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Bacteria were some of the first forms of life that we know of and have continued to thrive throughout the numerous mass extinctions and changes the earth has undergone. They are found in almost every known environment on earth, from the deep ocean depths to the arctic tundra. One of the areas where bacteria are most prominent is in soil environments where the number of species found here are numerous. Bacteria living these soil environments provide a number of services to the ecosystem. Decomposers generally break down decaying organic matter in the soil and thereby return to the soil organic matter useful for other organisms (Ingham 2010). Others are nitrogen-fixing and are associated with plant roots. Here they take nitrogen from the air and provide plants with nitrogen in useable forms. Other services include chemoautotrophs which use non-carbon compounds for energy and are important in nitrogen cycling and degradation of pollutants (Ingham 2010).
With so many bacteria found in these soil environments it is important to distinguish the type of bacteria you have isolated. By examining the isolated culture and performing several biochemical and environmental factor tests, one can narrow down possible identities of the isolated culture. When examining the culture one can describe the culture macroscopically by its pigment, form, elevation, margin, appearance, and texture (Egger 2010). Microscopically one can characterize the cells by their size, shape, and gram staining. Some biochemical tests that can be performed include tests for H2S production, nitrification, ammonification, denitrification, presence of oxidase, presence of catalase, and the optimal temperature, pH, and, salt concentration for growth (Egger 2010). Using these results one can determine the cultures identity using known properties of bacterial species.
This experiment pursued the interest of isolating a bacterial colony grown from forest soil and identifying this isolate through a number of biochemical and environmental factor tests. Through these tests and by describing the cell and culture morphology, the bacterial isolate was able to be narrowed down to a single genus. The isolate was determined to be of genus enterobacter due to the similarities of negative gram staining, both being mesophiles, facultative anaerobes, non-halophiles, and alkaliphiles, positive ammonification, and lack of H2S production. The methods used in this identification process can be used to determine the identity of other bacterial cultures.
The following methods were adapted from Biology 203 Lab Manual 2010 (Egger 2010). Dilutions of 10-2, 103, 10-4, 10-5, 10-6, 10-7 (w/v) of agricultural and forest soil were made. Broths, slants, deeps, spread, and streak plates were made using 10-2 dilutions of agricultural and forest soil using aseptic technique. Four pour plates were also made using 10-4 to 10-7 for both the agricultural and forest soil samples. These were grown over a week and the bacterial growth was recorded. A specific culture was chosen and a streak plate and slant were prepared to isolate the bacterial culture. The culture was observed under a microscope for form, appearance, colour, texture, and other observations. The cells were then gram stained and observed.
The isolated streak plate was then used to prepare a number of plates and tubes to determine biochemical properties. A starch agar plate was prepared to test for starch hydrolysis, a sulphide, indole, and motility (SIM) deep to test for motility and hydrogen sulphide production, a peptone broth tube to test for ammonification, an ammonium sulphate and nitrate broth tube to test for nitrification, a nitrate broth to test for denitrification, a thioglycollate broth tube to test for oxygen tolerance, and a TSA streak plate to test for presence of oxidase and catalase. Four TSA streak plates were made to be grown at 4, 22, 37, and 50oC to test for optimal temperature. Four TSB tubes at pH 3, 5, 7, and 9 were grown to test for optimal pH. Four TSA plates at 0, 0.5, 2, and 5% NaCl (w/v) concentration were prepared to test for optimal salt concentration.
After the first plates were made from the forest and agricultural soils, a beige, smooth colony from the forest soil was chosen, isolated, and used for the remainder of the study. After viewing the colony macroscopically it was found to have circular form, umbonate elevation, entire margin, shiny appearance, and smooth texture (Table 1).
Table 1. Summary of qualitative observations, biochemical tests, and environmental factors to identify unknown bacteria
Beige/Brown, Circular, Umbonate, Shiny, Translucent, Smooth, 10mm Diameter
Hydrogen Sulphide Reduction
Positive ââ‚¬" High amounts of ammonia
Denitrification (NO3- to NO2-)
Denitrification (NO3- to NH4+ or N2)
Nitrification (NH4+ to NO2-)
Nitrification (NO2- to NO3-)
22oC - Mesophile
9 - Alkaliphile
Optimal Salt Concentration
0% (w/v) - Non-halophile
Microscopically it was found that the cells were rod shaped and had dimensions of approximately 1.25um (Table 1). The cells were found to be gram negative, hydrolyze starch, have positive ammonification, and denitrification from NO3- to NO2- (Table 1). The cells were also found to contain catalase, no oxidase, and grew optimally at 22oC, pH of 9, and 0% NaCl (w/v) concentration (Table 1). The isolate had a temperature range of growth from 4oC to 37oC, growth at pH from 7 to 9, and growth at salt concentrations from 0 to 0.5% NaCl (w/v).
The results of the biochemical and environmental factor tests were used to give a possible identity for the bacteria. The negative gram staining was detected due to the pink colour under a microscope and positive starch hydrolysis was detected due to the absence of a dark colour when iodine was added (Table 1) (Egger 2010). The bacteria was found to be negative for hydrogen sulphide reduction because of the lack of black precipitate and positive for ammonification because of a brown colour present when Nesslerââ‚¬â„¢s reagent was added (Table 1) (Egger 2010). It was negative for motility as the growth was limited to the entry point of the inoculating needle found in the deep and positive for denitrification from NO3- to NO2- as after the addition of reagents A and B, the sample turned red (Table 1) (Leboffe and Pierce 2005). The bacteria showed positive nitrification from NO2- to NO3- as deep blue colour was observed when diphenylamine and H2SO4 were added, but no nitrification from NH4+ to NO2- as no colour change was seen when Trommdorfââ‚¬â„¢s reagent and H2SO4 were added (Table 1) (Egger 2010). The bacteria also showed presence of catalase as a bubbles were seen when H2O2 was added, but no presence of oxidase as no colour change was seen (Table 1) (Leboffe and Pierce 2005). The bacteria was determined to be a facultative anaerobe as there was growth throughout the thioglycollate tube, but best growth near the top (Table 1) (Leboffe and Pierce 2005). It was also found to grow optimally at 22oC making it a mesophile, pH of 9 making it an alkaliphile, and at 0% NaCl (w/v) concentration making it a non-halophile (Table 1) (Egger 2010).
Using these results I was able to come up with a possible identity of Family enterobacteriaceae and a possible identity of genus enterobacter. This family of bacterium has some strong distinguishing characteristics similar to the unknown isolate, those being both found in soil, length being between 1.2 and 3.0 um, being facultative anaerobes, H2S not being produced, both being mesophiles (growing best between 20 and 30oC), and similar cell morphology (Holt 1984). This particular genus from this family is chosen as it had the most similar results with neither it nor the unknown being motile (Holt 1984). The positive nitrification did not compare with many suspected bacteria genus. This may be a result of heterotrophic nitrifiers in the soil providing a positive result.
The genus enterobacter consists of many bacteria which are pathogens, affecting the urinary and respiratory tracts. Members of this genus are found worldwide throughout a number of environments including water, soil, plants, insects, and humans (Pontes et al. 2007). Recently members of this genus have become increasingly popular due to their clinical importance (Pontes et al. 2007).
To help confirm the identity of the unknown bacterial isolate many other tests could be performed, some specifically useful for enterobacteriaceae. These include tests for urease, lysine decarboxylase, and gluconate which would help distinguish from other gram negative facultative anaerobes (Holt 1984). Some limitations of the tests performed include the lack of precise measurement for addition of substrates for tests and possible contamination of bacterial cultures resulting in poor test results.
Through the progressive isolation of a single bacterial colony from a mixture of colonies from soil, a single bacterial colony was able to be isolated for characterization. Through a variety of biochemical and environmental factor tests, as well as qualitative observations, this bacterial isolate was able to be characterized to a particular genus (Table 1). The results of these experiments showed that this unknown bacterial isolate was most likely to be a part of the genus enterobacter (Holt 1984). Members of the genus enterobacter are known as pathogens and are found in numerous environments including soil, another suggestion that this is the correct bacteria (Pontes et al. 2007). Other tests including the detection of urea, lysine decarboxylase, and gluconate could be used to further confirm the identity of this unknown isolate (Holt 2984). The methods and tests used in this experiment can be used in other research to help determine the identity of an unknown bacterial isolate.