CN110412028A - A kind of method of counting of insect PuGV - Google Patents
A kind of method of counting of insect PuGV Download PDFInfo
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- CN110412028A CN110412028A CN201810390398.2A CN201810390398A CN110412028A CN 110412028 A CN110412028 A CN 110412028A CN 201810390398 A CN201810390398 A CN 201810390398A CN 110412028 A CN110412028 A CN 110412028A
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- 238000000034 method Methods 0.000 title claims abstract description 43
- 241000238631 Hexapoda Species 0.000 title claims abstract description 27
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 28
- 239000012128 staining reagent Substances 0.000 claims abstract description 26
- 238000004043 dyeing Methods 0.000 claims abstract description 17
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 33
- 241000701451 unidentified granulovirus Species 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 229960000583 acetic acid Drugs 0.000 claims description 16
- 239000012895 dilution Substances 0.000 claims description 15
- 238000010790 dilution Methods 0.000 claims description 15
- 241000500437 Plutella xylostella Species 0.000 claims description 13
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 claims description 13
- 239000012362 glacial acetic acid Substances 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000000399 optical microscopy Methods 0.000 claims description 9
- 238000012545 processing Methods 0.000 claims description 6
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 239000012153 distilled water Substances 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 7
- 241000607479 Yersinia pestis Species 0.000 abstract description 3
- 239000000575 pesticide Substances 0.000 abstract description 3
- 239000008187 granular material Substances 0.000 abstract description 2
- 241001635274 Cydia pomonella Species 0.000 description 9
- 210000002845 virion Anatomy 0.000 description 9
- 241000700605 Viruses Species 0.000 description 8
- 238000004364 calculation method Methods 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 238000005259 measurement Methods 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 238000001914 filtration Methods 0.000 description 4
- 210000003474 viral inclusion body Anatomy 0.000 description 4
- 210000003000 inclusion body Anatomy 0.000 description 3
- 239000002917 insecticide Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000537222 Betabaculovirus Species 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 241000508269 Psidium Species 0.000 description 1
- 241000711931 Rhabdoviridae Species 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 102000017941 granulin Human genes 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- YIEDSISPYKQADU-UHFFFAOYSA-N n-acetyl-n-[2-methyl-4-[(2-methylphenyl)diazenyl]phenyl]acetamide Chemical compound C1=C(C)C(N(C(C)=O)C(=O)C)=CC=C1N=NC1=CC=CC=C1C YIEDSISPYKQADU-UHFFFAOYSA-N 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 210000004777 protein coat Anatomy 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 241000701366 unidentified nuclear polyhedrosis viruses Species 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
Abstract
The present invention provides a kind of method of counting of insect PuGV, the described method comprises the following steps: 1) insect PuGV sample being diluted 2~1000 times using staining reagent and make its dyeing；2) sample after step 1) dyeing is diluted 2~1000 times using decoloration reagent again and is still bleached；3) using bateria chamber to the sample count after step 2) decoloration, and according to the granule density of formula calculating insect PuGV.Compared with prior art, method provided by the invention is easy to operate, is easy to judge, as a result accurately.Not only can be with quantitative detection insect PuGV, but also the accuracy of detection, reliability can be improved.Method provided by the invention is easy to operate, and the instrument used is simple, can widely promote the use of the fields such as pesticide producing, control of agricultural pest.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of quantitative detecting method of virus, in particular it relates to one
The method of counting of kind insect PuGV.
Background technique
Insect PuGV is subordinate to Rhabdoviridae Beta baculoviral category (Beta baculovirus), with protein
The form of inclusion body exists, and 1 virion is contained inside each viral inclusion body, and viral inclusion body is in oblong particle
Shape is about 300-511nm, wide about 119-350nm.Virus infectivity is mainly determined by the integrality of DNA and protein coat, viral
The granulin that fine and close lattice has been wrapped up outside particle forms the inclusion body of virus.Viral inclusion body can be processed into environmentally friendly peace
Full biological insecticides, for preventing and treating host insect.
In the preparation of biological insecticides, the assay of viral inclusion body is always that the key of control of product quality refers to
Mark.But insect PuGV inclusion body is significantly less than nuclear polyhedrosis virus, it cannot be with hemocytometer plate in optical microscopy
Lower counting.It is complicated for operation using the method that electron microscope counts and based on the molecular biology method of fluorescent quantitation, and need
Specific equipment, instrument price are expensive.The method for determining virus concentration by bioassay determines virus according to insect mortality
The method time and effort consuming of concentration.Using spectrophotometry, what it is due to formation is that non-uniform solution leads to unstable result, error compared with
Greatly.That is, the generally existing operation of the method for counting of the insect PuGV of institute's style of calligraphy part is multiple in currently practical production
It is miscellaneous, incubation time is long, result poor repeatability or the problems such as depend on expensive instrument unduly.
Therefore, currently to simple and efficient, experience dependence is low, the method for counting of reproducible insect PuGV is deposited
In demand.
Summary of the invention
Therefore, the purpose of the present invention is to provide a kind of method of counting of insect PuGV, the method for the present invention includes
It the sample pretreatment of insect PuGV and is counted using bateria chamber.Method of counting provided by the invention is easy to operate,
It is easy to judge, as a result accurately, can widely promotes the use of the fields such as pesticide producing, control of agricultural pest.
The contents of the present invention include following technical scheme:
A kind of method of counting of insect PuGV, the described method comprises the following steps:
1) insect PuGV sample is diluted 2~1000 times using staining reagent and makes its dyeing；
2) sample after step 1) dyeing is diluted 2~1000 times using decoloration reagent again and is still bleached；
3) using bateria chamber to the sample count after step 2) decoloration, and insect PuGV is calculated according to formula
Granule density.
Preferably, the insect PuGV is plutella xylostella granulosis virus or carpocapsa pomonella granulosis virus
Preferably, in step 1), the staining reagent is selected from trypan blue, methylenum careuleum Yihong or Coomassie brilliant blue G250.
It is highly preferred that the staining reagent is Coomassie brilliant blue G250 in step 1)；It is further preferred that described examine
The concentration of Mas bright blue G250 is 0.1~10g/L；It is further preferred that the concentration of the Coomassie brilliant blue G250 be 0.5~
5g/L；Most preferably, the concentration of the Coomassie brilliant blue G250 is 1g/L；
Preferably, in step 1), the dyeing is included at 95~100 DEG C, handles insect particle using staining reagent
Precursor virus sample at least 5min；Preferably, the staining reagent processing time is at least 5min, at least 6min, at least 7min, extremely
Few 8min, at least 9min, at least 10min；It is highly preferred that the staining reagent processing time is at least 10min；
Preferably, in step 1), it is further preferably 2-50 times, most preferably that the extension rate, which is 2-100 times,
It is 10 times.
Preferably, in step 2), the decoloration reagent is selected from distilled water, glacial acetic acid or methanol, glacial acetic acid and water
Mixed solution；
Preferably, the decoloration reagent is the mixed solution of methanol, glacial acetic acid and water；
It is highly preferred that the volume ratio of methanol, glacial acetic acid and water is (1~5): (1~5) in the mixed solution: (1~
10)；
It is further preferred that the volume ratio of methanol, glacial acetic acid and water is 1:1:(1~10 in the mixed solution)；
It is further preferred that the volume ratio of methanol, glacial acetic acid and water is 1:1:8 in the mixed solution.
Preferably, in step 2), the decoloration reagent dilutions time is 1-10 minutes.
Preferably, in step 2), it is further preferably 2-50 times that the decoloration reagent dilutions multiple, which is 2-100 times,
Most preferably 10 times.
Preferably, in step 3), described count uses optical microscopy, more preferably difference optical microscopy；
Preferably, in step 3), the microscopical amplification factor is 4~20 times of eyepiece, 40-100 times of object lens；It is more excellent
Selection of land is 20 times of eyepiece, 63 times of object lens.
Calculation formula:
16 lattice × 25 lattice cell counting board calculation formula: cell number/ml=100 small lattice inner cell number/100 × 400
× 10000 × extension rate；
1,25 lattice × 16 lattice cell counting board calculation formula: cell number/ml=80 small lattice inner cell number/80 × 400
× 10000 × extension rate.
It was found by the inventors of the present invention that in conventional method, when counting, judges whether the particle under microscope is virus
Grain, needs based on practical experience, the dependence of experience is very big.And method of the invention is used, use the dye of certain concentration
After color reagent handles viral sample, and use decoloration reagent carries out specific factor dilution, virion is coloured, and is easy to judge,
So as to directly carry out quantitative detection to insect PuGV sample under an optical microscope.
Compared with prior art, the invention has the following advantages that
1) method provided by the invention is easy to operate, is easy to judge, as a result accurately.
2) method provided by the invention not only can be with quantitative detection insect PuGV, but also the standard of detection can be improved
True property, reliability.
3) method provided by the invention is easy to operate, and the instrument used is simple, can widely promote the use of pesticide
The fields such as production, control of agricultural pest.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 is plutella xylostella granulosis virus sample to be handled using Coomassie brilliant blue G250 staining reagent, and use decoloration examination
After dilution agent, the picture counted under the microscope using bateria chamber, from figure 1 it appears that using method of the invention
The plutella xylostella granulosis virus sample of processing is more easily observable under the microscope；
Fig. 2 is the figure that untreated plutella xylostella granulosis virus sample uses bateria chamber to count under the microscope
Piece, from figure 2 it can be seen that untreated plutella xylostella granulosis virus sample is not easy to observe under the microscope；
Fig. 3 is the plutella xylostella granulosis virus sample after being dyed using staining reagent, is made after being diluted with water under the microscope
The picture counted using bateria chamber, from figure 3, it can be seen that the sample in the picture is not as good as use decoloration reagent dilutions
Sample is apparent visible.
Fig. 4 is to use bacterium under the microscope using after method processing carpocapsa pomonella granulosis virus sample of the invention
The picture that tally counts, figure 4, it is seen that the carpocapsa pomonella granulosis virus sample handled using method of the invention
It is more clear under the microscope visible.
Specific embodiment
The present embodiment is implemented under the premise of the technical scheme of the present invention, gives detailed embodiment and mistake
Journey, but protection scope of the present invention is not limited to following embodiments.
Unless stated otherwise, the reagent used in the present invention is that experiment is pure or analysis is pure, and can be commercially available.
The method of counting of 1 plutella xylostella granulosis virus sample of embodiment
1) taking concentration is 1 × 1012The plutella xylostella granulosis virus sample (sample 1) of OB/ml is tried using dyeing of the invention
Sample 1 is diluted 10 times by agent, that is, takes bulk samples 0.1ml, and 0.9ml staining reagent is added.The staining reagent formula: coomassie
Brilliant blue G250 concentration is 1g/L, 0.22 μm of film filtering.Sample 1 is chosen three samples and is repeated.
Then, sample is cooled to room temperature in 100 DEG C of placement 10min.
2) step 1) treated sample is counted with after 100 times of reagent dilutions of decoloration.The decoloration reagent is methanol: ice
Acetic acid: water, volume ratio 1:1:8.
3) measurement is counted under an optical microscope
Using bateria chamber, 0.02 μm of counting chamber thickness.
Difference optical microscopy, 20 times of eyepiece, 40 times or 63 times of object lens.
Sample concentration is calculated by the following formula
According to extension rate and sample to be tested concentration calculation formula:
The sum of bulk samples concentration (OB/ml)=80 lattice virion number × 5 × 50000 × extension rate
1.4 interpretation of result
Plutella xylostella granulosis virus sample after dyeing, and using decoloration reagent dilutions after it is high-visible under the microscope
(Fig. 1) differs greatly (Fig. 2) with not stained specimens visibility, also smaller (Fig. 3) using water-reducible sample visibility.Virus
After particle is using the staining reagent of this method, decoloration reagent number concentration is reused are as follows: 6.5 × 1011OB/ml is not dyed not
It the use of decolorising agent number concentration (estimation) concentration is 1.23 × 1011OB/ml-1.5×1011OB/ml uses Guava
The measurement of easyCyte microcapillary cytoanalyze is 3.86 × 1010OB/ml.This is because not dyeing without using decolorising agent meter
Several methods, due to virion diameter only 200nm, and the counting chamber height of bateria chamber is 0.1mm, differs nearly 200 times,
And the error that the imaging factors such as focusing generate.And sample needs to be filtered to remove impurity when capillary cell analysis-e/or determining, with
And instrument automatic identification is more than the virion group that default size is sticked together, and causes particle measurement final concentration relatively low.
The quantitative detection of 2 carpocapsa pomonella granulosis virus sample of embodiment
1) taking concentration is 1 × 1012The staining reagent of the carpocapsa pomonella granulosis virus sample (sample 2) of OB/ml, invention will
Sample 2 dilutes 2 times, that is, takes bulk samples 0.1ml, and 0.1ml staining reagent is added.The staining reagent formula: Coomassie brilliant blue
G250 concentration is 5g/L, 0.22 μm of film filtering.Sample 2 is chosen three samples and is repeated.
Then, sample is cooled to room temperature in 95 DEG C of placement 5min.
2) step 1) treated sample is counted with after 1000 times of reagent dilutions of decoloration.The decoloration reagent is methanol:
Glacial acetic acid: water, volume ratio 1:1:10.
3) measurement is counted under an optical microscope
Using bateria chamber, 0.02 μm of counting chamber thickness.
Difference optical microscopy, 20 times of eyepiece, 40 times or 63 times of object lens.
Sample concentration is calculated by the following formula
According to extension rate and sample to be tested concentration calculation formula:
The sum of bulk samples concentration (OB/ml)=80 lattice virion number × 5 × 50000 × extension rate
1.4 interpretation of result
Carpocapsa pomonella granulosis virus sample after dyeing, and using decoloration reagent dilutions after it is high-visible under the microscope
Virion after dyeing is high-visible under the microscope (Fig. 4).Without using decoration method number concentration 7.0 × 1010OB/ml is low
Concentration 2.1 × 10 after dyeing11OB/ml-2.6×1011OB/ml。
The method of counting of 3 plutella xylostella granulosis virus sample of embodiment
1) taking concentration is 1 × 1012The plutella xylostella granulosis virus sample (sample 1) of OB/ml is tried using dyeing of the invention
Sample 1 is diluted 1000 times by agent, that is, takes bulk samples 0.1ml, and 99.9ml staining reagent is added.The staining reagent formula: platform
Expect that blue concentration is 0.1g/L, 0.22 μm of film filtering.Sample 1 is chosen three samples and is repeated.
Then, sample is cooled to room temperature in 95 DEG C of placement 6min.
2) step 1) treated sample is counted with after 2 times of reagent dilutions of decoloration.The decoloration reagent is methanol: ice vinegar
Acid: water, volume ratio 1:1:1.
3) measurement is counted under an optical microscope
Using bateria chamber, 0.02 μm of counting chamber thickness.
Difference optical microscopy, 20 times of eyepiece, 40 times or 63 times of object lens.
Sample concentration is calculated by the following formula
According to extension rate and sample to be tested concentration calculation formula:
The sum of bulk samples concentration (OB/ml)=80 lattice virion number × 5 × 50000 × extension rate
1.4 interpretation of result
Plutella xylostella granulosis virus sample after dyeing, and using decoloration reagent dilutions after it is high-visible under the microscope,
It differs greatly with not stained specimens visibility.
The quantitative detection of 4 carpocapsa pomonella granulosis virus sample of embodiment
1) taking concentration is 1 × 1012The staining reagent of the carpocapsa pomonella granulosis virus sample (sample 2) of OB/ml, invention will
Sample 2 dilutes 50 times, that is, takes bulk samples 0.1ml, and 4.9ml staining reagent is added.The staining reagent formula: Coomassie brilliant blue
G250 concentration is 0.5g/L, 0.22 μm of film filtering.Sample 2 is chosen three samples and is repeated.
Then, sample is cooled to room temperature in 95 DEG C of placement 5min.
2) step 1) treated sample is counted with after 10 times of reagent dilutions of decoloration.The decoloration reagent is methanol: ice
Acetic acid: water, volume ratio 1:1:10.
3) measurement is counted under an optical microscope
Using bateria chamber, 0.02 μm of counting chamber thickness.
Difference optical microscopy, 20 times of eyepiece, 40 times or 63 times of object lens.
Sample concentration is calculated by the following formula
According to extension rate and sample to be tested concentration calculation formula:
The sum of bulk samples concentration (OB/ml)=80 lattice virion number × 5 × 50000 × extension rate
1.4 interpretation of result
Carpocapsa pomonella granulosis virus sample clearly may be used after reagent dilutions under the microscope after dyeing, and using decolourizing
See.
Finally illustrate, feature that the above examples are only used to illustrate the technical scheme of the present invention, those skilled in the art
The modification or equivalent replacement that technical characterstic based on this programme carries out, do not depart from the objective and range of this programme, should all cover
In claims of the present invention.
Claims (10)
1. a kind of method of counting of insect PuGV, the described method comprises the following steps:
1) insect PuGV sample is diluted 2~1000 times using staining reagent and makes its dyeing；
2) sample after step 1) dyeing is diluted 2~1000 times using decoloration reagent again and is still bleached；
3) using bateria chamber to the sample count after step 2) decoloration, and according to of formula calculating insect PuGV
Grain concentration.
2. according to the method described in claim 1, wherein, the insect PuGV is plutella xylostella granulosis virus or apple
Moth-eaten moth PuGV.
3. according to the method described in claim 1, wherein, in step 1), the staining reagent be selected from trypan blue, methylenum careuleum she
Red or Coomassie brilliant blue G250.
4. according to the method described in claim 3, wherein, in step 1), the staining reagent is Coomassie brilliant blue G250；
Preferably, the concentration of the Coomassie brilliant blue G250 is 0.1~10g/L；
It is highly preferred that the concentration of the Coomassie brilliant blue G250 is 0.5~5g/L；
It is further preferred that the concentration of the Coomassie brilliant blue G250 is 1g/L.
5. being used according to the method described in claim 1, wherein, in step 1), the dyeing is included at 95~100 DEG C
Staining reagent handles insect PuGV sample at least 5min；Preferably, the staining reagent processing time be at least 5min,
At least 6min, at least 7min, at least 8min, at least 9min, at least 10min；It is highly preferred that the staining reagent processing time is
At least 10min；
Preferably, in step 1), it is further preferably 2-50 times that the extension rate, which is 2-100 times, most preferably 10
Times.
6. according to the method described in claim 1, wherein, in step 2), the decoloration reagent be selected from distilled water, glacial acetic acid or
The mixed solution of person's methanol, glacial acetic acid and water；
Preferably, the decoloration reagent is the mixed solution of methanol, glacial acetic acid and water；
It is highly preferred that the volume ratio of methanol, glacial acetic acid and water is (1~5): (1~5): (1~10) in the mixed solution；
It is further preferred that the volume ratio of methanol, glacial acetic acid and water is 1:1:(1~10 in the mixed solution)；
It is further preferred that the volume ratio of methanol, glacial acetic acid and water is 1:1:8 in the mixed solution.
7. according to the method described in claim 1, wherein, in step 2), the decoloration reagent dilutions time is 1-10 minutes.
8. in step 2), the decoloration reagent dilutions multiple is 2-100 times according to the method described in claim 1, wherein,
It is further preferably 2-50 times, most preferably 10 times.
9. according to the method described in claim 1, wherein, in step 3), described count uses optical microscopy, it is therefore preferable to
Differ optical microscopy.
10. according to the method described in claim 9, wherein, in step 3), the microscopical amplification factor be eyepiece 4~
20 times, 40-100 times of object lens；Preferably 20 times of eyepiece, 63 times of object lens.
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