nitrogen cycle through the processes of nitrification and denitrification, and are also important in decomposition and digestion. However, bacteria are also used for other purposes. These include things such as the use of bacteria for food production, chemical production, pharmaceuticals, waste disposal, and pest control. Lastly, some bacteria are pathogens, causing disease in both plants and animals (Willey et al. 2008).
The goal of this experiment was to identify the bacteria collected from soil samples, based on specific tests and observations.
Materials and Methods
The experiment was carried out as according to the Biology 203 Lab Manual. All individual test procedures and reagents used can be found in the lab manual (Robertson and Egger 2008).
Two different samples containing the bacteria were collected, and diluted to 10-9 to 10-2 samples. Using proper technique, various test tubes and plates were inoculated with the samples. These cultures were incubated in aerobic and anaerobic environments, and growth observations were made. The number of colony forming units was then determined by enumeration. Four distinct colonies were then selected, and colony morphology was noted. These colonies were then sub-cultured, again using proper aseptic technique. Using the microscope, measurements and observations were made. Gram stain tests were then performed on the subcultures. Next, starch hydrolysis, sulfur and motility, ammonification, nitrification, denitrification, and catalase presence were all tested for with their respective tests and reagents, and observations were noted. Lastly, the bacteria were incubated in varying temperatures, pH, and salt concentrations. Optimal temperature, pH, and osmotic pressure were noted for each of the bacteria.
Circular, raised, smooth
(NO3- to NO2-)
(NO3- to NH3 or N2)
(NH3 / NH4+ to NO2-)
(NH3 / NH4+ to NO3-)
22 Degrees Celsius
Optimal Salt Concentration
Figure 1 ââ‚¬" Observations for Unknown Bacteria
The bacterial colony, after incubation, was found to be circular, raised, shiny and opaque, with a smooth texture and black in color. The cells of the colony were observed to be rod shaped (bacilli), and arranged in a singular pattern. The cell was approximately 7 um in diameter. The bacterium was also found to be positive for gram staining. The bacterium was also found to negative for any H2S reduction or motility as indicated by the SIM tubes. It was observed the unknown bacterium did not hydrolyze starch. Using proper reagent tests, it was found that the bacterium did not undergo denitrification, but did undergo nitrification. The bacterium was also found to be positive for catalase presence, indicating aerobic respiration. Optimal growth conditions for the bacterium included a temperature of 22 degrees Celsius, a pH of 5, and a salt concentration of 2%.
From the results of our tests, as well as our observations, a possible genus for our unknown bacteria is Lactobacillus (Bergey, S. 1984). Bacteria from the lactobacillus genus are rod ââ‚¬" shaped, and gram-positive. They undergo nitrification, but do not undergo denitrification. Optimal growth conditions for lactobacillus include temperatures in the 22 degrees Celsius range, a slightly acidic pH, and salt concentrations approximately 2 %. All of these factors correspond with the unknown bacteria. Also, although many members of the lactobacillus genus test negative for catalase presence, there are some that respire aerobically as well (Bergey, S. 1984). The unknown tested positive for catalase presence, indicating that it is either a facultative anaerobe or aerotolerant anaerobe. Lactobacilli that test positive for catalase presence are usually classified as facultative anaerobes (Willey et al. 2008).
In nature, the genus lactobacillus is a large constituent of the lactic acid bacteria group. The bacteria of this genus carry out homolactic fermentation using the Embden-Meyerhof pathway or heterolactic fermentation using the pentose phosphate pathway (Willey et al. 2008). By this fermentation, the bacteria convert lactose and other sugars to lactic acid, with lactic acid being the only product of the reaction (Willey et al. 2008). Members of the genus lactobacillus make the natural flora of the human body, and many of them in areas such as the mouth and digestive tract (Willey et al. 2008). Although most members of this genus are harmless, some have been linked to dental caries (Nase et al. 2001).
Lactobacillus is also used heavily in food production. Products that include the use of lactobacilli include cheese, beer, wine, fermented vegetable foods, and yogurt (Willey et al. 2008). Some lactobacilli, however, may provide unwanted results and spoil foods such as meat and milk due to their metabolic end products (Willey et al. 2008). Some lactobacilli are also used as probiotics that can provide health benefits such as protection from gastrointestinal diseases (Marteau et al. 2001).
To further aid in identification of our bacteria, more tests could have been used, such as a carbohydrate fermentation test. With this test, acid and/or gas would have been produced during fermentative growth with sugar or sugar alcohols. This would have worked, because lactobacilli rely on sugar fermentation for energy (Willey et al. 2008). Another test that could have been used is the cytochrome test, which detects the presence of cytochrome c oxidase. Lactobacillus would have tested negative for this, as they lack cytochromes because they obtain energy from substrate level phosphorylation rather than oxidative phosphorylation (Willey et al. 2008). Limitations of the tests that we performed include the fact that they were not specific enough to fully determine the species of our unknown product. Our tests provided only a broad view.
Sources of error that may have affected results include the possibility that we may not have used proper aseptic technique during an inoculation, and also that some of our tests provided results that were not clear. For example, the gram staining test did not provide completely clear-cut results; there was difficulty in determining whether the test was positive or negative. Also, results for the ammonification test were inconclusive.