Lauria bertani broth

This study was carried out in Central Campus of Technology Hattisar Dharan and Regional Agricultural Research Station (RARS) Tarhara from October 2018 to March 2019. Soil samples were collected from different altitudes ranging from Itahari (374 ft.), Tarhara (418 ft.), Dharan (1272 ft.) and Vedetar (5140 ft.) of Koshi Zone, Nepal. From each geographical region 25 different soil samples were collected and in total 100 different soil samples from four regions were collected. Soil samples weighing 10gm was collected in sterile plastic bags from 3-5cm depth and transported into the laboratory at 4°C. Materials used in this experiment were Sodium acetate (HiMedia, India), Lauria Bertani (LB) broth (HiMedia, India). Nutrient Agar (HiMedia, India), Coomassie brilliant blue (HiMedia, India), Phosphate buffer saline (HiMedia, India), Grams staining reagents (HiMedia, India), MR-VP medium and reagents (HiMedia, India), Indole medium (HiMedia, India), Citrate agar medium (HiMedia, India), Gelatin agar medium (HiMedia, India), Starch agar medium (HiMedia, India), Egg yolk agar medium (HiMedia, India), Blood agar medium (HiMedia, India), Carbohydrate fermentation broth (HiMedia, India), SIM medium (HiMedia, India), Triple sugar iron agar (HiMedia, India).

B. thuringiensis was isolated by Acetate selection method as described by Travers et al. (1987) with slight modification. One gram of soil sample was taken in a sterile conical flask containing 1mL of 0.25M sodium acetate (pH 6.8) and 9mL of Lauria Bertani (LB) broth (HiMedia, India). The broth was incubated for overnight at 30°C. After incubation the broth was heated at 100°C for 5 minute. After heat treatment, 0.1mL of sample was taken and spread on nutrient agar (HiMedia, India) and plates were incubated at 30ºC for overnight. Bt like colonies white, large, nearly circular with fine irregular margins and may be glossy, less glossy or rough were selected and further sub cultured on nutrient agar (HiMedia, India (Sneath, 1986) . For presence of parasporal crystal staining the bacterial culture incubated for 72 hours in a nutrient broth was used. Smear was stained with 0.25% coomassie brilliant blue for 3 minutes and washed with distilled water and observed under light microscope at oil immersion (Muniady et al., 2011) . The bacteria were identified as Bacillus thuringiensis based on colony morphology, Gram's staining, biochemical test and parasporal body staining. In this study B. thuringiensis DOR Bt-1 strain was used as a positive control.