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Soil is inhabited by thousands of different species of microorganisms Torsvik and Ovreas 2002. Bacteria, archaea fungi, and protists are all found in soil and play critical roles to the soil environment (Willey et al. 2008). The diversity in soil microbial communities seems to be greater than those found in most aquatic environments (Willey et al. 2008). Soil bacteria are essential for the biogeochemical cycles involving nitrogen, carbon, sulfur, iron, and manganese (Willey et al. 2008). Microbial communities degrade and recycle organic material causing soil formation and nutrient cycling (Willey et al. 2008). The activity of microbial life in soils is related to plant life in an soil environment (Willey et al. 2008). Only a small number of the bacteria living in soil will produce growth on microbiological media therefore growth media selection is critical to isolation of bacterial cultures (Davis et al. 2004).
The objective of this study was to isolate mixed populations of microorganisms from a forest soil sample using multiple culturing techniques. Upon isolation of colonies a pure culture was to be obtained and processed through various biochemical tests to determine the identity of a single bacterium in the soil.
MATERIALS AND METHODS
As outlined in Robertson and Egger (2009), the experiment required a forest soil sample of 1 gram to be suspended in 99 milliliters of deionized water. Serial dilutions from 10-2 to 10-7 were made with the soil sample and then cultured on sterile media using aseptic technique. Enumeration was done on the bacterial soil dilutions to acquire Colony-Forming Units. Streak plate sub-cultures were made from the colonies in order to obtain pure cultures. A selection of a distinct bacterial colony was made and examined for morphology and Gram stain testing. The bacterial isolate was studied for environmental effects on growth by sub-culturing it onto media of various temperature, pH and solute concentration. Starch hydrolysis, hydrogen sulfide production, motility, ammonification, nitrification, denitrification and catalytic activity were biochemical tests conducted on the bacterial isolate to distinguish its involvement in cycling of carbon, nitrogen and sulfur.
The isolation of bacterial colonies for a mixed population of microorganisms found in the forest soil resulted in the colony morphology outlined in Table 1. The colonies observed were prominently irregular in form and rose off the media (Table 1). The colonies were shiny yellow (Table 1). The examination of single bacterial cells under microscope demonstrated single bacillus arrangement (Table 1). The rod-shaped bacterial cells were approximately 4 micrometers in length (Table 1).
Table 1. Colony and cellular morphology of bacterial isolate from forest soil 10-4 culture.
Colony and Cellular Morphology
Cell shape at 1000x magnification
The isolated soil bacterial culture was notably gram positive when examined under microscope on the prepared slide (Table 2). The summary of biochemical tests found in Table 2 show the bacterium to be motile, catalase positive, incapable of starch hydrolysis or denitrification but able to perform nitrification (Table 2).
Table 2. Biochemical test results of forest soil 10-4 bacterial isolate.
Results and Observations
Negative, black with iodine
Hydrogen sulfide H2S reduction
Negative for H2S production
Denitrification (NO3- to NO2)
Denitrification (NO3- to NH3 or N2)
N/A no denitrification of NO3- to NO2-
Nitrification (NH3/ NH4+ to NO2-)
Nitrification (NH3/ NH4+ to NO3-)
Optimal pH / pH classification
pH 9 / alkalophile
Optimal salt concentration
The bacterium isolated from the forest soil sample could be Curtobacterium. This genus of bacteria is distinguished by its irregular rods and gram positive nature (Sneath et al 1986). It is possible for Curtobacterium to be confused with the genus of Cellulomonas (Sneath et al 1986). Both these genera are gram positive, irregular rods, positive for catalase activity, demonstrate motility and found in soil environments (Sneath et al 1986). These genera do not have a common oxygen use since Curtobacterium are strictly aerobic while most strains in Cellulomonas are facultatively anaerobic (Sneath et al 1986). There are differences in menaquinone, polar lipid and fatty acid composition between Curtobacterium and Cellulomonas as well.
The biochemical tests that most strongly aided the identification of the isolated bacterium included the gram staining, starch hydrolysis, motility, catalase activity, and the temperature effect on growth. Morphology of both the colonies and cellular levels impacted the identification process. The cell arrangements and dimensions obtained through examination of the bacterium aligned with those listed in Bergeyâ€™s Manual of
Systematic Bacteriology (Sneath et al 1986).
The natural habitat for Curtobacterium is plant and soil environments (Dworkin and Falkow 2006). This genus is known to dominate microbial communities of grass mulching and decaying litter (Dworkin and Falkow 2006). Curtobacteria has been isolated from plants, herbs, shrubs and trees which indicates that it must have a variety of functions within plants and soils (Dworkin and Falkow 2006). It can be inferred that this genus has nutrient cycling capabilities.
Further testing could be done to aid in the identification of bacterial isolates. Possible tests could include menaquinone composition, lipid and fatty acid composition, urease production and cell wall peptidoglycan properties (Sneath et al 1986). This study did not deeply investigate the oxygen use of the bacteria but further testing of this characteristic could be useful in identifying both genus and species. A polymerase chain reaction (PCR) amplification can be used to sequence DNA and detect microorganisms that may be difficult to isolate in vitro, however this can be a very expensive biochemical test (Picard et al. 1992).
The results obtained could have been erroneous from a number of sources. Improper isolation of colonies is a possibility as streak plates to obtain pure culture may have been contaminated. In testing motility it was essential to inoculate the slants correctly otherwise the results may become invalid. The tests performed offer some distinguishing characteristics but tend to lack detail, for instance the bacteria isolated was gram positive but very little information about the components of its cell wall and peptidoglycan composition was found.