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Microorganisms including fungi and bacteria can be found in forest and agricultural soils. These microoganisms can have many functions in the microenvironments of the soil. They can be decomposers: breaking up dead organic matter (even speeding up the rate or decomposition), recycling nutrients back into the soil: such as thymidine, growth nutrients, nitrogen (through nitrification and de-nitrification), can also perform methane oxidation. (Griffiths et al. 2000) They can be parasitic: decomposing living organic matter, or helpful, forming symbiosis partnerships: plant roots fixing nitrogen in exchange for a carbon source of glucose sugar, or plants exudatimg growth hormones from roots influencing the metabolism and growth of fungi and fungi releasing hormones that influence root morphology, metabolic changes and the growth of the plant. (Gogala 1991) These bacterium that can be found in soil are not only part of the biodiversity of the soil sample but they also help make up that biodiversity by adding and taking nutrients from the soil.
These microorganisms can be found throughout the soil layers depending on what type they are (aerobic, anaerobic, nitrogen oxidizing, ammonia oxidizing, etc.), their size and the type of soil they are found in. Bacteria found in soil often occur in colonies, usually located in micropores or areas associated with substantial deposits of organic matter and nutrients. (Foster 1988) These colonies can contain a mixture of microbial populations or only one type of micrograms; these colonies can then become constituted protection sites where the microorganisms can survive temporary adverse conditions. (Foster 1988)
As stated above different types of bacteria can be found in soil and using the differences between these different types the identity/genus of a soil bacteria differential testing of various biochemical/metabolic pathways and environmental conditions the identity of a particular micoorganism may be able to be found out.
(Robertson and Egger 2010)
Preparing Soil Samples, Sub-Culturing:
Soil samples that were collected from the agricultural soil outside UNBC of Prince George BC and prepared through dilution, 10-2 to 10-6; aseptic inoculation of these dilutions were done on TSA streak plates and incubated (48 hours at 22°C, 5 days at 4°C). From the 10-2 streak plate, a distinct colony was isolated and separated for further testing and possible identification.
Biochemical Testing and Environmental Factors:
Gram staining was performed.
A starch agar plate was inoculated and incubated (48 hours at 28°C, then refrigerated 5 days); a few drops of iodine were place to the edge of each culture and a halo was looked for to determine if starch was not present. Kovac's reagent was added, any colour change noted for presents of alpha-amylase outside the isolate.
A SIM (Sulfide, Indole, Mobility) deep was inoculated and incubated (48 hours at 28°C, then refrigerated 5 days); a black precipitate (ppt) was looked for to see if sulfur had been reduced and also any movement away for the stab line was noted.
A peptone broth was inoculated and incubated (7 days at 28°C); a drop of Nessler's reagent was added and a yellow to brown ppt was looked for to see if ammonia had been released.
An ammonium sulfate and a nitrite broth tube were inoculated and incubated (7 days at 28°C); on a spot plate Nessler's was added to a spot of isolate, as well as Trommsdorf's reagent and dilute H2SO4 (a blue-black colour was noted if nitrite was present), and phenylamine and concentrated H2SO4 (a deep blue colour was noted if nitrate was present).
A tube of nitrate broth was inoculated and incubated (7 days at 28°C); reagents A and B (sulfanilic acid and N,N-dimethly-1-1-naphthyamine) were added to the broth (pink/red colour, nitrite ions present); when no red colour was observed reagent C (zinc powder) was added to see if nitrate was not reduced (no colour) or if nitrite was reduced to ammonium ions or nitrogen gas (pink/red colour).
A tube of thioglycollate medium was inoculated and incubated (7 days at 22°C); the location of growth of the isolate and the presences of a blue colour were noted (to test for aerob-isity).
A TSA plate was inoculated and incubated (48 hours at 22°C, 5 days at 4°C); H2O2 added and any bubble release was noted (O2 released). Oxidase was added and any colour change was noted.
Environmental factors were tested through: inoculation of five TSA plates incubated at different temperatures: 4, 22, 37, 50°C for 48 hours (growth patterns were noted); inoculation of four TSB tubes at pHs 3, 5, 7 incubated for 48 hours (growth measured using a spectrophotometer); and inoculation of four TSA plates at 0.0, 0.5, 2.0, 5.0% NaCl incubated for 48 hours 9growth patterns were noted).
An initial observation of the chosen isolated was that it appeared as an orange, flat circle with a lightly rough edge and smooth surface, with about a 1mm diameter; when further isolated and re-plated it diffused a purple-orange pigment into the surrounding agar and gave of a slight odour.
The individual cells, when stained, appeared as rods in chains with the dimensions of about 2x1 ïm.
In Table 1 the results of the biochemical test and environmental tests that were preformed to try and identify the isolate are summarized.
Table 1: Summary of all biochemical and environmental tests done on the chosen isolate
(NO3- to NO2-)
(NO3- to NH3 or N2)
(NH3/NH4+ to NO2-)
(NH3/NH4+ to NO3-)
Optimal salt concentration
The first identification that could be made was that the chosen isolate could be a Staphylbacillis because, when observed under the microscope at 1000x magnification, chains of rods were observed. (Robertson and Egger 2010)
A second inisal identification is that the siolate could be a Proteus or Salmonella species because it can reduse sulfur to H2S. (Table 1, Robertson and Egger 2010)
A third observation was that the chosen isolate could be a facultative anaerobe because during the oxygen tolerance test the isolate was observed to grow through out the tube but the majority of growth was at the tope of the tube and their was still blue pigment indicating oxygen presence at the top of the tube. When looking at facultative anaerobes that were gram negative in the table from appendix one, which indicates a sample of these such organisms, the isolate could be a Spirochaeta, or a contaminate of Enterobacteriaceae (Table 1, Robertson and Egger 2010).
When comparing the results of the same tests to those done on the isolate however the contaminate Enterobacteriaceae is the more likely identification, but not a perfect match; as they both have similar cell and colony morphology, both hydrolyse starch, are ammonificators, and have negative results for any de/nitrification tests, positive for catalse, and are both neutophiles and nonhalophiles (Table 1, Appendix 1).
When comparing the isolate test results, summarized in Table 1, to facultative anaerobic gram-negative rods from the Bergey's Manual volume one, Enterobacteriaceae, Vibrionaceae, and Pastereurellaceae were the possible candidates at first glance (Baumann, et. al. 1984). On further study, however, it is only Enterobacteriaceae that stands out as a possible identification since Vibrionaceaes is highly motile, which the isolate was not, and Pastereurellaceaes can reduce nitrites to nitrates, again, which the isolate was observed not to do (Table 1, Baumann, et. al. 1984). As found in Bergey's was that Enterobacteriaceae can be slightly aerobic so other gram-negative aerobic rods and cocci were studied and compared to the isolate (Baumann, et. al. 1984).
These gram-negative aerobes the compared to the isolate in showing the same results to various testes as the isolate included: Pseudomonas, Xanthobacter, Flavobacterium, Azotobacteraceae (a possible contaminate), Halobacteriaceae and Neiseriaceae (Appendix 1, Brinley-Morgan et al 1984).
When comparing the isolate test results, summarized in Table 1, to these gram-negative aerobes, the two that standout and compare the most similarly to the isolate were Flavobacterium and Neiseriaceae (Appendix 1, Brinley-Morgan et al 1984). The isolate could not be Pseudomonas, Xanthobacter, Azotobacteraceae or Halobacteriaceae because the first three are capable of fixing nitrogen and performing denitrification which the isolate was observed not to do; and it could not be latter because those organisms require an very high slat concentrations in order to grow and survive (8-23% NaCl), levels at which the isolate would not be able to grow given that it is a nonhalophile (Appendix 1, Brinley-Morgan et al 1984).
Flavobacterium and Neiseriaceae, on the other hand, do compare in these respects, and it that they are all positive for oxidase, catalase and anhydrides, they all have little to no motility; but where the former and the isolate are psychrotophs with an optimal temperature for growth around 22 °C, the latter is a mesophile with an optimal growth temperature around 32-36 °C (Appendix 1, Brinley-Morgan et al 1984). A further classification of the isolate, if it is a Neiseriaceae, could be that it is a Moraxella because this type of neiseriaceae is smaller in cell diameter (0.6-1.0um) and does not reduce nitrites, as the isolate was observed to not do this as well (Brinley-Morgan et al 1984).
Enterobacteriaceae, Flavobacterium and Neiseriaceae are all pathogens found in soil cultures and can infect humans as well as animals; Flavobacterium also grow and are usually found around plant roots concentrated in the rhizosphere (1, 2, 3).
Some other tests that might have been do on the isolate sample include: more differential staining, DNA testing, and amino acid testing; the samples could also have been cultured on TSA plates with limiting nutrients to see which were essential for the growth of the organism.
Some common sources or error that were found included: because the bacteria samples were heat fixed before they were stained and observed under the microscope their dimensions could have been altered and the stain might have given the wrong result, the samples could have been contaminated through not completely sterile inoculation, and human error is also a factor.