Sampling And Isolation Of Bacteria From Soil.docx

SAMPLING AND ISOLATION OF BACTERIA FROM SOIL

INTRODUCTION Microorganisms are frequently present in soil, manure and decaying plant tissues which are able to degrade wastes that are correlated with the substrate organic matter. Agriculture soil is a dynamic medium in which a large number of pathogenic and nonpathogenic bacterial and fungal floras live in close association. Microbes in the soil are the key to carbon and nitrogen recycling. Microorganisms produce some useful compounds that are beneficial to soil health, plant growth and play an important role in nutritional chains that are important part of the biological balance in the life in our planet.

COLLECTION OF SOIL SAMPLES Four Soil samples were collected from different places of Lahore 30cm to 40cm deep from the earth with sterilized spatula in sterilized plastic bags. 1st sample (M) was collected from a potted plant of Riphah International University, 2nd sample (K) was collected from a ground nearby, 3rd sample (F) was collected from the area near bus stand, and 4th sample (S) was collected from the place near huts. Then the samples were moved to microbiology lab for bacterial identification.

PREPARATION OF NUTRIENT AGAR Solution of nutrient agar was prepared by adding 28 gram of nutrient agar in 1000ml of distilled water. The solution was autoclaved. 40 Petri plates were prepared. In each Petri plate 25ml of nutrient agar was added. One Petri plate was taken as control and 24 Petri plates were for the inoculation of microbes from the suspensions of soil samples prepared by serial dilution method. 15 Petri plates were for the isolation of colonies obtained after inoculation and incubation.

INOCULATION OF MICROBES Different soil samples treated for bacterial isolation by serial dilution method by using nutrient agar medium. Soil samples were prepared by dissolving 1 gram of each soil collection in 10 ml of sterilized water and mix well for 15 minutes and vortexed. Each suspension was serially diluted 10^-1 to 10^-6. The samples from these suspensions were inoculated in prepared nutrient agar plates with the help of sterilized wire loop. Then these

plates were incubated at 37C. The microbial growth was observed after 24 hours.

ISOLATION OF BACTERIA The colonies of bacteria and fungi were identified by morphological examination. The bacterial colonies were selected for further isolation needed for the identification of the gram positive and gram negative bacteria in the samples. The fungal colonies were not isolated.

ISOLATED BACTERIA

IDENTIFICATION OF BACTERIA GRAM STAINING This test was performed for the identification of bacteria by using different biochemical test.

CHEMICALS Primary stain

( crystal violet )

Mordent

( iodine )

Decolourizer

( 95% ethanol )

Counter stain

( saffranine )

PROCEDURE A clean glass slide was taken and then a drop of distilled water was transferred in center of the slide. A loop full of bacterial culture was mixed with the drop of water. A smear was formed by the edge of another slide and was heated over the flame to fix the smear. Four drops of crystal violet was poured on the smear. After 30 seconds, excess stain was rinsed with distilled water; 2-3 drops of iodine solution were added on the smear. After 60 seconds, the slide was again rinsed with distilled water. For decolourization, smear was rinsed with ethanol. Then 3-4 drops of saffranine solution was poured for 30 seconds. Then slide was washed and air dried. The slide was then observed under a microscope.

RESULT The colonies isolated from samples M5, K2, and S1 are gram negative because they appeared pink when observed under the microscope. The colonies isolated from samples M2, M3, K1, K3, F2, F3, F5, S, S1, S2, S3, and S5 are gram positive because they appeared purple/blue when observed under the microscope.

Figure i M2

Figure ii M3

Figure iii M5

Figure iv K1

Figure v K2

Figure vii F2

Figure viii F3

Figure x S

Figure xi S1

Figure xiii S3

Figure xiv S4

Figure vi K3

Figure ix F5

Figure xii S2

Figure xv S5

CONCLUSION We conclude that selected soil samples contain more number of gram positive bacteria than gram negative bacteria.

REFERENCES Collins CH, Lyne PM, Grange GM (1989). Collins and Lyne Microbiological methods, 6th Edition. Butterworth, London. Harold JB (2002). Microbiological Applications. Laboratory Manuals in General Microbiology, 8th Edition. McGraw-Hill Higher Education. Lyengar S, Behave PP (2005). In-vessel compositing of household wastes. Waste Management, p. 1-11.