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Microbial Analysis of Soil Essay

Abstract: soil samples were collected fortnightly from area near Dahisar River, A river in suburb of Mumbai. laboratory analysis started from July 2010 to September 2010. Total bacterial and fungal count were estimated by standard spread plate isolation. Isolated bacteria were subject to colony characterization and were estimated by their morphological and biochemical characters. As being a monsoon the occurrence of variation of different species were high. The microorganisms isolated from the soil were of staphylococcus strain and were gram positive, aerobic, coccus shaped bacteria. The fungal species were also identified, of which Aspergillus and Penicillium were dominant, followed by mucur, as sub dominant .This project aims to find out the water and soil quality of River and as it is flowing through an industrial area, to find out if it is getting affected by the Industrial pollutants.

Introduction:

Soil is the region on the earth’s crust where geology and biology meet, the land surface that provides a home to plant animal and microbial life (Pelczar et al., 1993). Soil teems with microscopic life (bacteria, fungi, algae, protozoa and viruses) as well as macroscopic life such as earthworms, nematodes, mites, and insects, and also the root systems of plants. The numbers and kinds of micro- organisms present in soil depend on many environmental factors: amount and type of nutrients available, 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 (O’Donnell et al., 2001). Soil is generally a favorable habitat for the proliferation of microorganisms, with micro colonies, developing around soil particles.

Numbers of micro organism .

In soil habitats normally are much higher than those in fresh water or marine habitats (Atals and Bartha, 1998). Bacteria make up the most abundant 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 protozoa (1.0 x 103- 5.0 x 105) and nematodes (50 – 200) counts per gram of soil are wide differences in the relative proportions of individual bacteria genera found in particular soils (Atals and Bartha, 1998). Soil fungi may occur as free-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). They grow and carry out active metabolism when conditions are favorable which implies adequate moisture, adequate aeration and relatively high concentrations of utilizable substrates (Postage, 1994; Miyanoto et al., 2002). In this research we isolate culturable heterotrophic bacteria and fungi from different top soil samples MATERIALS AND METHODS

Laboratory analysis
Preparation of materials

The materials needed for this experiment include; glass wares (conical flasks, bijou bottles, pipettes, petri-dishes) and they were washed with detergents. These glass wares were rinsed thoroughly with clean distilled portable water and left to air dry before sterilizing them in the autoclave at 15◦C for 1 hour. Also, the laboratory cabinets on which the work would be carried out was swabbed with cotton wool soaked in methylated spirit to sterilize it before any microbiological analysis was carried out to avoid the growth and isolation of other organisms not present in the samples. After sterilization, the plates were allowed to cool to about 45 degrees before they were used.

Microbiological evaluation

Ten (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 original 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 each sample and the bottles labeled according to date of collection Isolation and Enumeration of Micro-organisms.

1gram of the samples was homogenized in 9mls of distilled water to obtain a ratio of 1:9 and the second diluted of each sample was plated using the pour plate technique. Sterile molten nutrient agar (NA), potato dextrose agar (PDA), macconky’s agar,(MA) manitol salt agar (MSA) and deoxycholate astrate agar (DCA) were used{the potato dextrose agar (PDA) was acidified). These agars were then added and left to solidify undisturbed. These plates were incubated 37oC for 24hours (incubation was aerobic) and the procedure was repeated using 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 using nutrient agar. The plates were incubated between 24hours at 370C and all colonies appearing on the end of the incubation period were counted using 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 culture was obtained.

Preparation of Pure Culture

It is necessary to isolate organisms in pure culture before studying and identifying them because a pure culture originates from one cell. Characteristics colonies from the original culture on the plates were picked with a sterile wire loop (using surface streaking method) and this loop was used to make streak of the colony on the surface of newly prepared 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 prepared agar slants, labeled and incubated for growth after which they were kept in the refrigerator for future use and identification.

Identification of Isolates

These 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 fermentation, 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 Techniques

A 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 stained 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 running 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 primary reagent and it was washed immediately with distilled water.

The slide finally was flooded with a counter stain safranine (a secondary stain) for 60secons and also washed off with distilled water and allow to air dry. The slide was covered with a cover slide and observed under the microscope using oil immersion x 100 objective lens with immersion oil. The gram reaction 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.

Motility

Motility was determined by hanging drop techniques. Using loop, a little part of the colony of the organisms were grown in peptone water for 18hours and then placed in the grease free slide 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 Mold Isolates

Mold 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 Staining

A 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 seconds before observing under x40 under the microscope. The microscope examination of actively growing mold was on the basis of structures bearing spores, presence or absence of septate.

BIOCHEMICAL TESTS

Catalase Test

Catalase test demonstrates the presence of catalase enzyme by aerobic microorganisms. Catalase is an enzyme that catalysis the release of oxygen from hydrogen 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 contact with the hydrogen peroxide. The production of gas bubbles however indicates a positive reaction and this shows that catalase enzyme is produced.(Fawole&Oso, 2001)

Oxidase test

This 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 quantity 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 al, 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 test

Olutiola et al, 1991, describes the test as one which is important in the differentiation 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 tube containing decarboxylase medium becomes violet. An uninoculated test tube serves as a control (i.e. remained yellow)

Sugar fermentation test

The 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 Durham 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 Durham’s 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)

Discussion:

The 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 possible that a number of bacteria and fungi may be missed in this study, the isolates could be readily assigned dominant (e.g. Bacillus sp, Aspergillus sp) or transient/succession 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 population 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 overall picture of event. It would require more modern technology (nuclei acid probes) to obtain such detailed overview of microbial diversity. This should be a subject of extension of this investigation in future.

Conclusion

Through this project, if emphasis is made 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 great risk of typhoid. Health inspector and municipal authorities should look into this matter for further investigation and if possible improvement

Acknowledgement

Investigators are grateful to the Principal & Management of S.V.K.M’s Mithibai College for constant encouragement & support. And head of department of zoology Prof. V.V. Dalvie for providing me opportunities and Prof. Radhika D’souza, under whose guidance the project was successfully completed

References

1 .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). Lignin–degradation 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 fungi

4. O’ Donnell AG, Seasman M, Macrae A, Waite I, Davies JT (2001). Plants 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): 137–150. 7. Fawole and Oso, 2001


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