Sample collection: Soil samples of Sara and Mirpur series were collected from two different locations of Kotwali thana of Jessore district. The general description of the locations is discussed.
Soil preparation: Top (0-15 cm) soil samples were collected from the field and taken into laboratory using thermo flask. Fresh soil samples were used for this study.
Isolation of bacteria: At first sample was prepared by soil and physiological water (dw + 0.9% NaCl solution) as described by Dubey and Maheshwari (1999). Serial dilution of sample was performed as described by Joklik et al. (1992). Then nutrient agar media was prepared as described by Prescott and Harley (2002). Nutrient agar media and equipment used in bacterial culture were sterilized by exposure to steam at 1210C temperature and 15 lbs (pound per square inch) of pressure as described by Prescott and Harley (2002). Then petriplate for bacterial culture was prepared as described by Joklik et al. (1992). From isolated colony obtained by spread plate technique pure culture was prepared by streak plate technique as described by Prescott and Harley (2002). Three replications were adopted for spread plate technique and streak plate technique.
Viable count: Viable Count was made by colony count method as described by Prescott and Harley (2002). The plates with 25 to 250 colonies were selected for counting. The following formula was used.
Total bacteria per gram soil = no. of colonies x dilution factor / Volume of sample (ml)
Characterization: Colony characteristics and morphological characteristics were determined. Well-isolated colonies of nutrient agar plates were evaluated in size, pigmentation, form, margin and elevation as described by Cappuccino and Sherman (1999). Shape and arrangement were determined by simple and negative staining as described by Shaha (2003). Staining characteristics were determined by Gram stain, capsule stain, spore stain and acid fast stain as described by Cappuccino and Sherman (1999).
Simple staining: Heat fixed bacterial smear was prepared on a glass slide. The smear was flooded with crystal violate for 20 to 60 seconds. Then the smear was washed with tap water to remove excess stain. After drying, the slide was examined under oil immersion.
Negative staining: A drop of nigrosin was placed close to one end of a clean slide. Using sterile technique, a loopful of inoculum from the culture was placed and mixed in the drop of nigrosin. The mixture was pushed with the edge of a second slide held at a 30 0 angle and placed in front of bacterial suspension to form a thin smear. After air drying, the slide was examined under oil immersion.
Capsule stain: Air dried bacterial smear was prepared on a glass slide. The smear was flooded with crystal violate and kept for 5 to 7 minutes. Then the smear was washed with 20% copper sulfate solution. After air drying, the slide was examined under oil immersion. Spore stain: Heat fixed bacterial smear was prepared on a glass slide. The smear was flooded with malachite green and placed on a warm hot plate, allowing the preparation to steam for 2 to 3 minutes. The stain was prevented from boiling. The slide was removed from hot plate, cooled and washed with tap water. The smear was counterstained with safranin for 30 seconds. Then the smear was washed with tap water. After air drying, the slide was examined under oil immersion. Acid fast stain: Heat fixed bacterial smear was prepared on a glass slide. The smear was flooded with carbol fuchsin and placed on a warm hot plate, allowing the preparation to steam for 5 minutes. The stain was prevented from boiling. The slide was removed from hot plate, cooled and washed with tap water. Acid alcohol was added drop by drop till carbol fuchsin failed to wash from smear. The smear was washed with tap water. Then the smear was counterstained with methylene blue for 2 minutes. Then the smear was washed with tap water. After air drying, the slide was examined under oil immersion.
Gram stain: Heat fixed bacterial smear was prepared on a glass slide. The smear was flooded with crystal violate and kept for 1 minute. Then the smear was washed with tap water. After that the smear was flooded with Gram’ s iodine and kept for 1 minute. The smear was washed with tap water. Ethyl alcohol 95% was added drop by drop till crystal violate failed to wash from smear. Again the smear was washed with tap water. After that the smear was counterstained with safranin for 45 seconds. Again the smear was washed with tap water. After air drying, the slide was examined under oil immersion.
Capsule stain: Air dried bacterial smear was prepared on a glass slide. The smear was flooded with crystal violate and kept for 5 to 7 minutes. Then the smear was washed with 20% copper sulfate solution. After air drying, the slide was examined under oil immersion. Spore stain: Heat fixed bacterial smear was prepared on a glass slide. The smear was flooded with malachite green and placed on a warm hot plate, allowing the preparation to steam for 2 to 3 minutes. The stain was prevented from boiling. The slide was removed from hot plate, cooled and washed with tap water. The smear was counterstained with safranin for 30 seconds. Then the smear was washed with tap water. After air drying, the slide was examined under oil immersion. Acid fast stain: Heat fixed bacterial smear was prepared on a glass slide. The smear was flooded with carbol fuchsin and placed on a warm hot plate, allowing the preparation to steam for 5 minutes. The stain was prevented from boiling. The slide was removed from hot plate, cooled and washed with tap water. Acid alcohol was added drop by drop till carbol fuchsin failed to wash from smear. The smear was washed with tap water. Then the smear was counterstained with methylene blue for 2 minutes. Then the smear was washed with tap water. After air drying, the slide was examined under oil immersion.