芽孢杆菌筛选分离（Isolation and isolation of Bacillus）.doc

芽孢杆菌筛选分离（Isolation and isolation of Bacillus） Experimental method: Samples: purchased bacteria, commercial microbial preparations (solid or liquid), soil, water or deadwood, rotten leaves (record, sampling location, PH vegetation, etc.). Medium: peptone, 10g/L beef extract, 3-5g/L, NaCl5g/L, PH7.2 Enrichment culture: Take the 5g sediment or gut soluble 0.5g, with 45 (50) mL /250mL (sterile water flask with a glass bead), oscillator 15-20min, 5mL bacterial suspension or 5mL samples adding 45mL bud medium /250mL Erlenmeyer flask, 75 DEG -80 DEG 15-20min water bath to 35 DEG -37 DEG 150-200r/min oscillation 24h. Staining for two to three times, training reserve. Or take 1g sediment (soil) in 20mL broth medium, /250mL triangle bottle, eight layers of gauze sealing, 75 -80 24h water bath, 15-20min, and then 150-200r/min oscillation. Microscopic examination of sporulation, spore colonies were selected with crossed cultured to obtain single colonies. Isolation and purification: The enrichment of bacteria in 75 -80 OC water bath 15-20min, 10-3, 10-4, gradient dilution of 10-5, 10-6, 10-7, select three suitable gradient mixed bacteria or coated on nutrient agar malt juice medium or directly coated on the casein medium screening, can also be crossed separation (crossed the first flat plate is arranged on the 30 C (24-48 h) or 37 C (18-24h), sterility test, surface water condensation drying). Each gradient is two plates, and the plate is inverted at 35 -37 24-48h. The single colonies were numbered and the colonies were fast growing, the surface was dry and rough, and the opaque colonies were transferred to the slope. Take a little sand smear, do spore stain to determine whether bacillus. Screening of protease producing strains: Operating procedures: sample dilution - heat treatment (75-80 C) - - gradient dilution spread plate single colony number (transfer inclined) - microscopy (spore staining smear) - screening (pick - casein flat sieve (pick) enzyme production medium, centr