Isolation Of Common Soil Bacteria From Agricultural Soil Biology Essay
Dirt is one of three natural resources, and is indispensable for a broad scope of life. It is made of three chief constituents ; minerals, organic affair from dead workss and animate beings, and the life organisms that reside in the dirt. These micro-organisms can include everything from angleworms and roundworms, arthropods, and fungi, to protozoa and bacteriums. Important procedures maintained by these beings include decomposition, mineralization, and alimentary cycling ( Kaye and Hart 1997 ) . The immense diverseness of bacteriums found in dirt histories for the saving of really of import activities.
Some bacteriums form symbiotic associations with workss, populating in nodules on the roots. These bacteriums work to repair atmospheric N into ammonium hydroxide, while others convert ammonium to nitrates, a signifier readily available for workss ( Yoshida and Ancajas 1971 ) . Many environmental factors can act upon the distribution of bacteriums in the dirt, including O and salt concentrations, pH, and temperature. It is so of import to understand these effects on bacteriums, and to analyze the beings that are so indispensable to biochemical rhythms. As such, soil beings from wood and agricultural dirts will be studied utilizing several techniques including biochemical testing, gram-staining, and environmental factor proving in an attempt to place certain isolates.METHODS ( Egger 2010 ) :Consecutive dilutions were prepared from agricultural dirt samples ( 10-2 – 10-7 ) . Using the sterile technique, spread and run home bases were inoculated with the 10-2 dilution every bit good as stocks, deeps, and slant tubings.
Pour home bases were made utilizing 10-4 and 10-7 dilutions, and a spread home base was prepared on Brewer ‘s anaerobiotic medium. Bacterias were sub-cultured from the aerophilic home bases and inoculated onto TSA ( Tryptic Soy Agar ) home bases and angles. From these civilizations, settlement morphology was observed. Cell morphology was analysed utilizing a microscope with oil submergence lens ( 1000X ) and gram-stained slides.
Drops of H2O on the microscope slides were inoculated with the bacterial isolates and heat-fixed. They were so stained with crystal violet, treated with I mordant, decolorized with 95 % ethyl alcohol, and counterstained with saffranine. Several Biochemical trials were conducted to assist place the isolates. Starch hydrolysis was tested for by dropping I onto settlements, and detecting reaction.
SIM deeps were inoculated to look into for motility and Sulfide production, every bit good as the presence of Indole utilizing Kovac ‘s reagent. Ammonification was examined utilizing inoculated Peptone stock tubings, and Nessler ‘s reagent. Nitrification was tested utilizing Nessler ‘s reagent ( ammonium hydroxide ) , Trommsdorf ‘s reagent ( nitrite ) , and aniline ( nitrate ) . Nitrate decrease 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 Zn.
Assorted environmental factors were besides studied. Oxygen tolerance was determined by culturing in Thioglycollate medium and proving for Catalase utilizing H peroxide. Optimum temperature was observed by turning civilizations at different temperatures ( 4, 22, 37, and 50oC ) , and optimum osmotic force per unit area was determined by detecting growing on TSA home bases ( 0, 0.5, 2, and 5 % NaCl concentrations ) . Using a spectrophotometer, the turbidness of inoculated TSB tubing of changing pH ( 3, 5, 7, and 9 ) was measured to find pH of optimum growing.
Consequence:The stray sub-cultured settlements from agricultural dirt were observed to be non-pigmented, umbonate in signifier, and slimy textured, with an undulating border and motility abilities ( Table 1 ) . Individual cells were Gram-negative rod or ovoid-shaped, 2I?m in length. The settlements tested negative for starch hydrolysis, H2S decrease, and the production of Indole. Using Nessler ‘s reagent, the bacterium was negative for ammonification. The stray bacteriums tested positive for all nitrification and denitrification trials, bespeaking presence of ammonium hydroxide, nitrite, nitrate, and nitrite ions. The O tolerance was observed to be aerophilic, and the bacteriums tested positive for the enzyme Catalase, involved with aerophilic respiration.
Out of the four temperatures selected, the bacteriums expressed most optimum growing on the home base incubated at 22oC. Optimum pH was observed to be impersonal ( pH 7 ) , while there was no differentiation in growing between the changing osmotic force per unit area home bases ; all showed optimum growing ( Table 1 ) .Table 1. Summary of Observations, Biochemical Tests, and Environmental Factor Tests for Isolated Bacterium from Agricultural Soil.Colony MorphologyNon-pigmented, Slimy, Umbonate, Undulating border, FilamentousCell morphologyRod-Bacillus, 2I?mGram StainNegativeStarch HydrolysisNegativeHydrogen Sulfide ReductionNegativeMotilityPositiveAmmonificationNegativeIndole ProductionNegativeDenitrification ( nitrate-nitrite )PositiveDenitrification ( nitrate-ammonia/N2PositiveNitrification ( ammonia-nitrite )PositiveNitrification ( nitrite-nitrate )PositiveCatalasePositiveOxygen ToleranceObligate AerobeOptimum Temperature~ 22oC MesophileOptimal pH7 – NeutrophilOptimal Osmotic PressureOptimum growing at all concentrations testedDiscussion:Harmonizing to the informations obtained, a logical categorization of the stray bacteriums would be within the genus Acetobacter. This genus includes Gram-negative acetic acid bacteriums, with the ability to change over intoxicant to acetic acid under aerophilic conditions.
The cells are spheroidal to bacillar, normally arranged in braces or ironss and are frequently filiform. They can be motile or non-motile, depending on the species. Acetobacter bacteriums are obligate aerobes, incapable of agitation. These chemoorganotrophs have optimum temperatures of 25-30oC, and turn most expeditiously in medium of pH 5.
4 – 6.3. They test negative for the enzyme Oxidase, the production of H2S and Indole nevertheless, they test positive for the enzyme Catalase ( Krieg and Holt 1984 ) . All these features are consistent with the stray bacterium from agricultural dirt. However, incompatibilities arise with the nitrification and denitrification biochemical trials. Acetobacter bacteriums are characterized by proving negative for all nitrification and denitrification trials, while the stray bacterium was observed to prove positive for all.
However, these trials are known to demo false positives ( Egger pers. Comm. ) .In nature, Acetobacter bacteriums are of import dirt micro-organisms, lending to the mineralization of complex micro-organisms such as aromatic compounds ( Kaye and Hart ) . Their characteristic ability to oxidise ethyl alcohol to acetic acid makes them an interesting constituent of the assorted bio-life of dirts. Industrially, this feature is utilized in the production of vinos and acetums ( Sokollek and al. 1998 ) .
In future surveies, farther biochemical testing may be in order to accurately place the bacterial isolate. The nitrification and denitrification trials can be refined and replicated to obtain more dependable information. To contract the designation, more extended pH testing may be helpful, concentrating in the scope of pH 4-8. Furthermore, Acetobacter can easy be distinguished in the lab by growing on a medium incorporating about 7 % ethyl alcohol, and adequate Ca carbonate to render the medium partly opaque. Because the bacteriums can change over the ethyl alcohol to acetic acid, this merchandise reacts with the Ca carbonate and produces a really distinguishable clear zone around the settlement ( Takemura et al. 1993 ) .Possible beginnings of mistake could hold occurred during techniques such as gm staining, the sterile technique, and biochemical testing. Beginnings of mistake during gram staining can include overheating during heat arrested development, over-decolourization with ethyl alcohol, over-washing with H2O, or the fact that some bacteriums are more able to retain the colour than others.
Contamination while utilizing the sterile technique can go on if it is non performed right. Besides, an over-heated rod will kill bacteriums and cause false negatives during biochemical testing. Mistakes are most likely to hold occurred with the nitrification and denitrification trials, demoing false positives.In decision, the informations obtained indicates a relation to the genus Acetobacter ; nevertheless some informations ( nitrification and denitrification trials ) were contradictory to this hypothesis. Subsequently, survey aims were partly attained and further biochemical testing is required for an accurate designation.LITERATURE CITED: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.
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