Industrial production technology of Suyun Jinli bacteria (Bt) emulsion.

First, to outline Bt, short for Bacillus thuringiensis, is a kind of spores that can produce spore crystals that can cause insect morbidity and death in insects, and its preparations are widely used in pest control in agriculture, agriculture, fruits, vegetables, urban gardens.
II, the production process operating procedures (i) sand tube bacteria preparation 1, sand treatment: take construction sand or river sand after 60-80 mesh screening with industrial Hc soaked 48h after washing with clean water, and then use 0.2NNao neutral, drying with magnets to remove the magnetic metal particles, loaded into a clean container for backup.
2, sand control readiness: the above-treated sand and soil in a small test tube of 1.2 x 12 cm, each tube about 2 grams, stuffed with cotton plugs, first with 2.0kg/cm2 high pressure extinguished Bacteria 1.5 hours, intermittent sterilization three times, and then 160 degrees C dry heat sterilization 2 hours, after sterile testing, that is, to extract a bit of sand placed in the oblique interview tube 36 degrees C culture 24 hours before checking for sterile after spare.
3, the production of sand spores: choose to grow well, after the production capacity of qualified excellent test tube oblique bacteria species one added 5 ml of sterile water, with inoculation needle scraped off the spores to make a high concentration of spore suspension with sterile straw absorption 0.2-0.3 ml is connected to the sand pipe, and then placed in the vacuum dryer, with the vacuum pump intermittently drained, sealed with paraffin, and later placed in a refrigerator at 0-5 degrees C, in a drying situation.
(ii) preparation of oblique bacteria species 1, culture base: beef paste 0.3%, protein 1%, agar 2%, adjust 7.5-8.0 melting after the sub-pack test tube, plug into the test tube wrapped in sterilization.
2, sterilization: in the disinfection pot 1.1kg/cm2 steam sterilization for 30 minutes, such as cooling to 60-70 oC when removed into a slope, blank culture for 2 days to observe sterile-free use.
3, inoculation culture: put the strain test tube slope in the inoculation room or sterile operating box, turn on the ultraviolet light sterilization after 30 minutes off.
With the inoculation ring according to the operating procedures to scrape a little seed moss, go to the fresh sloped culture base to line up inoculation, and then put in a 30 degrees C culture box for 3-4 days, after checking normal, put in the refrigerator spare.
(iii) flat bottle expansion seed preparation 1, culture base: the same slope culture composition, plus glucose 1%, dissolved and loaded into the flat bottle.
2, sterilization, inoculation and culture are the same as oblique bacteria.
(iv) Fermentation 1, culture-based cottonseed cake powder 3.5%; Jade rice flour 2.0%; fish powder 1.0%; yeast powder 0.2%; CaCO 30.1%; bubble enemy 0.03% with NoaH ph8.5 disinfection after about 7.0.
2, sterilization: fermentation tank before feeding clean, according to the feeding coefficient of 50% feed, sub-filter to 2kg/cm2 sterilization 30 minutes, solid tank sterilization 1.1kg/cm2, tank temperature 121-125c, maintain 30 minutes (according to the sterilization procedure).
3, spore suspension preparation: take cultured flat bottle seeds 3-4 added sterile water, the moss washed into suspension, and then poured into the inoculation bottle, with injection needles.
the spore suspension for 20 minutes in a water bath at 75 degrees C.
4, vaccination: through the fermentation tank inoculation hole using pressure difference to inject spore suspension into the fermentation tank.
5, culture: tank pressure to maintain 0.3-0.5kg/cm2, stirring 220 ext./min, the amount of air after vaccination 4 hours before smaller, gradually increased to 1:1, the temperature of 30 degrees C to 1 degree C.
Under the above conditions, the growth and development period of bacteria is: 0-4 hours, adjust the growth and growth period, 5-12 hours, growth and reproduction period, 13-20 hours, spore crystal formation period, 21-can, spore crystal maturation period.
6, testing: culture process 4, 8, 12, 16, 20 ... Each sample detects growth, PH value, and the last sample is used to determine the number of bacteria.
(v) after-treatment of fermentation liquid 1, the fermented fermentation liquid with industrial hydrochloric acid to PH5.0-6.5.
2, vacuum concentrate with a thin film concentrator (or centrifuge).
3, testing: according to the number of fermentation liquid bacteria and concentration multiply estimated the number of bacteria 10 billion / ml sampling test, testing in line with product quality standards can be terminated concentration, pressure into the storage tank.
4, additives: according to the provisions of the addition of NaCl1%, sodium benzoate 0.3% stirred evenly.
5, finished product inspection: (1) the finished product by the quality inspection department to test the indicators of product quality standards, issued packaging notice.
(2) packaging: the use of 1 liter plastic bottle packaging, bottles have internal and external lids, do not leak, package packaging should meet the technical and safety needs of product inventory, handling and transportation process, the box should have a product certificate and instruction manual.
4, Bt emulsion main production equipment list: Annex I: Bt emulsion quality test method: A, Bt emulsion spores determination: A1 principle using blood cell counting plate in the optical microscope, counting spores, and then using formula to calculate the number of spores per 1 ml of products.
A2 instrument blood cell counting board, optical microscope, etc.
A3 operation A3.1 crystal treatment will be the sample to be tested shaken and quickly absorbed 10 ml in the equivalent amount of 0.5N NaoH solution, intermittent shaking, processing for 1-2 hours, so that the crystal all self-dissolving, until the microscope to check for no crystal.
A3.2 dilution In 500 ml triangular bottle (inner glass beads), filled with 99.5 ml of sterile water, the NaoH treated sample shaken and quickly used a straw to absorb 0.5 ml in the triangulation bottle (i.e. a total dilution of 400 times).
A3.3 production with sterile straw from the full shake of 400 times dilution to draw a little, from one end of the cover glass into the blood cell counting plate, so that the bacteria liquid along the slide between the seepage, and with filter paper suction tank flow out of excess bacteria liquid, the operation process of the blood cell counting plate must not produce air bubbles, otherwise should be redoed.
15 minutes before counting.
A3.4 observation read out the number of spores after the production of a good film with a low multiplile mirror observation, with a 25 x 16 counting board, the number of four corners of the four Chinese grid and the middle of a Chinese grid of spores, and then converted into 1 ml of bacteria in the total number of spores.
A4 calculation of spores per 1 ml product is calculated as follows: A -B x C x 5 x 104, A is the sample 1 ml of spores (100 million per ml).
B is the total number of spores contained in five Chinese grids.
C is a dilution multiply.
5 x 104 is the constant i.e. the number of bacteria per grid volume of the blood cell count board is converted to 1 ml of bacteria.
note: If a 16 x 25 blood cell count board count is used, the following formula is used for calculation: A-B x C x 3.2 x 104 B, Bt emulsion and toxicity determination: B1 for testworm collection: artificial feeding Or field harvesting (preferably within a specific area, on the same plant), the same age (one or two years old), healthy insects with good growth (commonly used as green insects, but avoid taking on crops that are not short of spraying), and standby after 1-2 days of indoor observation.
B2 appliances, sterilise with 1kg/cm2 steam for 30 minutes, spare.
B3.1 dilutes the sample 500, 1000, 1500, 2000 times, with 3 repetitions per concentration.
B3.2 use a 300px petri dish or canned bottle as a container, wherein 2-3 pieces of cabbage leaves, the liquid is applied to the vegetable slices (front and back), dried and put into 20-25 two-year-old vegetable green insects, while a control group.
B3.3 was raised and tested in 27-28 degrees C light.
B4 results record 24, 48, 72 hours after the test to observe and record the number of dead insects and insect-infested larvae, and the observation results included in the table below Bt emulsion pathogenicity measurement table B5 calculation according to the table above The data listed are used to calculate the mortality rate of 72 hours of vegetable green vegetables, which is calculated according to the following formula: Death of insects Mortality Rate (%) x 100 test insects Note 1: In the process of calculation and statistics, the number of aphids does not participate in the mortality calculation.
Note 2: If the control group mortality rate of 5%, should be corrected its mortality rate, its correction method is as follows: control group live insect number - treatment group live insect number correction mortality rate (%) x x 100 control group live insect number Note 3: If there are no vegetable green insects, can be used for two-year-old silkworm generation, the test method remains unchanged.
C and PH values: using precision PH test paper determination.
Ii: Bt Preparation Instructions for Use : This product is an efficient microbial insecticide.
has the characteristics of harmless to people and animals, no injury to natural enemies, no pollution, low price and so on.
can be widely used in agriculture, for forests, fruits, vegetables, urban garden pest control, in the establishment of pollution-free vegetables, tobacco, tea cash crop areas, especially has a very good promotion value.
practice has proved that the Bt emulsion produced by our factory not only has a significant insecticidal effect and a long duration, but also brings a wide range of social and production benefits.
can completely replace some highly toxic pesticides such as potassium amine phosphorus.
the "sex" liquid dosage form (sticky milky liquid) "Regulation" per milliliter containing more than 10 billion spores, spores than 1:1.
the use of the use of mu is generally 2 male and two.
spray with water.
refer to the following table: Note: (1) adding a small amount of chemical pesticides can increase efficiency;
avoid exposure to the sun and high temperatures.