Bacillus thuringiensis strain separating, purifying and domesticating method and use
A technique for the separation and purification of Bacillus thuringiensis, applied in the field of separation and purification of a strain of Bacillus thuringiensis and its domestication and application, can solve problems such as high salinity, high organic content, environmental pollution, etc., and achieve high research and application value Effect
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Embodiment 1
[0027] Example 1: Isolation and purification of Bacillus thuringiensis ODPY, the specific steps are:
[0028] A. Under aseptic conditions, add water to the sludge of the pesticide production oxadiazon wastewater treatment tank and mix it and let it stand for 0.5~2h. Take 1mL of the supernatant and dilute it 10 with 0.8% normal saline. -1 ~10 -8 , Get 8 parts of diluted bacteria liquid;
[0029] B. On a sterile workbench, take 8 plates, pour into each plate 15-20 mL of beef extract peptone solid medium that has been melted and cooled to about 40-50 ℃, and wait for it to solidify;
[0030] C. Take 0.1~0.2mL of the diluted bacteria solution in A and apply them to 8 plates evenly with a sterilized triangular coating rod, and number them in sequence;
[0031] D. Place the 8 plates prepared above in an incubator at 30°C for 18-60 hours;
[0032] E. Pick single colonies with clearer shapes from each plate, place them on the solidified beef extract peptone solid medium on the other 8 plates acc...
Embodiment 2
[0036] Example 2: Identification of Bacillus thuringiensis ODPY
[0037] Colony observation, staining, optical microscope observation, electron microscope observation, and physiological and biochemical experiments were performed on the obtained strain ODPY. The colony of this strain is round, with small jagged edges, milky white in color, moist and shiny. The surface morphology of the colony is convex and the upper surface is smooth. Observed under an optical microscope, the bacterial colonies are rod-shaped, facultatively anaerobic, with flagella, and spores; Gram staining is positive and can use a variety of organic compounds as the sole carbon source for growth. According to the "Berger's Bacteria Identification Manual" 》Preliminary identification is obtained, and the strain is judged to belong to the genus Bacillus.
[0038] The 16S rDNA sequence of the strain was sequenced, and the obtained sequence was compared in the NCBI database by BLAST. The results showed that the spec...
Embodiment 3
[0040] Example 3: Optimization of ODPY culture conditions of Bacillus thuringiensis
[0041] 1. Determination of the optimum pH value for growth
[0042] To investigate the growth of Bacillus thuringiensis ODPY in the environment of different initial pH value culture media, the specific operation method is: follow the principle of single variable, fix other culture conditions such as constant temperature shaker temperature 30℃, rotation speed 150rpm, adjust The pH of the beef extract peptone liquid medium (the formula is the same as the isolation and purification medium) to make the initial pH value of 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, respectively, shake culture for 24h, take samples, and determine the bacterial concentration OD by absorbance method 600 . The test results showed that the optimal growth pH of Bacillus thuringiensis ODPY was 8.5.
[0043] 2. Optimization of carbon source
[0044] To investigate the utilization of several carbon sources such as glucose, sucrose, starch, et...
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