Microbial Analysis of Soil Essay Example for Free

Microbial Analysis of Soil EssayAbstract landed estate samples were collected fortnightly from eye socket near Dahisar River, A river in suburb of Mumbai. laboratory analysis started from July 2010 to September 2010. tote up bacterial and fungous count were estimated by standard spread plate isolation. Isolated bacteria were subject to colony portrait and were estimated by their morphological and biochemical characters. As being a monsoon the occurrence of variation of different species were high. The microorganisms degage from the dirty were of staphylococcus strain and were gram constructive, aerophilic, coccus shaped bacteria. The fungal species were in addition identified, of which Aspergillus and genus Penicillium were dominant, followed by mucur, as sub dominant .This project aims to find out the pissing and soil caliber of River and as it is flowing through an industrial ara, to find out if it is getting affected by the industrial pollutants.IntroductionSoil i s the region on the earths crust where geology and biology meet, the land protrude that provides a home to instal animal and microbial life (Pelczar et al., 1993). Soil teems with microscopic life (bacteria, fungus kingdom, algae, phylum Protozoa and viruses) as well as macroscopic life such as earthworms, nematodes, mites, and insects, and also the root systems of plants. The numbers game and kinds of micro- organisms present in soil depend on many environmental factors amount and type of nutrients operative, available moisture, degree of aeration, pH, temperature etc (Prescott et al., 1999). Soil bacteria and fungi play pivotal roles in various biochemical cycles and are responsible for the recycling of organic compounds (Wall and Virginia, 1999). Soil microorganisms also influence above- ground ecosystems by contributing to plant nutrition, plant health, soil structure and soil fertility (ODonnell et al., 2001). Soil is generally a favorable home ground for the proliferatio n of microorganisms, with micro colonies, developing around soil particles.Numbers of micro organism .In soil habitats usually are much higher than those in scrawled water or marine habitats (Atals and Bartha, 1998). Bacteria progress to up the most rampant group of micro- organisms in the soil (3.0 x 106 5.0 x 108) per gram of soil, followed by the actinomycetes (1.0 x 106 2.0 x 107), fungi (5.0 x 103 9.0 x 106), yeast (I.0 x 103 1.0 x 106), algae and phylum Protozoa (1.0 x 103- 5.0 x 105) and nematodes (50 200) counts per gram of soil are wide differences in the relative pro portions of individual bacteria genera found in busy soils (Atals and Bartha, 1998).Soil fungi may occur as plain-living organisms or in mycorrhizal association with plant roots. Fungi are found primarily in the top 10 cm of the soil and are rarely found below 30 cm. They are most abundant in well-aerated and acidic soils (Domsch et al., 1980). Most fungi in soil are opportunistic (zymogenous). The y grow and post out active metabolism when conditions are favorable which implies adequate moisture, adequate aeration and relatively high concentrations of utile substrates (Postage, 1994 Miyanoto et al., 2002). In this research we isolate culturable heterotrophic bacteria and fungi from different top soil samples MATERIALS AND METHODSresearch laboratory analysis dressing of materialsThe materials needed for this experiment include codswallop wares (conical flasks, bijou bottles, pipettes, petri-dishes) and they were water-washed with detergents. These glass wares were rinsed thoroughly with fresh distilled portable water and left to air dry onward sterilizing them in the autoclave at 15C for 1 hour. Also, the laboratory cabinets on which the work would be carried out was swabbed with cotton wool awry(p) in methylated spirit to sterilize it before any microbiological analysis was carried out to avoid the branch and isolation of other organisms not present in the samples. Aft er sterilization, the plates were allowed to cool to about 45 degrees before they were used.Microbiological evaluationTen (10) grams of the soil sample for microbiological evaluation was weighed into 9ml of sterile water. Preparation of serial dilution goes thus 1ml of the authentic stocks solution was poured into 9ml sterile distilled water and mixed thoroughly to give 10-2 of the original sample and this was done for separately sample and the bottles labeled according to date of collectionIsolation and Enumeration of Micro-organisms.1gram of the samples was homogenized in 9mls of distilled water to obtain a ratio of 19 and the second diluted of individually sample was plated victimization the pour plate technique. Sterile molten nutrient agar (NA), potato dextrose agar (PDA), macconkys agar,(MA) manitol salt agar (MSA) and deoxycholate astrate agar (DCA) were usedthe potato dextrose agar (PDA) was acidified). These agars were then added and left to solidify undisturbed. These p lates were incubated 37oC for 24hours (incubation was aerobic) and the procedure was repeated exploitation 10-2 finally the number of colonies per plates were counted and recorded. The acidified PDA was incubated at 25C for 3-7 days for microbial growth.Total Bacterial counts (Cfu/g)The total bacteria count for each sample was determined with the pour plate techniques use nutrient agar. The plates were incubated between 24hours at 370C and all colonies appearing on the end of the incubation period were counted employ digital unlimited colony counter and the counts were expressed in colony forming unit per gram CFU/g of the sample. Colonies of bacteria developing on the plates were observed, isolated and reisolated on a fresh media until pure nuance was obtained.Preparation of Pure CultureIt is necessary to isolate organisms in pure refinement before athletic fielding and identifying them because a pure culture originates from one cell. Characteristics colonies from the origina l culture on the plates were picked with a sterile wire grommet (using surface streaking method) and this grummet was used to make streak of the colony on the surface of newly disposed(p) sterile agar plates of NA,MA MSA. These streak will space out the inoculants and discrete colony of a particular specie of organism and then incubated at 35-37oC for 24hours to enhance microbial growth. Distinct colonies were re-inoculated on another fresh agar plates in order to obtain a pure culture. The isolates were picked with sterile loop and streaked into ready agar slants, labeled and incubated for growth after which they were kept in the refrigerator for future use and identification. realisation of IsolatesThese isolated bacteria were identified using both morphological culture characteristics (i.e. the color, shape, elevation, capacity, consistency, edge) and biochemical test (i.e. citrate, oxidase, indole, sugar tempestuousness, test etc.)and the bacteria were identified based on the results obtained from the above mentioned biochemical characterization results and the procedures include.Grams Staining TechniquesA drop of distilled water was placed on a clean glass slide. The inoculating wire loop was sterilized by flaming until it was red hot (this is to prevent the invasion of unwanted micro- organisms that might be inhabiting the wire loop) in the blue flame of a Bunsen burner. The loop was allowed to cool and the small portion of each colony of microorganisms to be gram asperseed was picked and smeared in the drop of water (distilled) on the glass slide and then spread into a thin smear along the slide. The smear was air dried and passed through the blue flame. The smear was stained with 1%crystal violet and left for 1minutes (60secs) and then washed with travel rapidly distilled water it was then stained again with Lugols iodine for another 60secs and also washed with running distilled water.The slide was decolorized rapidly with 75% alcohol in order to present the organism from having the color of the immemorial reagent and it was washed immediately with distilled water. The slide finally was flooded with a counter stain safranine (a lowly stain) for 60secons and also washed off with distilled water and allow to air dry. The slide was overlayed with a cover slide and observed under the microscope using oil immersion x 100 impersonal genus Lens with immersion oil. The gram reply of the isolated arrangement and the shape of the cell were observed and recorded. Gram positive (+ve) bacterial were characterized by a purple color (i.e. the primary stain) while the gram negative (-ve) bacteria were characterized by red color (i.e. the secondary stain) .This procedure is actually used to ascertain the component of each organisms cell wall.MotilityMotility was determined by hanging drop techniques. Using loop, a teeny part of the colony of the organisms were grown in peptone water for 18hours and then placed in the grease free s lide and covered with a Vaseline bound cover slip and then observed under x100 objective lens. A motile organism is then seen moving in the drop of liquid.Identification Of couch IsolatesMold isolated was identified using cultural and morphological characteristics and according to (Fawole and Oso, 2001), microscopic observation was carried out using lacto phenol blue stain.Procedure for Mold StainingA drop of lacto phenol blue stain was dropped on a clean grease free sterilized glass slide and after this a sterile inoculating wire loop was used to pick the mycelium unto the glass slide from the mold culture .The mycelium was spread evenly on the slide. Teasing was carried out to separate the mycelium in order to get a homogenous mixture and the mixture was then covered with cover slips gently and then allowed to stay for some(prenominal) seconds before observing under x40 under the microscope. The microscope examination of actively growing mold was on the alkali of structures bea ring spores, presence or absence of septate.BIOCHEMICAL TESTSCatalase TestCatalase test demonstrates the presence of catalase enzyme by aerobic microorganisms. Catalase is an enzyme that catalysis the release of oxygen from total heat peroxide (H2O2). To test for catalase, a drop of 3% hydrogen peroxide solution was added to a slide and the organism to be tested for catalase production is brought in touching with the hydrogen peroxide. The production of gas bubbles however indicates a positive reaction and this shows that catalase enzyme is produced.(FawoleOso, 2001)Oxidase testThis was carried out by placing a clean filter paper on the working bench or petri dishes and 2-3 drops of freshly prepared oxidase reagent was added to the isolate using a sterile inoculating wire loop. After this, a few measuring stick of oxidase reagent was added and a purple coloration was observed within 10-15minutes which indicated that the organisms is oxidase positive and according to Olutiola et a l, 1991, a positive reaction is dependent on the presence of cytochrome. This test is also useful for the separation of Neisseria in mixed culture and in differentiating Pseudomonas from enteric bacteria.Indole testOlutiola et al, 1991, describes the test as one which is important in the note of colonies and it depends on the production of indole from tryptophan by the organism. An inoculating loop was used to inoculate the organism into a test furnish containing decarboxylase mass medium becomes violet. An uninoculated test tube serves as a control (i.e. remained yellow)Sugar fermentationation testThe ability of the isolates to utilize certain sugar as energy source was tested. If the organism does ferment a particular sugar, acid will be produced and gas may be produced or not. acid production is indicated by color change of the medium from red to yellow and acid presence could also be detectable with a ph. indicator in the medium while the production of gas is indicated by a void produced in a shorthorn tube. The fermentation medium was prepared by 0.1g of sodium chloride and 0.1g of fermentable sugar (glucose) in 10ml of distilled water.An amount of 9ml of the medium was pipette into a test tube containing Durhams tubes in replicates. 5ml of phenol red indicator was immediately discharged into the test tubes. The test tubes containing medium were sterilized in an autoclave at 121 o for 15minutes.After sterilization, each isolate were incubated in glucose Medium. An uninoculated test tube was also incubated for glucose to serve as a control. The test was also carried out using maltose, lactose, galactose, manitol, sucrose, fructose and mannose.(Olutiolaet al., 1991) wordThe abundance of bacteria and fungi in this study were typical of environment with high species richness and functional diversity. Despite the fact that it is viable that a number of bacteria and fungi may be confused in this study, the isolates could be readily assigned dominant (e.g . Bacillus sp, Aspergillus sp) or transient/ period roles in the isolation of organisms form different seasons, which form the basis of this study. In additions to the implications of the determination of the number of microorganisms during soil sampling, one should consider the qualitative aspect of the preservation of important species and groups of microorganisms and of the changes in these biochemical characteristics resulting from the variations in these counts.Although the results of this study would not be considered to be exhaustive, as it was done within the limits of facilities available in the laboratory, an insight into the macrocosm dynamics and distribution of culturable aerobic bacteria and fungi diversity has been elucidated. This is without prejudice to the possible influence which a substantial proportion of bacteria and fungi that are not culturable in vitro could have on the boilers suit picture of event. It would require more modern technology (nuclei acid pro bes) to obtain such detailed overview of microbial diversity. This should be a subject of extension of this investigation in future.ConclusionThrough this project, if emphasis is do on public health, the observation and findings show striking predominance of Salmonella typhi. And E.coli. E.coli being an enterobacter cause dysentery and S.typhi poses a swell risk of typhoid. Health inspector and municipal authorities should look into this matter for further investigation and if possible improvementAcknowledgementInvestigators are grateful to the Principal Management of S.V.K.Ms Mithibai College for constant encouragement support. And liberty chit of department of zoology Prof. V.V. Dalvie for providing me opportunities and Prof. Radhika Dsouza, under whose guidance the project was successfully completedReferences1 .Atals RM, Bartha R (1998). Microbial Ecology Fundamentals and Applications. 4th Edition. Benjamin Cummings Publishing Company Inc. Addison Wesley Longman Inc. pp. 300 350. 2. Miyanoto T, Igaraslic T, Takahashi K (2002). Lignindegradation ability of litter decomposing basidomycetes from picea forest of Hokkaida Myco.sci. (41) 105 110. 3. Domsch KH, Gaws W, Anderson TH (1980). Compendium of soil fungi4. O Donnell AG, Seasman M, Macrae A, Waite I, Davies JT (2001). forms and Fertilizers as drivers of change in microbial community structure and function in soil. Plant Soil (232) 135 145. 5. Pelczar MJ, Chan ECS,krieg NR (1993). Microbiology Concept and Application International edition McGraw-Hill, USA. Pp 281-324. 6. Wall DH, Virginia RA (1999). Controls on soil biodiversity insights from extreme environments. Appl. Soil Ecol. (13) 137150. 7. Fawole and Oso, 2001