Bacillus Thuringiensis - Quantification-Heavy Metal Analysis Method PDF

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17 / F, S eav i ew Pla za, No . 2 83 S h a u Ke i Wa n Ro a d, S h au K e i Wa n

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ISO 9001:2008
METHOD OF ANALYSIS FOR HEAVY METALS (Pb , cadmium

(Cd),arsenic (As), chromium (Cr))
、Cd、
Study on the determination of As、、Pb、
、Cr by ICP-AES
The heavy metal elements in - were analyzed by means of microwave-assisted digestion

with AFS and AAS. - samples were digested completely with 3 ml HNO3, and 1ml HF, and it
showed that there wasn’t big difference to analyze the standard soul with AFS,AAS and ICP-
MS.
Instruments
microwave dissolver (ethos one, Italy milestone)
USA PE optima 7300V Plasma Automic Emission Spectrometer , Quartz Torch, Scott double
spray chamber, Meinhard Concentric nebulizer, three channel Wriggle pump ,
SCD(Subsection Charge-Coupled Detector)
Reagent
HNO3, HF, H2O2, A.R.
Pb,As,Cr, Cd standard solution, 1.0mg/ml
deionized water;
Procedure (- Pre-treatment: )
Bake - in 105℃oven to dry, grind to small granule, then dry in 105℃oven again. Cool and
transfer it to a mortar and grind to a fine powder. Weighing and through 100mesh
microwave dissolve:
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ISO 9001:2008
Weigh 0.2g(accurate to 0.0001g) to Ceramic digestion tank, also parallel sample and the
blank sample. Add 7ml H3NO3, 1ml H2O2 and 1ml HF to three separate tank, put three
tanks in draught cupboard till the reaction finished, cover and transfer to microwave
dissolver. Program increase temperature to 200 within 5mins, and hold for 15mins, cool
and take out. Open the tank, filter the digestion solution to 250ml volumetric flask, dilute
with deionized water to scan. The solution analyzed with ICP-AES.
ICP-AES working condition:

High-frequency power: 1150W
carrier gas pressure: 25Psi
auxiliary gas flow rate:1.0L/min
bi-survey Plasmas mode
wavelength: 228．812nm
Sample test
Pre-treat - sample as above, then analyzed by ICP-AES
Calibration curve
Dilute the standard solution of 1.0ml/ml to serious standard solution
Pb: 0, 1.00, 5.00, 10.00mg/l
Cr: 0, 1.00, 5.00, 10.00mg/l
Cd: 0, 0.50, 2.50, 5.00mg/l
As: 0, 2.00, 5.00, 10.00mg/l
Then analyzed by ICP-AES to get the fitting equation of each element
Pb Y=0.8732+0.0057X R=0.99999
Cd Y=0.0947+57.3093X R=0.99997
Cr Y=0.4362+11.1921X R=0.99999
As Y=-0.0644+1.3247X R=0.99989
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ISO 9001:2008
Quantification of Bacillus thuringiensis subs aizawai
Treat the parasporal crystal of bacillus thuringiensis TC with alkaline solution to get the
protoxin, then according to the differences of protein molecular weight , through the SDS-
PAGE to isolate the protoxin away from the bacillus thuringiensis strains protein,
quantitative the content according to the protein zone area by Electro phoretic pattern
scanning
Instruments
Electrophoresis system,
double-sided vertical cell(1.5mm Concave grooved rubber mold), gel area:170mm
170mm(1.5mm、20 hole sample mold)
Gel Documentation system
Centrifuge:10000r/min
Balance: accurate to 0.0001g
Reagents and solutions

ammonium persulfate(APS), Sodium dodecyl sulfate(SDS),
tetramethylethylenediamine (TEMED), NaOH(sodium hydroxide)
30% acrylamide gel stock solution: weigh 30g acrylamide, 0.8g N,N'-
Methylenebisacrylamide(MBA) to 100ml distilled water, filter, store at 4℃in dark place
SEPARATING GEL BUFFER: dissolved Tris(hydroxymethyl)aminomethane(Tris) 18.17g
and SDS 0.4g in distilled water, adjust pH to 8.8 with concentrated hydrochloric acid, then
dilute with distilled water to 100ml.
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Stacking gel Buffer:weigh 6.06g Tris and 0.4g SDS, dissolved with distilled water, adjust pH
to 6.8 with concentrated hydrochloric acid, then dilute with distilled water to 100ml
electrode buffer: weigh 3.036g Tris,14.42g Glycine and 0.4g SDS, dissolved with distilled
water to 1000ml.
3×Sample Diluent: 1mol/L、pH6.8 Tris-HCl 18.75ml,SDS 6g, Glycerin Glycerol 30ml,
Mercaptoethanol 15ml, few bromophenol blue, dilute with distilled water to 100ml
Fixing solution: 500ml 95% ethanol, 160ml acetic acid, dilute with distilled water to
1000ml
Staining solution:weigh Coomassiebrilliantblue (CBB) R-250 1g,add 250ml 95% ethanol,
80ml acetic acid, dilute with distilled water to 1000ml, filter for use
Destaining solution: 250ml 95% ethanol, 80ml acetic acid, dilute with distilled water to
1000ml
Protoxin standard: protoxin(molecular weigh 130kDa) 8.0% TC
Sample treatment
Weigh standard, sample separately 20.0mg(accurate to 0.1mg), transfer to 1.5ml centrifuge
tube, suspension with 1ml water. Pipette 100μL to another 1.5ml centrifuge tube, add 25μL
0.5mol/L NaOH(get the NaOH 0.1mol/L), stratify for 5min, add 75μL 3×Sample Diluent, the
total volume is 200μL . Boiled for 6min in 100℃water, centrifuge at 2000r/min for 10min,
collect the upper layer for Electrophoresis sampling.
SDS-PAGE isolate the protoxin
1. Preparing the 8%-10% Polyacrylamide Gel by discontinuous buffer system
2.Sampling: sample standard solution on the polyacrylamide gel hole with
6,8,10,12,14µL(protoxin approximately 3-7µg) to get a calibration curve. Then sample the
sample solution upper layer(approximately 5µg) to the hole, inject the electrode buffer,
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ISO 9001:2008
turn on the current.

3. Electrophoresis: the voltage of the initial stage is 100V,when the sample reach the
SEPARATING GEL, increase to 170V, continue till the indicator reach the bottom of 1cm,
stop and take away the gel, soak in 7.5% acetic acid for 30mins.
4. Staining: stain SEPARATING GEL with CBB R-250 overnight
5. Destaining: discard the staining solution, wash gel with rinsing solution, then add
destaining solution, destain at 37 , replace the destaining solution till the background is
clear.
Determination
The 130kDa protein zone is clear after destaining, analyzed the gel by by gel imaging
system
M: the calculated protein content from the calibration curve

C: sample concentrations
V: sample volume
n: sample dilute times

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WILLOWOOD LIMITED

17 / F, S eav i ew Pla za, No . 2 83 S h a u Ke i Wa n Ro a d, S h au K e i Wa n

METHOD OF ANALYSIS FOR HEAVY METALS (Pb , cadmium

The heavy metal elements in - were analyzed by means of microwave-assisted digestion

USA HONG KONG INDIA AFRICA CHINA CENTRAL AMERICA

ICP-AES working condition:

USA HONG KONG INDIA AFRICA CHINA CENTRAL AMERICA

Quantification of Bacillus thuringiensis subs aizawai

Reagents and solutions

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USA HONG KONG INDIA AFRICA CHINA CENTRAL AMERICA

USA HONG KONG INDIA AFRICA CHINA CENTRAL AMERICA

turn on the current.

M: the calculated protein content from the calibration curve

5 WILLOWOOD GROUP OFFICES WORLD-WIDE

USA HONG KONG INDIA AFRICA CHINA CENTRAL AMERICA

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