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[HG/T 3616-1999] Bacillus thuringiensis technical
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HG 3616-1999: PDF in English HG 3616-1999
HG
CHEMICAL INDUSTRY STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
Bacillus thuringiensis technical
APPROVED ON: JUNE 16, 1999
IMPLEMENTED ON: JUNE 01, 2000
Approved by: National Bureau of Petroleum and Chemical Industry
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative references ... 4
3 Requirements ... 4
4 Test methods ... 5
5 Inspection rules ... 10
6 Marks, labels, packaging, storage and transportation... 10
Annex A (informative) Preparation of electrophoretic gel ... 11
Annex B (standard) Determination of virulence potency ... 13
Bacillus thuringiensis technical
Bacillus thuringiensis (B. t.) is the most widely used microbial insecticide. Its main
insecticidal component is the toxin protein in the parasporal crystal. The relative
molecular mass of the protein that is toxic to Lepidoptera is 130000.
1 Scope
This Standard specifies the requirements, test methods, marks, labels, packaging,
storage and transportation of Bacillus thuringiensis raw powder.
This Standard applies to the original powder of Bacillus thuringiensis for the control of
Lepidoptera pests.
2 Normative references
The following standards contain provisions which, through reference in this Standard,
constitute provisions of this Standard. At the time of publication, the editions indicated
are valid. All standards are subject to revision. The parties who are using this Standard
shall explore the possibility of using the latest version of the following standards.
GB/T 1250-1989, Rules for expression and judgement of limiting values
GB/T 1600-1979 (1989), Determination of the moisture content in pesticides
GB/T 1601-1993, Determination method of pH value for pesticides
GB/T 1604-1995, Commodity pesticide regulations for acceptance
GB/T 1605-1979 (1989), Sampling method for commercial pesticides
GB 3796-1983, General rule for packing of pesticides
GB/T 16150-1995, Sieve test for dustable and wettable powders of pesticides
3 Requirements
3.1 Appearance: Off-white to tan powder.
3.2 The raw powder of Bacillus thuringiensis shall meet the requirements in Table 1.
4.3.1.2 Instruments, equipment
Electrophoresis.
Sandwich type vertical electrophoresis tank (1.5mm concave rubber frame with groove),
gel plate area 145mm × 100mm (1.5mm, 12-well sample tank mold).
High-speed thin-layer tomography scanner or electrophoretic image scanner.
Centrifuge: 10000r/min.
Analytical balance: accurate to 0.0001g.
4.3.1.3 Reagents and solutions
Ammonium persulfate (AP).
Sodium dodecyl sulfate (SDS).
Tetramethylethylenediamine (TEMED).
Sodium hydroxide.
30% acrylamide: Weigh 30g of acrylamide, 0.8g of methylene bisacrylamide (formerly
known as: methylene bisacrylamide). Dissolve in 100mL of distilled water. Filter. Store
at 4°C in a dark place for future use.
1mol/L, pH8.8 trishydroxymethylaminomethane-HCl buffer solution: Weigh 30.25g of
trishydroxymethylaminomethane and dissolve in distilled water. Adjust to pH 8.8 with
concentrated hydrochloric acid. Use distilled water to set volume constant to 250mL.
1mol/L, pH6.8 trishydroxymethylaminomethane-HCl buffer solution: Weigh 12.10g of
trishydroxymethylaminomethane and dissolve in distilled water. Adjust to pH 6.8 with
concentrated hydrochloric acid. Use distilled water to set volume constant to 100mL.
Electrode buffer solution: Weigh 3.03g of trishydroxymethylaminomethane, 14.42g of
glycine, 1g of sodium lauryl sulfate. Use water to dissolve and set volume constant to
1000mL.
3X sample diluent: 18.75mL of 1mol/L, pH6.8 trishydroxymethylaminomethane-HCl,
6g of sodium lauryl sulfate, 30mL of glycerin, 15mL of mercaptoethanol, a little
bromophenol blue. Use distilled water to set volume constant to 100mL.
Staining solution: Weigh 1g of Coomassie Brilliant Blue (CBB) R-250. Add 450mL of
methanol, 100mL of glacial acetic acid, 450mL of distilled water. Dissolve and filter
for future use.
Decolorizing solution: Measure 100mL of methanol, 35mL of glacial acetic acid. Use
distilled water to set volume constant to 1000mL.
Rinse solution: Measure 30mL of absolute ethanol, 10mL of glacial acetic acid, and
60mL of distilled water. Mix well before use.
Toxin protein standard sample: Raw powder of which toxin protein (relative molecular
mass is 130000) content is 9.3%.
4.3.1.4 Specimen processing
Weigh 20mg of standard sample and specimen (accurate to 0.1mg) respectively.
Transfer to a 5mL centrifuge tube. Add 2mL of water to fully suspend. Then add 0.45mL
of 0.55mol/L sodium hydroxide solution (so that the final concentration of sodium
hydroxide solution is 0.1mol/L). Place for about 5min. Then add 1.30mL of 3X sample
diluent. Make the final volume to 3.75mL. Boil for 6min in 100°C boiling water.
Centrifuge (at 2000 r/min) for 10min. Take the supernatant, so as to prepare samples
for electrophoresis.
4.3.1.5 SDS-PAGE separation of toxin proteins
a) Prepare 8%~10% polyacrylamide gel
Use the discontinuous buffer system. For the gel making method, see Annex A
(informative).
b) Sample loading
Take the supernatant of the above-mentioned standard sample solution. Load 6,
8, 10, 12, and 14μL of samples into the sample wells of the polyacrylamide gel
respectively (toxin protein content is about 3~7μg), as the standard curve. Then
take a certain volume of the supernatant of the specimen solution (toxin protein
content is about 5μg). Add to the sample well. After injecting the electrode buffer,
turn on the power.
c) Electrophoresis
The initial voltage of electrophoresis is controlled at about 100V. After the
specimen enters the separation gel, increase the voltage to 120V and continue
electrophoresis. Stop electrophoresis when the front of the indicator reaches about
1cm from the bottom. Take out the gel plate. Soak in 7.5% (volume percent) acetic
acid for 30min.
d) Dyeing
Remove the separation gel. Stain overnight with Coomassie brilliant blue (CBB)
R-250 staining solution.
e) Decolorizing
Pyridine.
Others are the same as 4.3.1.3.
4.3.2.4 Specimen processing
Same as 4.3.1.4.
4.3.2.5 SDS-PAGE separation of toxin proteins
a) Prepare 8%~10% polyacrylamide gel
Same as 4.3.1.5a).
b) Sample loading
Take the supernatant of the above standard solution. Load 15, 20, 30, 40, 50μL of
samples into the sample wells respectively (toxin protein content is about
7.5~25μg), as the standard curve. Then take a certain volume of the supernatant
of the specimen solution (toxin protein content is about 15μg). Add to the sample
well. After injecting the electrode buffer, turn on the power.
c) Electrophoresis
Same as 4.3.1.5c).
d) Dyeing
Same as 4.3.1.5d).
e) Decolorizing
Same as 4.3.1.5e).
4.3.2.6 Determination
Scrape off the zone to be tested with a scalpel. Put in a glass test tube. Then add 3.0mL
of 25% pyridine (volume fraction). Shake at 37°C to elute the Cox Brilliant Blue (CBB)
R-250 adsorbed by the toxin protein. After equilibration use a spectrophotometer. With
reference to 25% pyridine, at 605nm, measure the absorbance of the solution. The toxin
protein content is calculated by formula (1).
4.3.2.7 Allowable difference
Take the arithmetic mean value as the determination result. The relative deviation of
the two parallel determination results is less than or equal to 8%.
4.4 Determination of virulence potency
Annex A
(informative)
Preparation of electrophoretic gel
A1 Plate preparation
Plate electrophoresis is used in this experiment. The specific operation depends on the
laboratory conditions. The basic operation is generally to select two glass plates of the
same size according to the height of the electrophoresis tank. One of the plates has a
2~3cm high groove at one end. After the two glass plates are washed and dried, place a
"gap strip" on both sides of the glass plate without grooves (plastic strips and rubber
strips are acceptable, and its width and thickness are determined according to needs).
Then put the grooved glass plate. Secure the two glass plates with clips. In this way, a
certain gap is formed between the two glass plates. The lower end of the formed gap
shall be closed to prevent the poured gel from leaking out. Usually seal with tape. After
the poured gel is solidified, tear off the geld paper. Or block with 1%~1.5% agar. The
method is: Add electrode buffer or distilled water to the agar. Heat to dissolve in a
boiling water bath. Place the glass plate device with a gap vertically in a small tank with
a height of 3cm and a width of 3cm, which is wider and longer than the glass plate
(commercial electrophoresis tanks have matching devices). Pour the dissolved agar gel
into the small tank while it is hot. After cooling, take out the glass plate device. The
lower end is sealed tightly. Polyacrylamide gel can be perfused.
A2 Separation gel preparation
Take out the gel-making reagent from the refrigerator and equilibrate to room
temperature. Prepare the separation gel according to Table A1. The separation gel
concentration in this experiment is 10%.
After the gel is prepared and mixed, quickly inject it into the gap between the two glass
plates until the gel surface is about 3.5cm away from the groove of the glass plate. Then
gently spread 1cm high distilled water on the gel surface. When adding distilled water,
it is usually added slowly along the glass plate. Do not disturb the gel surface. Place the
gel plate vertically at room temperature for about 20~30min to allow it to condense. At
this point, a very clear interface can be seen between the gel and the distilled water.
Then suck out the distilled water on the gel surface.
A3 Stacking gel preparation
Its dosage depends on the actual situation. The preparation method is according to Table
A1.
Annex B
(standard)
Determination of virulence potency
B1 Method One for the determination of virulence potency - Determination
method using Plutella xylostella as test insect (arbitration method)
B1.1 Reagents or materials
Reference substance: CS-95, , potency is 20000IU/mg.
Larvae of Plutella xylostella: Plutella xylostella.
Edible rapeseed oil.
Yeast powder: for industrial use.
Vitamin C: medical, analytically pure.
Agar powder: the gel strength is greater than 300g/cm2.
Dipotassium hydrogen phosphate: analytically pure.
Potassium dihydrogen phosphate: analytically pure.
Polysorbate-80: viscosity is 3.5×10-4~5.5×10-4m2/s.
Vegetable leaf powder: Brassica napus leaves are dried at 80°C, ground, and passed
through an 80-mesh sieve.
Sucrose: analytically pure.
Cellulose powder CF-11.
Potassium hydroxide: analytically pure.
Sodium chloride: analytically pure.
15% Paraben: methylparaben (chemically pure) is dissolved in 95% ethanol.
10% formaldehyde solution: formaldehyde (analytically pure) is dissolved in distilled
water.
Casein solution: 2g of casein (BR biological reagent) with 2mL of 0.001mol/L
preparation method of the reference substance.
c) Suspended concentrate sample
Oscillate the sample for 20min. Shake well. Take 10.00mL of sample with a
pipette. Add to a ground-mouth Erlenmeyer flask filled with 90mL of sterile
distilled water. Pipette three times. Shake well to obtain a mother solution
containing 100μL/mL. Dilute the mother solution into six dilutions with contents
of 5.000, 2.500, 1.250, 0.625, 0.313 and 0.156μL/mL.
For some samples whose potency is too high or too low, before the determination,
it is necessary to do a preliminary test with 3 concentrations with large distance
differences. Estimate the range of LC50 values (half lethal intermediate
concentration). The dilution concentration is designed accordingly.
B1.3.2 Preparation of infected feed
Feed formula: 0.5g of vitamin C, 1.0mL of casein solution, 3.0g of vegetable leaf
powder, 1.5g of yeast powder, 1.0g of vitamin powder, 2.0g of agar powder, 6.0g of
sucrose, 0.2mL of rapeseed oil, 0.5mL of 10% formaldehyde solution, 1.0mL of 15%
paraben, and 100mL of distilled water.
Add sucrose, yeast powder, casein solution, and agar powder to 90mL of distilled water
and mix thoroughly. Stir and boil to completely melt the agar. Add paraben and stir well.
Blend the other ingredients into a paste with the remaining 10mL of distilled water. Mix
well with the agar when it has cooled to around 75°C. Stir well. Put it in a 55°C water
bath to keep warm for later use. Take seven 50mL beakers. Label them. Preheat in the
55°C water bath. Add 1mL of the corresponding concentration of infection solution to
each beaker. Use buffer as blank control. Add 9mL of molten infected feed to each
beaker. Stir with an electric stirrer for 20s to fully mix the infection solution and feed
in each beaker.
Let the beaker rest. After being cooled and solidified, use a medical scalpel to cut the
infected feed into 1cm × 1cm feed blocks. Take 4 feed blocks for each concentration
and put them into 4 insect tubes respectively. Put one block in each tube and write the
label.
B1.3.3 Infestation
Randomly take the insect tube that has been placed with the feed. Put 10 third-instar
early larvae of Plutella xylostella into each tube. Take 4 tubes of each concentration.
Put on tampons. Write labels. Feed under the same feeding conditions.
B1.4 Result checking and calculation
48h after infection, check the death of the test insects. Criteria for judging dead insects:
Gently touch the insect body with a thin stick. Those who do not respond are deemed
Constant temperature incubator.
B2.3 Determination steps
B2.3.1 Feed preparation
Feed formula: 12g of yeast powder, 24g of soybean powder, 1.5g of vitamin C, 0.42g
of sodium benzoate, 3.9mL of 36% acetic acid, 300mL of distilled water.
Put soybean powder, yeast powder, vitamin C, sodium benzoate and 36% acetic acid
into a large beaker. Add 100mL of distilled water to moisten. Add the remaining 200mL
of distilled water to the agar powder. Heat till boil in the microwave to completely melt
the agar. Take out and cool to 70°C. Mix with other ingredients immediately. Stir at
high speed in an electric mixer for 1min. Quickly move to a 60°C water bath and cover
to keep warm.
B2.3.2 Preparation of infection solution
Weigh 100.0~150.0mg of wettable powder sample. Put it into a ground Erlenmeyer
flask with a stopper filled with glass beads. Add 100mL of phosphate buffer. Soak for
10min. Oscillate on the oscillator for 1min to prepare the mother solution. Or after fully
shaking the suspension sample evenly, draw 1.00mL to a ground Erlenmeyer flask with
a stopper filled with glass beads. Add 99mL of phosphate buffer. Soak for 10min.
Oscillate on the oscillator for 1min to prepare the mother solution. Weigh
150.0~300.0mg of reference substance (accurate to 0.1mg) on the analytical balance.
Make it into mother solution according to the above method. Dilute the sample and
standard mother solution with phosphate buffer in a certain ratio. At least 5
concentrations are diluted for each sample and reference substance. Set a buffer solution
as a control. Pipette 3mL to 50mL of each concentration of infection solution into a
small beaker for use. As a control, pipette 3mL of phosphate buffer.
B2.3.3 Mixing and packaging of feed and infection solution
Pipette 27mL of feed with a syringe. Pour into the above-mentioned beaker that has
been placed with the sample or reference substance infection solution. Stir with an
electric mixer at high speed for 0.5min. Quickly pour it into each small hole on the
tissue culture plate (the pouring amount does not need to be consistent; the bottom of
the hole shall prevail). Solidify and set aside.
B2.3.4 Infestation
At room temperature of 26~30°C, shake the newly hatched larvae (within 12h after
hatching) without feeding into a specimen jar with a diameter of 20cm. Wait for a few
minutes to select healthy larvae that climb up the mouth of the tank as test insects. Use
a brush to gently move them into the small holes in the tissue discs where the infected
feed has been placed. One insect per hole. For each concentration and blank control,
put 48 insects. Cover with foil. Then stack the tissue culture plates one on top of the
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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