Soil is one of three natural resources, and is essential for a wide range of life. It is made of three main components; minerals, organic matter from dead plants and animals, and the living organisms that reside in the soil. These microorganisms can include everything from earthworms and nematodes, arthropods, and fungi, to protozoa and bacteria. Important processes maintained by these organisms include decomposition, mineralization, and nutrient cycling (Kaye and Hart 1997). The huge diversity of bacteria found in soil accounts for the preservation of very important activities. Some bacteria form symbiotic associations with plants, living in nodules on the roots. These bacteria work to fix atmospheric nitrogen into ammonia, while others convert ammonium to nitrates, a form readily available for plants (Yoshida and Ancajas 1971). Many environmental factors can influence the distribution of bacteria in the soil, including oxygen and salt concentrations, pH, and temperature. It is then important to understand these effects on bacteria, and to study the organisms that are so indispensable to biochemical cycles. As such, soil organisms from forest and agricultural soils will be studied using several techniques including biochemical testing, gram-staining, and environmental factor testing in an effort to identify certain isolates.
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METHODS (Egger 2010):
Serial dilutions were prepared from agricultural soil samples (10-2 - 10-7). Using the aseptic technique, spread and streak plates were inoculated with the 10-2 dilution as well as broths, deeps, and slant tubes. Pour plates were made using 10-4 and 10-7 dilutions, and a spread plate was prepared on Brewer's anaerobic medium. Bacteria were sub-cultured from the aerobic plates and inoculated onto TSA (Tryptic Soy Agar) plates and slants. From these cultures, colony morphology was observed. Cell morphology was analysed using a microscope with oil immersion lens (1000X) and gram-stained slides. Drops of water on the microscope slides were inoculated with the bacterial isolates and heat-fixed. They were then stained with crystal violet, treated with iodine mordant, decolorized with 95% ethanol, and counterstained with safranin. Several Biochemical tests were conducted to help identify the isolates. Starch hydrolysis was tested for by dropping iodine onto colonies, and observing reaction. SIM deeps were inoculated to check for motility and Sulfide production, as well as the presence of Indole using Kovac's reagent. Ammonification was examined using inoculated Peptone broth tubes, and Nessler's reagent. Nitrification was tested using Nessler's reagent (ammonia), Trommsdorf's reagent (nitrite), and phenylamine (nitrate). Nitrate reduction was tested with sulfanilic acid and N.N-dimethyl-1-1-naphylamine for the presence of nitrite ions, and if negative, tested for the presence of nitrate with zinc. Various environmental factors were also studied. Oxygen tolerance was determined by culturing in Thioglycollate medium and testing for Catalase using hydrogen peroxide. Optimal temperature was observed by growing cultures at different temperatures (4, 22, 37, and 50oC), and optimal osmotic pressure was determined by observing growth on TSA plates (0, 0.5, 2, and 5% NaCl concentrations). Using a spectrophotometer, the turbidity of inoculated TSB tubes of varying pH (3, 5, 7, and 9) was measured to determine pH of optimal growth.
The isolated sub-cultured colonies from agricultural soil were observed to be non-pigmented, umbonate in form, and slimy textured, with an undulating margin and motility abilities (Table 1). Individual cells were gram-negative rod or ovoid-shaped, 2Î¼m in length. The colonies tested negative for starch hydrolysis, H2S reduction, and the production of Indole. Using Nessler's reagent, the bacteria was negative for ammonification. The isolated bacteria tested positive for all nitrification and denitrification tests, indicating presence of ammonia, nitrite, nitrate, and nitrite ions. The oxygen tolerance was observed to be aerobic, and the bacteria tested positive for the enzyme Catalase, involved with aerobic respiration. Out of the four temperatures selected, the bacteria expressed most optimal growth on the plate incubated at 22oC. Optimal pH was observed to be neutral (pH 7), while there was no distinction in growth between the varying osmotic pressure plates; all showed optimal growth (Table 1).
Table 1. Summary of Observations, Biochemical Tests, and Environmental Factor Tests for Isolated Bacterium from Agricultural Soil.
Non-pigmented, Slimy, Umbonate, Undulating margin, Filamentous
Hydrogen Sulfide Reduction
Always on Time
Marked to Standard
~ 22oC Mesophile
7 - Neutrophile
Optimal Osmotic Pressure
Optimal growth at all concentrations tested
According to the data obtained, a logical classification of the isolated bacteria would be within the genus Acetobacter. This genus includes gram-negative acetic acid bacteria, with the ability to convert alcohol to acetic acid under aerobic conditions. The cells are ellipsoidal to rod-shaped, usually arranged in pairs or chains and are often filamentous. They can be motile or non-motile, depending on the species. Acetobacter bacteria are obligate aerobes, incapable of fermentation. These chemoorganotrophs have optimal temperatures of 25-30oC, and grow most efficiently in medium of pH 5.4 - 6.3. They test negative for the enzyme Oxidase, the production of H2S and Indole however, they test positive for the enzyme Catalase (Krieg and Holt 1984). All these characteristics are consistent with the isolated bacteria from agricultural soil. However, inconsistencies arise with the nitrification and denitrification biochemical tests. Acetobacter bacteria are characterized by testing negative for all nitrification and denitrification tests, while the isolated bacteria was observed to test positive for all. However, these tests are known to show false positives (Egger pers. Comm.).
In nature, Acetobacter bacteria are important soil microorganisms, contributing to the mineralization of complex microorganisms such as aromatic compounds (Kaye and Hart). Their characteristic ability to oxidize ethanol to acetic acid makes them an interesting component of the various bio-life of soils. Industrially, this characteristic is utilized in the production of wines and vinegars (Sokollek and al. 1998).
In future studies, further biochemical testing may be in order to accurately identify the bacterial isolate. The nitrification and denitrification tests can be refined and replicated to obtain more reliable information. To narrow the identification, more extensive pH testing may be helpful, focussing in the range of pH 4-8. Furthermore, Acetobacter can easily be distinguished in the lab by growth on a medium containing about 7% ethanol, and enough calcium carbonate to render the medium partially opaque. Because the bacteria can convert the ethanol to acetic acid, this product reacts with the calcium carbonate and produces a very distinct clear zone around the colony (Takemura et al. 1993).
Possible sources of error could have occurred during techniques such as gram staining, the aseptic technique, and biochemical testing. Sources of error during gram staining can include overheating during heat fixation, over-decolourization with ethanol, over-washing with water, or the fact that some bacteria are more able to retain the color than others. Contamination while using the aseptic technique can happen if it is not performed correctly. Also, an over-heated rod will kill bacteria and cause false negatives during biochemical testing. Errors are most likely to have occurred with the nitrification and denitrification tests, showing false positives.
In conclusion, the data obtained indicates a relation to the genus Acetobacter; however some data (nitrification and denitrification tests) were contradictory to this hypothesis. Subsequently, study objectives were partially attained and further biochemical testing is required for an accurate identification.
Egger, K. 2010. Biology 203 Lab Manual, Prince George BC: University of Northern British Columbia: p4-32.
Egger, K. 2010. Head Lab Instructor. University of Northern British Columbia.
Kaye, J.P., S.C. Hart. 1997. Competition for nitrogen between plants and soil microorganisms. Trends in Ecology and Evolution 12: 139-143.
Krieg, N.R., J.G. Holt, 1984, The Bergey's Manual of Systematic Bacteriology. Lippincott Williams & Wilkins, Baltimore, MD.
Sokollek, S.J., C. Hertel, and W.P. Hammes. 1998. Description of Acetobacter oboediens sp. Nov. and Acetobacter pomorum sp. Nov., two new species isolated from industrial vinegar fermentations. Society for General Microbiology 48: 935-940.
Takemura, H., K. Kondo, S. Horinouchi, and T. Beppu. 1993. Induction by ethanol of alcohol dehydrogenase activity in Acetobacter pasteurianus. Journal of Bacteriology 175: 6857-6866.
Yoshida, T., R.R. Ancajas. 1971. Nitrogen fixation by bacteria in the root zone of rice. Soil Science Society of America 35: 156-158.