Some standard content:
ICS 65. 100. 10
National Standard of the People's Republic of China
GB/F 19567.2--2004
Bacillus thuringiensis suspension concentrate
Bacitlus thuringgiensis suspension concentrate2004-06-22Issued
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaAdministrative Committee of Standardization of the People's Republic of China
2004-12-01Implementation
GB/T19567.2--2004
Bacillus thuringiensis (Bt) is the most widely used microbial insecticide at present. Its main insecticidal ingredient is the toxin protein in the cytoplasm. Among them, the relative molecular mass of the toxin protein toxic to lepidopteran pests tested in this standard is 130 k[a. In the bioassay, beet armyworm is used to detect the toxicity of Bacillus thuringiensis suspensions with characteristics against lepidoptera and Spodoptera pests, while diamondback moth and cotton bollworm are used to detect the toxicity of Bacillus thuringiensis suspensions with activity against other lepidoptera pests. This standard is formulated based on the relevant materials of Bacillus thuringiensis industry and enterprise standards previously formulated by our country, combined with the actual situation of our country. This standard makes specific requirements and provisions for the requirements of Bacillus thuringiensis suspensions, test methods, sampling, packaging, transportation, etc., and provides a unified technical basis for the production of Bacillus thuringiensis. Appendix A of this standard is an informative record, and Appendix B is a normative appendix. This standard is proposed by the Ministry of Agriculture of the People's Republic of China. Drafting units of this standard: Pesticide Control Institute of the Ministry of Agriculture, National Engineering Research Center for Microbiological Pesticides of Huazhong Agricultural University, Chaobei Province Biological Bag Pesticide Engineering Research Center.
The main drafters of this standard are Jiang Hui, Dao Ziniu, Sui Wen, Wang Mei, Wang Xiaoqian, Shi Xinping, Gu Baoben. The Ministry of Agriculture Pesticide Inspection Institute is responsible for the interpretation. 1 Scope
Bacillus thuringiensis suspension
GB/T19567.2--2004
This standard specifies the requirements, test methods, and marking, labeling, packaging, storage and transportation of Bacillus thuringiensis suspension. This standard applies to Bacillus thuringiensis suspension prepared from Bacillus thuringiensis powder and adjuvants for preventing lepidopteran pests.
2 Normative references
The clauses in the following documents become the clauses of this standard through reference in this standard. For all dated referenced documents, all subsequent amendments (excluding errata) or revised versions are not applicable to this standard. However, parties to an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For any undated reference, the latest version shall apply. GB/T1250 Method for expressing and determining limiting values GB/T1600-2001 Method for determining moisture content of pesticides GB/T 1601 Method for determining pH value of pesticides GB/T1604 Rules for acceptance of commercial pesticides GR/T 1605-2001 Method for sampling commercial pesticides GB3796 General rules for pesticide packaging GB/T16150-1995 Method for determining fineness of pesticide powders and wettable powders GB/T5451-2001 Method for determining wettability of pesticide wettable powders 3 Requirements 3.1 Appearance: Brown or brown suspension. 3.2 Bacillus thuringiensis suspension concentrate shall meet the requirements of Table 1. Table 1 Control Items Index of Suspension Concentrate of Bacillus thuringiensis Toxin Protein (<130 kDa)/(%) Toxicity (P, Ha)/(IU/m1), (S. e.)/(H./mg) PH Value Suspension Rate (effective ingredient)/(%) Fineness (150μm)/(%) 1.5-~6.5 Note: P, Ha and Se are the condensation of Piuiella xyiastella, Heliothisannigem and Spodoptera triguz, respectively 4 Test Method Unless otherwise specified, all reagents used in this method are analytically pure and all solutions are aqueous solutions. 4.1 Sampling
The sampling is carried out in accordance with the "Sampling of Liquid Preparations" in GB/T1605-2001. The sampling packages are determined by the random number table method. The final sampling volume is not less than 250 mL.
GB/T 19567.2--2004
4. 2 Toxin protein content
The toxin protein content was determined by sodium dodecane sulfate-poly(ethyleneimine)amide (SDS-PAGE) gel image processing method. 4.2.1 Method summary
The parasporal crystals of Bacillus yunjinensis suspension were treated with alkaline solution to degrade them into toxin proteins, and then subjected to S1S-PAGE. The difference in the relative molecular mass of the proteins was used to separate the toxin proteins from other impurities. The protein zone area was scanned by electrophoresis image and quantified. 4.2.2 Instruments and equipment
a) Electrophoresis instrument;
b) Double vertical electrophoresis (1.5mm concave grooved rubber mold frame), gel plate area 170mm×170mm (1.5mm, 20-well sample mold)
Electrophoresis gel imaging system
d) Centrifuge: 10 00r/min:
e) Analytical balance: accurate to 0.0001g. 4.2.3 Reagents and solutions
4.2.3.1 Ammonium persulfate (APS).
4.2.3.2 Sodium dialkyl sulfate (SDS
4.2.3.3 Tetramethylethylenediamine (TFMED). 4.2.3.4 Sodium hydroxide.
4.2.3.5: 30% acrylamide gel mother solution, weigh acrylamide 30. Methylenebisacrylamide (formerly known as: acrylamide 4.2.3.6 Separation gel buffer: weigh 18.17 g of tris(hydroxymethyl)nitromethane (Tris) and 0.4 g of SDS, dissolve in distilled water, adjust to pH 8.8 with concentrated acid, and make up to 100 mL with distilled water: 4.2.3.7 Concentration gel buffer: weigh 6.06 g of Tris and 0.4 g of SDS, dissolve in distilled water, adjust to pH 6.8 with hydrochloric acid, and make up to 100 mL with distilled water. mL.
4.2.3.8 Electrode buffer: weigh 3.036g Tris, 14.42g glycine, 1g SDS, dissolve in water and dilute to 1000mL. 4.2.3.9 3× sample diluent: 1mol/T, pH 6.8 Tris-HC 18.75mL, SDS 6g glycerol 30mL, 15mL ethyl alcohol, dilute to 100mL with distilled water 4.2.3.10 Fixative: take 500mL 95% ethanol, 160ml glacial acetic acid, dilute to 1000mL with distilled water 4.2.3.11 Staining solution: weigh 1g Coomassie Brilliant Blue (CBB) R-250, add 250ml 95% ethanol, 80ml glacial acetic acid, dilute to 1000mL with distilled water mL, dissolve and filter before use. 4.2.3.12 Decolorizing solution: Measure 250mL of 95% ethanol and 80mL of glacial acetic acid, and dilute to 1000mL with distilled water. 4.2.3.13 Toxin protein standard sample: Toxin yellow white (relative molecular weight 130kDa) original powder with a base content of 8.0%. 4.2.4 Sample treatment
After shaking the sample, take 100μL and add it to a 1.5ml centrifuge tube, add 25μL of 0.5mol/L sodium hydroxide solution (so that the final concentration of the sodium hydroxide solution is 0.1mol/L), and let it stand for 5min. Weigh 20.0mg of the standard sample (accurate to 0.1mg), transfer it to a 1.5ml centrifuge tube, add 1mL of water to fully suspend it, take 100μL and add it to another 1.5ml centrifuge tube, add 25uL of 0.5mol/L sodium hydroxide solution (so that the total concentration of the sodium hydroxide solution is 0.7tnol/1.), and let it stand for about 5min. Add 75μL of 3× sample diluent to the standard sample and sample respectively, so that the final volume is 200pl., boil in 100℃ boiling water for 6min, centrifuge (2000T/min) for 10min, and take the upper clear liquid for electrophoresis.
4.2.5 SDS-PAGE separation of mycocystin
4.2.5.1 Preparation of 7.5% polyacrylamide gel using a discontinuous buffer system, gel preparation method see Appendix A (Informative Appendix). 4.2.5.2 Sample loading
Take the upper supernatant of the above standard sample solution and load 6, 8, 0, 12, 14L (mycocystin GB/T19567.2—2004
content is about 3ug--71g), as the standard curve, take a certain volume of the supernatant of the sample solution (the thin case protein content is about 5μg) and add it to the loading hole, inject it into the bottom of the electrode buffer, and turn on the power. 4.2.5.3 Electrophoresis
The initial voltage of electrophoresis is controlled at about 100V. After the sample enters the separation gel, the voltage is increased to 170V and the electrophoresis is continued. When the indicator front reaches about 1m from the bottom, the electrophoresis is stopped, the gel plate is taken out, and it is immersed in 7.5% (volume fraction) acid for 39min. 4.2.5.4 StainingWww.bzxZ.net
Partially remove the separation gel and stain it with Coomassie Brilliant Blue (CBB) R-250 staining solution overnight. 4.2.5.5 Decolorization
Pour off the staining solution, wash the gel with rinse solution first, then add decolorizing solution, decolorize at 37℃, and change the decolorizing solution several times until the background is clear.
4.2.6 Decolorization
After decolorizing the gel plate, the protein band with a molecular weight of 130kDa can be clearly seen. The gel is analyzed using a gel imaging system. The percentage of toxin protein in the sample (X) is calculated according to formula (1). XM
x×n×100%
M-~-the amount of protein calculated from the standard protein regression curve; C is the concentration of the sample;
V is the sample volume;
\ is the dilution multiple of the sample.
4.2.7 Allowable difference
The arithmetic mean is taken as the determination result, and the relative deviation of the results of two parallel determinations is less than or equal to 5%. 4.3 Determination of toxicity
Carry out according to Appendix B (Normative Appendix).
4.4 Determination of pH value
Determine according to GB/T1601.
4.5 Determination of fineness
Determine according to 2.2 of GB/T16150-~1995. 4.6 Determination of buoyancy
4. 6. 1 Apparatus and reagents
a) Volumetric siphon: 25() ml., with stopper;
b) Pipette:
e) Stopwatch:
d) Diagonal flask: 500mL100ml
Constant temperature water bath;
Standard hard water: The preparation method shall be in accordance with the standard hard water preparation method in GB/T5451-200°. 1
4.6.2 Operation steps
After spreading the sample evenly, take 5.00ml (accurate to 0.01ml) and put it in a 100ml.=angle bottle. Add 100ml of standard hard water. Oscillate 50 times with a flat plate. Transfer 250ml of the obtained suspension to a measuring cylinder with a stopper. Dilute it to 250ml with standard hard water. Put the measuring cylinder in a 30℃.11℃ constant overflow water bath. When the temperature of the suspension reaches 30℃, cover the measuring cylinder, pick up the measuring cylinder and gently shake the sediment first, then regularly move the measuring cylinder up and down with the middle of the measuring cylinder as the center to make a uniform suspension. Put the measuring cylinder in the constant temperature water bath, open the lid, let it stand for 30min, and use a pipette to extract 9/10 of the suspension in the upper part of the measuring cylinder by suction (complete within 15s to 30s). During the extraction process, the pipette should descend along the wall of the measuring cylinder as the liquid level drops, and do not stir the sediment at the bottom. The test suspension and the remaining 25 mL suspension in the recording tube shall be tested for toxicity according to Appendix B (Normative Appendix). 4.6.3 Calculation of the suspension rate (Y) in the sample shall be calculated according to formula (2): Y111.1x(CQ)×100%
Wu Zhong:
Central-toxicity of the test suspension:
Q--toxicity of the 25 mL suspension remaining at the bottom of the measuring tube. 4.6.4 Allowable difference
The difference between the results of two repeated measurements shall not exceed 10%. 5 Inspection rules
Conform to the relevant provisions of GB/T1604. The limit value shall be handled in accordance with GB/T1250. 6. Marking, labeling, packaging, storage and transportation
6.1 Product packaging should comply with the provisions of GB3796, and indicate the standard number used and the test insects used for testing. 6.2 The suspension concentrate is mainly packaged in plastic bottles and sealed. 6.3 When storing, it should be strictly protected from sunlight and pressure, and placed in a cool and dry place. 6.4 During transportation, be careful to handle it with care to prevent damage. 2
6.5 Guarantee period. Under normal storage and transportation conditions, the quality guarantee period of Bacillus thuringiensis suspension concentrate is 18 months from the production date. The content of toxicity titer and toxin protein of the product is less than 3.2 index when it leaves the factory. The content of toxicity titer and toxin protein of the product during the shelf life shall not be less than 60% of the 3.2 index.
A.1 Plate preparation
Appendix A
(Informative Appendix)
Preparation of electrophoresis gel
GB/T 19567.2--2004
This experiment uses a double vertical electrophoresis tank, and the specific operation depends on the laboratory conditions. The basic operation is to select two glass plates of the same size according to the size of the electrophoresis tank, one of which has a groove of 2 to 3 meters high at the end. After the two glass plates are washed and dried, a "spacer strip" (plastic strip or rubber strip can be used, and its width and thickness are determined according to needs) is placed on both sides of the glass plate without grooves, and then the glass plate with grooves is placed on top, and the two glass plates are fixed with clamps, so that a certain gap is formed between the two glass plates. The lower end of the gap should be sealed to prevent the injected glue from leaking out. It is usually sealed with adhesive tape. After the injected glue solidifies, the adhesive tape can be removed. Alternatively, it can be sealed with 1% to 1.5% agar. The method is: add electrode buffer or distilled water to the agar, heat dissolve in a boiling water bath, and vertically place the glass plate device with the gap in a small groove (commercial electrophoresis tanks have matching devices) with a height of 3 cm and a width of 3 cm, which is wider and longer than the glass plate. Then, heat the dissolved agar glue into the small groove, and take it out after cooling. The lower end of the glass plate device is sealed and the polyacrylamide glue can be injected. A.2 Preparation of separation gel
Take out the gel-making reagent from the ice, balance it to room temperature, and prepare the separation gel according to the formula in Table A.1. The concentration of the separation gel in this test is 7.5%. According to the formula in Table A.1, after the glue is well stirred and mixed, it is slowly injected into the gap between the two glass plates until the glue surface is about 3m away from the groove of the glass plate. Then gently spread distilled water m high on the gel space. When adding distilled water, usually add it slowly along the slope of the glass plate, do not disturb the gel surface. Place the gel plate vertically at room temperature for about 1 hour to make it coagulate. At this time, a very clear interface can be seen between the gel and the distilled water. Then pour out the distilled water with a small amount of distilled water on the gel surface.
A.3 Preparation of concentrated gel
The preparation method depends on the actual situation. The preparation method is prepared according to the formula in Table A.1. Mix the gel solution according to the formula in Table A.1, take a small amount and pour it into the glass separation plate, rinse and separate the gel surface. Then pour it out. Pour the remaining gel solution into the glass plate space, make the gel solution level and the groove of the glass plate, and then insert the "comb" (sample slot template), place it at room temperature for 20min~30min, and the concentrated gel can coagulate. After solidification, slowly take out the "comb". Be careful not to align the gel holes when taking it out. After taking out the "comb", add distilled water to the formed gel holes to rinse the uncondensed amide, pour out the distilled water in the hole, and then add electrode buffer. Fix the glass plate filled with gel vertically on the electrophoresis tank. The concave glass plate and the electrophoresis are close together to form a reservoir. Add electrode buffer to it so that it contacts the buffer in the gel holes. Add electrode buffer to the reservoir at the lower end of the electrophoresis tank. Table A, 1 SDS-PAGE gel formula
Storage solution
30% acrylamide mother solution
Separation gel buffer
Concentration gel buffer
Double distilled water
10% ammonium persulfate (APS)
Tetramethylethylenediamine (TFMEE)
Separation gel
Concentration gel
GB/T 19567.2---2004
(Normative Record)
Determination of toxicity
B.1 Method for determination of toxicity - Method for determination using Ptutetla xytostelta as test insect B. 1.1 Reagents or materials
Standard product: CS-1995, Hab, 20000IU/mg. Larvae of Plutelia zytostella: Edible rapeseed oil.
Yeast powder: Industrial use.
Vitamin C: Medical use, analytical grade.
Agar: Gel strength greater than 300g/cm.
Dipotassium hydrogen phosphate: Analytical grade.
Polysorbate-80: Viscosity 3.5×10m/s~5.5×10m/s. Leaf powder: Japanese blue rapeseed leaves, dried at 80℃, ground, and sieved through 80 mesh. Sugar: Analytical grade.
Cellulose powder CF-11,
Potassium hydroxide: analytical grade.
Sodium nitride: analytical grade
15% paraben: methyl parahydroxybenzoate (chemically pure) in 95° alcohol. 10% formaldehyde solution: formaldehyde (analytical grade) dissolved in distilled water, casein solution; add 2 g of casein (FR biological reagent), 0.1 mol/L potassium hydroxide 2 mL, 8 mL distilled water, sterilized. Phosphate buffer: 8.5 g of sodium chloride, 6.0 g of dipotassium hydrogen phosphate, 3.0 g of potassium monohydrogen phosphate, 0.1 mL of sorbitan-80 solution, 1 000 ml of distilled water..
B.1.2 Instruments and equipment
Grinding T-shaped flask: 250 mL, with stopper.
Analytical balance: accurate to 0.1 g.
Electric stirrer; stepless speed regulation, 100 t/min~6 000 r/min. Medical surgical inoculant.
Microwave oven or electric stove,
Medicine container.
Water bath.
Insect culture tube: 9 cmx2.5 cm.
Small beaker: 50mL,
Large beaker: 500mL.
Test tube: 18mm×180mm
Glass beads; diameter 5 mm.
Pipette: 10 mL, 5 mL, 2 mL, 1 ml
B. 1. 3 Determination steps
B. 1.3. 1 Preparation of infection solution
B. 1. 3. 1. Standard
GB/T 19567.2—2004
Use an analytical scale to accurately weigh 100.0 mg~15c.0 mg (accurate to 0.1 mg) of the standard and place it in a 250 mL triangular flask containing 10 glass beads. Add 100 ml of phosphate buffer, soak for 10 min, and shake in a sterilizer for 3 minutes to obtain a standard stock solution with a concentration of 1 mg/mL (the stock solution can be stored in a refrigerator for 10 days). Then dilute the standard stock solution into six diluted infection solutions with concentrations of 1.000, 0.500, 0.250, 0.125, 0.0625, and 0.0313 mg/mL. B.1.3.1.2 Suspension sample
Oscillate the sample for 20 min and shake it thoroughly. Pipette 10.00 mL of the sample (accurate to 0.01 mL). Add it to a 1.1 triangular flask containing 90 mL of sterile distilled water, pipette and rinse it for 10 times, and shake it thoroughly to obtain a 100 μL/mL stock solution. Dilute the stock solution to six gradient dilution solutions with concentrations of 4.000, 2.000, 1.000, 0.500, 0.250, and 10.125 μL/mL, respectively. For some samples with too high or too low titers, a preliminary test should be conducted with three concentrations with large differences before the determination to estimate the range of the lethal median concentration (1 value) and design the dilution concentration accordingly. B.1.3.2 Preparation of infected feed
Feed formula: 0.5g vitamin C, 10ml casein solution, 3.0g vegetable leaf powder, 1.5g motherwort powder, 1.0g cellulose powder, 2.0g agar powder, 6.0g cane sugar, 0.2mL rapeseed oil, 0.3mL 10% formaldehyde solution, 1.6ml 15% paraben, 100mL distilled water. Add sugarcane peel, yeast powder, casein solution and agar powder into 90% distilled water and mix well, stir and boil to dissolve agar completely, add paraben and stir, and mix other ingredients into paste with the remaining 10mL distilled water. When the agar temperature reaches about 75, mix it thoroughly, stir, and place it in a 55℃ water bath to keep warm for later use. Take 7 50 ml beakers, write labels, and preheat in 55°C water. Add 1 ml of infection solution of corresponding concentration to each beaker, and use buffer solution as blank control. Add 9 mL of dissolved infection solution to each beaker, and stir with an electric stirrer for 29 minutes to fully mix the infection solution and feed in each beaker. Let the beaker stand, wait for cooling and solidification, and cut the infected feed into 1 ti × 1 c feed blocks with a medical scalpel. Take 4 blocks of each concentration and put them into 4 insect tubes, one block for each meal, and write labels. B.1.3.3 Infection with insects
Randomly take insect tubes with feed placed. Put 10 first-instar larvae of diamondback moth into each tube, 4 tubes for each concentration, plug them with cotton wool, write labels, and raise them under the same feeding conditions.
B.1, 4 Result inspection and calculation
Check the death of the test insects 48 hours after infection. The standard for judging dead insects is to touch the insect body lightly with a thin swab, and those without any reaction are considered dead.
Calculate the mortality rate of the test insects at each concentration of the standard and sample, and look up the Allil011 table or calculate the corrected mortality rate (Xt). The blank control mortality rate needs to be corrected if it is below 10%, and the test result is invalid if it is greater than 10. The corrected mortality rate is calculated according to (B.1):
Where +
T—the mortality rate of the drug treatment;
C—the mortality rate of the blank control.
x100%
Convert each concentration of the infection solution into a logarithmic value, and convert the corrected mortality rate into a mortality rate value. Use the least squares method to respectively calculate the C value of the standard and the LC value of the sample to be tested, and calculate the toxicity titer (X) of the sample to be tested. Each potency is calculated according to formula (R.2):
S.--ICs-value of standard product;
P--potency of standard product:
GB/T 19567.2-2004
Y sample LC value.
B, ±. 5 Permissible difference
The relative deviation of the toxicity determination method is allowed, but the maximum relative deviation of the results of 3 repeated determinations of each sample shall not exceed 20%. The mortality rate caused by each concentration of the toxicity determination preparation should be between 10% and 90%, and there should be at least two concentrations above and below 50% mortality rate. B, 2 Toxicity titer determination method - determination method using cotton bollworm (Heliothis armigera) as test insect B.2.1 Reagents and materials
Standard product: CS-1995.Hb+200001U/mg. Cotton bollworm larvae: Heliothis armigera. Soybean powder: roast the soybeans and grind them to pass through a 60-mesh sieve. Flour: pass through a 60-mesh sieve.
Yeast powder: for industrial use.
36% acetic acid solution: acetic acid (chemically pure), dissolved in distilled water. Sodium benzoate: analytically pure.
Formaldehyde: analytically pure.
Vitamin C: for medical use, analytically pure.
Agar powder: gel strength greater than 300g/cm2. Phosphate buffer: same as 3.1.1.
E.2.2 Instruments and equipment
Analytical balance: accurate to 0.1ng.
Electric stirrer: stepless speed regulation, 100 r/min~-000 r/min microwave oven or electric stove,
vibrator.
Water boiler.
Tissue culture plate: 24 spores,
Porcelain plate: 30 cm×20 cm.
Grinded concave triangle bottle: 250 tnml., with stopper.
Large beaker: 1 000 ml.
Small beaker: 50m.
Test tube: 18mm×180mm
Glass beads; diameter 5 mm.
Syringe, 50 mL.
Specimen cylinder
Constant temperature incubator.
B.2. 3 Determination steps
B, 2.3.1 Simple material preparation
Feed formula: yeast powder 12 g, soybean powder 24 g, vitamin C 1. 5 g, sodium benzoate 0. 42 g, 36% acetic acid 3. 9 ml.. Distilled water 300 mL.
Put the yellow grass powder, yeast powder, vitamins, sodium benzoate and 36% acetic acid in a large beaker, add 100 ml distilled water to moisten and set aside: add the remaining 200 ml distilled water to the agar powder, heat in a microwave oven until boiling, dissolve the agar completely, take it out and cool it to 70, mix it with other ingredients, stir it in an electric stirrer at high reverse for 1 minute, and quickly move it to a 60 water bath pot and add heat to keep warm. B.2.3.2 Preparation of infection solution
After the suspension sample is fully shaken and homogenized, draw 1.00 mL (accurate to 0.01 mL) into a ground-mouth stoppered triangular flask GB/T 19567.2--2004
containing glass beads, add 99.0 mL of phosphate buffer, soak for 10 minutes, and shake it on an oscillator for 1 minute to form the mother solution. On an analytical balance: weigh 150.0 mg to 300.0 mg of standard (accurate to 0.1 nig),Prepare the mother solution as above. Dilute the sample and standard product stock solution with phosphate buffer in a certain ratio. Dilute each sample and standard product to at least 5 concentrations, and set buffer as a control. Pipet 3 mL of each concentration of infection solution into a 50 mL beaker for use. Pipet 3 mL of phosphate buffer for control. B. 2. 3. 3 Mixing and dispensing of feed and infection solution Pipet 27% feed into the artificial beaker containing sample or standard product infection solution with a syringe, stir at high speed for 0.5 min with an electric stirrer, and pour into each small hole on the tissue culture plate at a low speed (the amount poured does not need to be consistent, and the bottom of the hole is the best). Solidify and set aside. B.2.3.4 Inoculation rack
At room temperature of 26℃:~30℃, shake the newly hatched larvae (within 12 hours after hatching) that have not been fed into a specimen tank with a diameter of 20 cm, wait for a few minutes, select healthy larvae that have grown fat in the tank as test insects, and gently transfer them to the small holes of the tissue plate with infected feed with a brush, one insect in each hole. For each concentration and blank control, 48 insects are collected, covered with a plastic sheet, and then the tissue culture plates are stacked one by one, tied with a rubber band, and placed upright in a 30℃ constant temperature incubator for 72 hours. .2.4 Result inspection and statistical analysis
Use the naked eye or a magnifying glass to check the number of dead and live insects. Touch the insect body with a fine stick, and the insects that have no reaction at all are dead insects, and the mortality rate is calculated. If there is death in the control, the corrected mortality rate can be calculated by checking the Abhtt correction value table or formula (B.1). The control mortality rate does not need to be corrected if it is below 6%, but needs to be corrected if it is between 6% and 15%. If it is greater than 15%, the test is invalid. The concentration is converted into logarithmic values, and the mortality rate or corrected mortality rate is converted into rate value. The LC5 values of the standard product and the sample are calculated by the least square method, and the toxicity titer is calculated according to formula (R.2). B.2.5 Allowable relative deviation
The allowable relative deviation requirements for the toxicity determination method are the same as those in B.1, 5. B.3 Toxicity titer determination method - determination method using Spodoprera xigua as test insect B.3.1 Materials and reagents
Standard: CS-2002.Ib, 20000IU/mg beet armyworm larvae, Spodopterutrigua, soybean powder: fried soybeans and ground through 60 mesh. Yeast powder (200 mesh): for industrial use.
Vitamin C: medical, analytical grade,
15% paraben: methyl paraben (chemically pure) dissolved in 95% alcohol. 10% formaldehyde: formaldehyde (analytical grade) dissolved in distilled water. Agar: gel strength greater than 300/cm.
Distilled water.
B.3.2 Instruments
Analytical test, accuracy 0.1mg
Electric stirrer: stepless speed regulation, 100r/min-~6000r/min microwave oven.
Oscillator.
Water bath.
Tissue culture plate: 24 wells:
Porcelain plate: 30cm×20cm
Ground-mouth triangle bottle, 250 ml, cold.
Beaker: 1000mL.
Small beaker: 50ml.
Test tube: 18mm×180mm
GB/T19567.2-2004
Glass beads: 5tm in diameter.
Syringe: 50 mL.
Specimen preparation.
Constant vortex incubator.
B. 3. 3 Determination steps
B. 3. 3. 1 Material formula
Soybean powder 32g (60 days), yeast powder 16g (200 mesh), agar 6g, vitamin C 2g + 15% paraben 6.7mL, 10% formaldehyde 4mL, water 400mLa
Pour soybean powder, yeast powder, vitamin C, paraben and 10% formaldehyde into a large beaker, add 150mL water, mix and set aside. Add the remaining 2501ml of water to agar, heat to boiling in a microwave oven to completely dissolve the agar, remove and cool to 70℃. Mix with other ingredients, stir at high speed in an electric stirrer for 1min, quickly move to a 60% water bath and cover to keep warm. B.3.3.2 Preparation of infection solution
Weigh (Szab-2002 standard 150.0ng~300.0mg accurate to 0.1mg), put it into a ground-mouth stoppered triangular flask containing glass beads, add 100ml of phosphate buffer, soak for 10min, and shake on a shaker for 1min to obtain the standard mother solution. After the suspension sample is fully shaken and evenly mixed, draw 1.00mL (accurate to 0.01mL) into a ground-mouth stoppered triangular flask containing glass beads, add 99.00mL of phosphate buffer, soak for 10min, and shake on a shaker for 1min to obtain the sample mother solution. Dilute the mother solution to a certain concentration gradient by two-fold dilution method. Each sample should be diluted at least 5 times. Pipette 3 mL of infection solution of each concentration into a 50 mL beaker for use. Pipette 3 mL of phosphate buffer for control.
B.3.3.3 Mixing and dispensing of feed and infection solution Use a syringe to draw 27 mL of feed and inject it into the beaker containing the above sample or standard infection solution. Stir with an electric stirrer for 0.5 min and quickly pour it into each small hole on the tissue culture plate (the amount poured does not need to be consistent, and the bottom of the hole should be covered). Solidify and set aside. B.3.3.4 Infection with insects
In a room temperature of 26℃~30℃, shake the newly hatched larvae (within 12 hours after hatching) that have not been fed into a specimen cylinder with a diameter of 30 cm, wait for a few minutes, select the healthy larvae that are fat on the cylinder mouth as test insects, and gently move them into the small holes of the tissue plate with infected feed with a brush. 48 worms were collected for each concentration and blank control, covered with a plastic sheet, and then the tissue culture plates were stacked one by one, tied tightly with rubber bands, and placed vertically in a 25℃ incubator for 72 hours. B.3.4 Result inspection and statistical analysis
Check the number of dead and live worms with the naked eye or a magnifying glass. Touch the body with a fine stick. The worms that do not react at all are dead worms. Calculate the mortality rate. If there are dead worms in the control, you can check the Abbott correction value table or calculate the corrected mortality rate according to formula (B.1). If the control mortality rate is below 6%, no correction is required. If it is between 6% and 15%, the test is invalid if it is greater than 15%. Convert the concentration into a logarithmic value, convert the death rate or corrected mortality rate into a probability value, and use the least multiplication method or a calculator with statistical functions to calculate the 1.L· of the standard and sample respectively. Calculate the toxicity titer according to formula (B.2). B.3.5 Allowable deviation
The allowable relative deviation requirements for the toxicity determination method are similar to those of B1.5. B.4 Detection of suspected samples containing other effective ingredients For some suspected samples that meet the analytical indicators in 3.2 of the main text, the toxicity contribution rate of the crystal mixture in the suspension can be determined to determine whether the sample contains other effective ingredients. B,4.1 Separation of different components in the suspension
Use internal suspension to make a 10mg/1nL bath solution. Centrifuge at 5000r/min for 10min at 4℃, retain the precipitated component, suspend it with an equal volume of acetone, centrifuge it again, wash it by centrifugation 3 times, and retain the precipitated component; suspend the precipitated component with an equal volume of distilled water, centrifuge and retain the precipitated component, wash it by centrifugation 3 times, and retain the precipitated component. The precipitated component is the crystal mixture in the suspension. B.4.2 Determination of toxicity potency
According to the method of R.1, 13.2 or B.3, the toxicity potency of the suspension concentrate and the precipitated components of the suspension concentrate (cell mixture) shall be determined respectively.1) Calculate the corrected mortality rate. If the control mortality rate is below 6%, no correction is required. If it is between 6% and 15%, correction is required. If it is greater than 15%, the test is invalid. Convert the concentration to logarithmic value, the mortality rate or corrected mortality rate to rate value, and use the least square method to calculate the LC5 value of the standard product and the sample respectively. Calculate the toxicity titer according to formula (R.2). B.2.5 Allowable relative deviation
The allowable relative deviation requirements for the toxicity determination method are the same as those in B.1, 5. B.3 Toxicity titer determination method - determination method using Spodoprera xigua as test insect B.3.1 Materials and reagents
Standard: CS-2002.Ib, 20000IU/mg beet armyworm larvae, Spodopterutrigua, soybean powder: fried soybeans and ground through 60 mesh. Yeast powder (200 mesh): for industrial use.
Vitamin C: medical, analytical grade,
15% paraben: methyl paraben (chemically pure) dissolved in 95% alcohol. 10% formaldehyde: formaldehyde (analytical grade) dissolved in distilled water. Agar: gel strength greater than 300/cm.
Distilled water.
B.3.2 Instruments
Analytical test, accuracy 0.1mg
Electric stirrer: stepless speed regulation, 100r/min-~6000r/min microwave oven.
Oscillator.
Water bath.
Tissue culture plate: 24 wells:
Porcelain plate: 30cm×20cm
Ground-mouth triangle bottle, 250 ml, cold.
Beaker: 1000mL.
Small beaker: 50ml.
Test tube: 18mm×180mm
GB/T19567.2-2004
Glass beads: 5tm in diameter.
Syringe: 50 mL.
Specimen preparation.
Constant vortex incubator.
B. 3. 3 Determination steps
B. 3. 3. 1 Material formula
Soybean powder 32g (60 days), yeast powder 16g (200 mesh), agar 6g, vitamin C 2g + 15% paraben 6.7mL, 10% formaldehyde 4mL, water 400mLa
Pour soybean powder, yeast powder, vitamin C, paraben and 10% formaldehyde into a large beaker, add 150mL water, mix and set aside. Add the remaining 2501ml of water to agar, heat to boiling in a microwave oven to completely dissolve the agar, remove and cool to 70℃. Mix with other ingredients, stir at high speed in an electric stirrer for 1min, quickly move to a 60% water bath and cover to keep warm. B.3.3.2 Preparation of infection solution
Weigh (Szab-2002 standard 150.0ng~300.0mg accurate to 0.1mg), put it into a ground-mouth stoppered triangular flask containing glass beads, add 100ml of phosphate buffer, soak for 10min, and shake on a shaker for 1min to obtain the standard mother solution. After the suspension sample is fully shaken and evenly mixed, draw 1.00mL (accurate to 0.01mL) into a ground-mouth stoppered triangular flask containing glass beads, add 99.00mL of phosphate buffer, soak for 10min, and shake on a shaker for 1min to obtain the sample mother solution. Dilute the mother solution to a certain concentration gradient by two-fold dilution method. Each sample should be diluted at least 5 times. Pipette 3 mL of infection solution of each concentration into a 50 mL beaker for use. Pipette 3 mL of phosphate buffer for control.
B.3.3.3 Mixing and dispensing of feed and infection solution Use a syringe to draw 27 mL of feed and inject it into the beaker containing the above sample or standard infection solution. Stir with an electric stirrer for 0.5 min and quickly pour it into each small hole on the tissue culture plate (the amount poured does not need to be consistent, and the bottom of the hole should be covered). Solidify and set aside. B.3.3.4 Infection with insects
In a room temperature of 26℃~30℃, shake the newly hatched larvae (within 12 hours after hatching) that have not been fed into a specimen cylinder with a diameter of 30 cm, wait for a few minutes, select the healthy larvae that are fat on the cylinder mouth as test insects, and gently move them into the small holes of the tissue plate with infected feed with a brush. 48 worms were collected for each concentration and blank control, covered with a plastic sheet, and then the tissue culture plates were stacked one by one, tied tightly with rubber bands, and placed vertically in a 25℃ incubator for 72 hours. B.3.4 Result inspection and statistical analysis
Check the number of dead and live worms with the naked eye or a magnifying glass. Touch the body with a fine stick. The worms that do not react at all are dead worms. Calculate the mortality rate. If there are dead worms in the control, you can check the Abbott correction value table or calculate the corrected mortality rate according to formula (B.1). If the control mortality rate is below 6%, no correction is required. If it is between 6% and 15%, the test is invalid if it is greater than 15%. Convert the concentration into a logarithmic value, convert the death rate or corrected mortality rate into a probability value, and use the least multiplication method or a calculator with statistical functions to calculate the 1.L· of the standard and sample respectively. Calculate the toxicity titer according to formula (B.2). B.3.5 Allowable deviation
The allowable relative deviation requirements for the toxicity determination method are similar to those of B1.5. B.4 Detection of suspected samples containing other effective ingredients For some suspected samples that meet the analytical indicators in 3.2 of the main text, the toxicity contribution rate of the crystal mixture in the suspension can be determined to determine whether the sample contains other effective ingredients. B,4.1 Separation of different components in the suspension
Use internal suspension to make a 10mg/1nL bath solution. Centrifuge at 5000r/min for 10min at 4℃, retain the precipitated component, suspend it with an equal volume of acetone, centrifuge it again, wash it by centrifugation 3 times, and retain the precipitated component; suspend the precipitated component with an equal volume of distilled water, centrifuge and retain the precipitated component, wash it by centrifugation 3 times, and retain the precipitated component. The precipitated component is the crystal mixture in the suspension. B.4.2 Determination of toxicity potency
According to the method of R.1, 13.2 or B.3, the toxicity potency of the suspension concentrate and the precipitated components of the suspension concentrate (cell crystal mixture) shall be determined respectively.1) Calculate the corrected mortality rate. If the control mortality rate is below 6%, no correction is required. If it is between 6% and 15%, correction is required. If it is greater than 15%, the test is invalid. Convert the concentration to logarithmic value, the mortality rate or corrected mortality rate to rate value, and use the least square method to calculate the LC5 value of the standard product and the sample respectively. Calculate the toxicity titer according to formula (R.2). B.2.5 Allowable relative deviation
The allowable relative deviation requirements for the toxicity determination method are the same as those in B.1, 5. B.3 Toxicity titer determination method - determination method using Spodoprera xigua as test insect B.3.1 Materials and reagents
Standard: CS-2002.Ib, 20000IU/mg beet armyworm larvae, Spodopterutrigua, soybean powder: fried soybeans and ground through 60 mesh. Yeast powder (200 mesh): for industrial use.
Vitamin C: medical, analytical grade,
15% paraben: methyl paraben (chemically pure) dissolved in 95% alcohol. 10% formaldehyde: formaldehyde (analytical grade) dissolved in distilled water. Agar: gel strength greater than 300/cm.
Distilled water.
B.3.2 Instruments
Analytical test, accuracy 0.1mg
Electric stirrer: stepless speed regulation, 100r/min-~6000r/min microwave oven.
Oscillator.
Water bath.
Tissue culture plate: 24 wells:
Porcelain plate: 30cm×20cm
Ground-mouth triangle bottle, 250 ml, cold.
Beaker: 1000mL.
Small beaker: 50ml.
Test tube: 18mm×180mm
GB/T19567.2-2004
Glass beads: 5tm in diameter.
Syringe: 50 mL.
Specimen preparation.
Constant vortex incubator.
B. 3. 3 Determination steps
B. 3. 3. 1 Material formula
Soybean powder 32g (60 days), yeast powder 16g (200 mesh), agar 6g, vitamin C 2g + 15% paraben 6.7mL, 10% formaldehyde 4mL, water 400mLa
Pour soybean powder, yeast powder, vitamin C, paraben and 10% formaldehyde into a large beaker, add 150mL water, mix and set aside. Add the remaining 2501ml of water to agar, heat to boiling in a microwave oven to completely dissolve the agar, remove and cool to 70℃. Mix with other ingredients, stir at high speed in an electric stirrer for 1min, quickly move to a 60% water bath and cover to keep warm. B.3.3.2 Preparation of infection solution
Weigh (Szab-2002 standard 150.0ng~300.0mg accurate to 0.1mg), put it into a ground-mouth stoppered triangular flask containing glass beads, add 100ml of phosphate buffer, soak for 10min, and shake on a shaker for 1min to obtain the standard mother solution. After the suspension sample is fully shaken and evenly mixed, draw 1.00mL (accurate to 0.01mL) into a ground-mouth stoppered triangular flask containing glass beads, add 99.00mL of phosphate buffer, soak for 10min, and shake on a shaker for 1min to obtain the sample mother solution. Dilute the mother solution to a certain concentration gradient by two-fold dilution method. Each sample should be diluted at least 5 times. Pipette 3 mL of infection solution of each concentration into a 50 mL beaker for use. Pipette 3 mL of phosphate buffer for control.
B.3.3.3 Mixing and dispensing of feed and infection solution Use a syringe to draw 27 mL of feed and inject it into the beaker containing the above sample or standard infection solution. Stir with an electric stirrer for 0.5 min and quickly pour it into each small hole on the tissue culture plate (the amount poured does not need to be consistent, and the bottom of the hole should be covered). Solidify and set aside. B.3.3.4 Infection with insects
In a room temperature of 26℃~30℃, shake the newly hatched larvae (within 12 hours after hatching) that have not been fed into a specimen cylinder with a diameter of 30 cm, wait for a few minutes, select the healthy larvae that are fat on the cylinder mouth as test insects, and gently move them into the small holes of the tissue plate with infected feed with a brush. 48 worms were collected for each concentration and blank control, covered with a plastic sheet, and then the tissue culture plates were stacked one by one, tied tightly with rubber bands, and placed vertically in a 25℃ incubator for 72 hours. B.3.4 Result inspection and statistical analysis
Check the number of dead and live worms with the naked eye or a magnifying glass. Touch the body with a fine stick. The worms that do not react at all are dead worms. Calculate the mortality rate. If there are dead worms in the control, you can check the Abbott correction value table or calculate the corrected mortality rate according to formula (B.1). If the control mortality rate is below 6%, no correction is required. If it is between 6% and 15%, the test is invalid if it is greater than 15%. Convert the concentration into a logarithmic value, convert the death rate or corrected mortality rate into a probability value, and use the least multiplication method or a calculator with statistical functions to calculate the 1.L· of the standard and sample respectively. Calculate the toxicity titer according to formula (B.2). B.3.5 Allowable deviation
The allowable relative deviation requirements for the toxicity determination method are similar to those of B1.5. B.4 Detection of suspected samples containing other effective ingredients For some suspected samples that meet the analytical indicators in 3.2 of the main text, the toxicity contribution rate of the crystal mixture in the suspension can be determined to determine whether the sample contains other effective ingredients. B,4.1 Separation of different components in the suspension
Use internal suspension to make a 10mg/1nL bath solution. Centrifuge at 5000r/min for 10min at 4℃, retain the precipitated component, suspend it with an equal volume of acetone, centrifuge it again, wash it by centrifugation 3 times, and retain the precipitated component; suspend the precipitated component with an equal volume of distilled water, centrifuge and retain the precipitated component, wash it by centrifugation 3 times, and retain the precipitated component. The precipitated component is the crystal mixture in the suspension. B.4.2 Determination of toxicity potency
According to the method of R.1, 13.2 or B.3, the toxicity potency of the suspension concentrate and the precipitated components of the suspension concentrate (cell crystal mixture) shall be determined respectively.01mL), add 99.00mL of phosphate buffer to a ground-mouth stoppered flask containing glass beads, soak for 10 minutes, and vibrate on a vibrator for 1 minute to form the sample mother solution. Dilute the mother solution to a certain concentration gradient using the two-fold dilution method. Each sample should be diluted to at least 5 concentration gradients. Pipette 3 mL of each concentration infection solution into a 50mL beaker for use. Pipette 3 mL of phosphate buffer for control.
B.3.3.3 Mixing and subpackaging of feed and infection solution Use a syringe to draw 27 ml of feed and inject it into the beaker containing the sample or standard infection solution. Stir with an electric stirrer for 0.5 min and quickly pour it into the small holes on the tissue culture plate (the amount poured does not need to be consistent, and the bottom of the hole should be covered). Solidify and set aside. B.3.3.4 Infection
In a room temperature of 26℃～30℃, shake the newly hatched larvae (within 12 hours after hatching) that have not been fed into a specimen cylinder with a diameter of 30 cm, wait for a few minutes, select healthy larvae that are fat on the mouth of the cylinder as test insects, and use a brush to gently move them into the small holes of the tissue plate with infected feed. 48 larvae per hole. Cover with a plastic sheet, then stack the tissue culture plates one by one, tie them tightly with rubber bands, and place them vertically in a 25℃ incubator for 72 hours. B.3.4 Result inspection and statistical analysis
Check the number of dead and live worms with the naked eye or a magnifying glass. Use a fine swab to touch the body. If there is no reaction at all, it is a dead worm. Calculate the mortality rate. If there is a dead T. in the control, you can check the Abbott correction value table or calculate the corrected mortality rate according to formula (B.1). If the control mortality rate is below 6%, no correction is required. If it is between 6% and 15%, correction is required. If it is greater than 15%, the test is invalid. Convert the concentration into a logarithmic value, convert the death rate or corrected mortality rate into a probability value, use the least square method or a calculator with statistical function to calculate the 1.L· of the standard and sample respectively. Calculate the toxicity titer according to formula (B.2). B.3.5 Allowable deviation
The allowable relative deviation requirements for the toxicity determination method are similar to those of B1.5. B.4 Detection of suspected samples containing other effective ingredients For some suspected samples that meet the analytical indicators in 3.2 of the main text, the toxicity contribution rate of the crystal mixture in the suspension can be determined to determine whether the sample contains other effective ingredients. B,4.1 Separation of different components in the suspension
Use internal suspension to make a 10mg/1nL bath solution. Centrifuge at 5000r/min for 10min at 4℃, retain the precipitated component, suspend it with an equal volume of acetone, centrifuge it again, wash it by centrifugation 3 times, and retain the precipitated component; suspend the precipitated component with an equal volume of distilled water, centrifuge and retain the precipitated component, wash it by centrifugation 3 times, and retain the precipitated component. The precipitated component is the crystal mixture in the suspension. B.4.2 Determination of toxicity potency
According to the method of R.1, 13.2 or B.3, the toxicity potency of the suspension concentrate and the precipitated components of the suspension concentrate (cell crystal mixture) shall be determined respectively.01mL), add 99.00mL of phosphate buffer to a ground-mouth stoppered flask containing glass beads, soak for 10 minutes, and vibrate on a vibrator for 1 minute to form the sample mother solution. Dilute the mother solution to a certain concentration gradient using the two-fold dilution method. Each sample should be diluted to at least 5 concentration gradients. Pipette 3 mL of each concentration infection solution into a 50mL beaker for use. Pipette 3 mL of phosphate buffer for control.
B.3.3.3 Mixing and subpackaging of feed and infection solution Use a syringe to draw 27 ml of feed and inject it into the beaker containing the sample or standard infection solution. Stir with an electric stirrer for 0.5 min and quickly pour it into the small holes on the tissue culture plate (the amount poured does not need to be consistent, and the bottom of the hole should be covered). Solidify and set aside. B.3.3.4 Infection
In a room temperature of 26℃～30℃, shake the newly hatched larvae (within 12 hours after hatching) that have not been fed into a specimen cylinder with a diameter of 30 cm, wait for a few minutes, select healthy larvae that are fat on the mouth of the cylinder as test insects, and use a brush to gently move them into the small holes of the tissue plate with infected feed. 48 larvae per hole. Cover with a plastic sheet, then stack the tissue culture plates one by one, tie them tightly with rubber bands, and place them vertically in a 25℃ incubator for 72 hours. B.3.4 Result inspection and statistical analysis
Check the number of dead and live worms with the naked eye or a magnifying glass. Use a fine swab to touch the body. If there is no reaction at all, it is a dead worm. Calculate the mortality rate. If there is a dead T. in the control, you can check the Abbott correction value table or calculate the corrected mortality rate according to formula (B.1). If the control mortality rate is below 6%, no correction is required. If it is between 6% and 15%, correction is required. If it is greater than 15%, the test is invalid. Convert the concentration into a logarithmic value, convert the death rate or corrected mortality rate into a probability value, use the least square method or a calculator with statistical function to calculate the 1.L· of the standard and sample respectively. Calculate the toxicity titer according to formula (B.2). B.3.5 Allowable deviation
The allowable relative deviation requirements for the toxicity determination method are similar to those of B1.5. B.4 Detection of suspected samples containing other effective ingredients For some suspected samples that meet the analytical indicators in 3.2 of the main text, the toxicity contribution rate of the crystal mixture in the suspension can be determined to determine whether the sample contains other effective ingredients. B,4.1 Separation of different components in the suspension
Use internal suspension to make a 10mg/1nL bath solution. Centrifuge at 5000r/min for 10min at 4℃, retain the precipitated component, suspend it with an equal volume of acetone, centrifuge it again, wash it by centrifugation 3 times, and retain the precipitated component; suspend the precipitated component with an equal volume of distilled water, centrifuge and retain the precipitated component, wash it by centrifugation 3 times, and retain the precipitated component. The precipitated component is the crystal mixture in the suspension. B.4.2 Determination of toxicity potency
According to the method of R.1, 13.2 or B.3, the toxicity potency of the suspension concentrate and the precipitated components of the suspension concentrate (cell crystal mixture) shall be determined respectively.
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