CN109141997A - A kind of flaking method of microexamination Aphytis - Google Patents
A kind of flaking method of microexamination Aphytis Download PDFInfo
- Publication number
- CN109141997A CN109141997A CN201810874528.XA CN201810874528A CN109141997A CN 109141997 A CN109141997 A CN 109141997A CN 201810874528 A CN201810874528 A CN 201810874528A CN 109141997 A CN109141997 A CN 109141997A
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- China
- Prior art keywords
- sample
- aphytis
- alcohol
- glass slide
- added
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 16
- 241001454985 Aphytis Species 0.000 title claims description 39
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000011521 glass Substances 0.000 claims abstract description 13
- 244000283070 Abies balsamea Species 0.000 claims description 14
- 235000007173 Abies balsamea Nutrition 0.000 claims description 14
- 239000004858 Canada balsam Substances 0.000 claims description 14
- 238000001035 drying Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 7
- 239000003292 glue Substances 0.000 claims description 4
- 239000006166 lysate Substances 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 3
- 238000006424 Flood reaction Methods 0.000 claims description 3
- 108091005804 Peptidases Proteins 0.000 claims description 2
- 102000035195 Peptidases Human genes 0.000 claims description 2
- 235000019833 protease Nutrition 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 241001124076 Aphididae Species 0.000 abstract description 4
- 239000010634 clove oil Substances 0.000 abstract 1
- 230000002934 lysing effect Effects 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 230000007935 neutral effect Effects 0.000 description 4
- 230000002688 persistence Effects 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- OINQDWUHUPWLHC-UHFFFAOYSA-N 2-hydroxypropanoic acid;phenol Chemical compound CC(O)C(O)=O.OC1=CC=CC=C1 OINQDWUHUPWLHC-UHFFFAOYSA-N 0.000 description 2
- -1 50% Chemical compound 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 241000317929 Chalcididae Species 0.000 description 2
- 241000257303 Hymenoptera Species 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000002224 dissection Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000003071 parasitic effect Effects 0.000 description 2
- 244000215068 Acacia senegal Species 0.000 description 1
- 241001547764 Aphelinidae Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 229920000084 Gum arabic Polymers 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 239000000205 acacia gum Substances 0.000 description 1
- 235000010489 acacia gum Nutrition 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229920001206 natural gum Polymers 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
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Sampling And Sample Adjustment (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明提供一种显微观察黄蚜小蜂的制片方法，通过组织裂解液、酒精、丁香油等处理，解剖后，通过加拿大树胶固定烘干等步骤后，完成制片。本发明方法既能够保证黄蚜小蜂标本能够被清晰观察到并胸腹节后缘扇状突的微观结构，并进行拍照鉴定，而且能够保证制作的玻片标本永久保存。The invention provides a production method for microscopic observation of the yellow aphid wasp, which is processed by tissue lysing solution, alcohol, clove oil, etc., dissected, fixed and dried by Canadian gum, etc., to complete the production. The method of the invention can not only ensure that the sample of the yellow aphid wasp can be clearly observed and the microstructure of the fan-shaped process of the rear edge of the thoracoabdominal segment, and be photographed for identification, but also can ensure the permanent preservation of the produced glass slide sample.
Description
Technical field
The invention belongs to aphid chalcid fly (parasitic wasp) microexamination technical fields, and in particular to microexamination Aphytis is (outstanding
It is that median segment rear fan-shaped is prominent) flaking method.
Background technique
Aphytis (Aphytis) it is subordinate to Hymenoptera (Hymenoptera) aphid Chalcididae (Aphelinidae), it is shell
The important parasitic wasp of worm, plays a significant role in biological control of insect pests.But Aphytis individual is small, needs to make slide
Sample carries out microexamination and taxonomic identification.The prominent shape of Aphytis median segment rear fan-shaped, arrangement are category classification mirror
Fixed most important feature, this feature need the microexamination of high quality that could obtain.And at present in relation to Aphytis slide mark
This main two kinds of production method:
1. conventional neutral gum mounting method: first sample being placed in 5% potassium hydroxide (or 5% sodium hydroxide) solution and is impregnated, so
After wash 20-30min, by the various concentrations dehydration of alcohol such as 50%, 70%, 95%, 100%, move into caryophyllus oil in case film-making.Film-making
When the sample in caryophyllus oil is chosen on coverslip (be inverted, i.e. the outside of belly upward), and drip a little caryophyllus oil, inhaled after dissection, whole appearance
Dry caryophyllus oil adds neutral gum mounting.The deficiency of conventional neutral gum mounting method: the sample of neutral gum mounting can be permanent
It saves, but unfortunately sample is easily deformed or shrinkage during dehydration, clarity and transparency are not ideal enough, almost very
Difficulty sees the prominent microstructure of Aphytis median segment rear fan-shaped clearly.
2. He Shi liquid method of tableting: sample first passes through the immersion liquid of acetic acid lactic acid phenol, and (lactic acid: it is molten that lactic acid phenol is made in crystalline phenol=10:5
Liquid, then lactic acid phenol solution: glacial acetic acid=5:7 is mixed) processing 6-24h, moved on to when polypide is transparent dissection on coverslip,
Whole appearance, then with He Shi liquid, (12g gum arabic, 20ml water-solubleization, add 20-40g chloraldurate and 10-20g glycerol is mixed
Close) mounting.The deficiency of He Shi liquid method of tableting: although slide sample clarity is preferable, the slide sample being fabricated to due to
He Shi liquid category water-soluble glue, easily moisture absorption are got damp, and the sample of institute's mounting cannot make persistence.
Summary of the invention
The purpose of the present invention is to provide a kind of flaking methods of microexamination Aphytis, solve and deposit in the prior art
It is easily deformed or shrinkage in product sample, clarity and transparency are not ideal enough, are difficult to see Aphytis median segment rear clearly
The disadvantages of prominent microstructure of fan-shaped, easily moisture absorption are got damp, and the sample of institute's mounting cannot make persistence.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of flaking method of microexamination Aphytis, described method includes following steps:
(1) 100% alcohol impregnates, cleans fresh Aphytis sample；
(2) Aphytis sample is put into 1.5ml centrifuge tube, 90ul Tissue lysates and 10ul Proteinase K is added, is put into 56
DEG C water-bath in, overnight；
(3) solution after staying overnight is sopped up, retains Aphytis sample, it is rear that 200ul100% alcohol is added；
(4) a spill glass slide is taken, Aphytis sample is drawn onto glass slide recess together with alcohol, it is clean simultaneously to alcohol volatilization
10ul pure water is added, sample back is upward；
(5) when the volatilization of Aphytis sample back skin pure water is clean, sample is placed in microscopic photography under microscope and is fanned
Shape lug structure；
(6) after shooting, extra pure water is sopped up, drop 20ul caryophyllus oil floods Aphytis sample；
(7) a common glass slide is taken, Aphytis sample is moved on on common glass slide together together with caryophyllus oil, is dissected, carefully
Head, feeler and wing are removed, the whole appearance of polypide is left；
(8) extra caryophyllus oil is sopped up with filter paper, adds a thin layer canada balsam, fix head, feeler, wing and polypide；
(9) it is dried 24 hours using 40 DEG C of baking piece machines；
(10) sample of above-mentioned drying is taken, is successively added canada balsam 2-3 times, head, feeler, wing and polypide difference are finally made
It is buried in canada balsam；
(11) before canada balsam does not solidify completely, round coverslip mounting is taken；
(12) it is dried 2 weeks using 40 DEG C of piece machine of baking until drying, to effectively remove the bubble in glue；
(13) sample after drying is placed in microscopically observation, shooting.
The Tissue lysates are 10 mM Tris-HCl, 100mM EDTA, 1wt.% SDS, pH 8.0.
The present invention has the advantages that
The application motion can either guarantee that Aphytis sample can be obviously observed prominent microcosmic of median segment rear fan-shaped
Structure, and identification of taking pictures is carried out, and can guarantee the slide sample persistence of production.
Detailed description of the invention
Fig. 1 Aphytis of the present inventionAphytis holoxanthus (♀) film-making figure, wherein upper figure is prior art system
Piece, the following figure are film-making result of the present invention；It is prominent for fan-shaped in frame.
Fig. 2 Aphytis of the present inventionAphytis holoxanthus (♀) film-making figure, wherein upper figure is prior art system
Piece, the following figure are film-making result of the present invention；It is prominent for fan-shaped in frame.
Specific embodiment
Embodiment 1
1. 100% alcohol impregnates, cleans fresh Aphytis sample；
2. Aphytis sample is put into 1.5ml centrifuge tube, addition 90ul Tissue lysates (10 mM Tris-HCl,
100mM EDTA, 1% SDS, pH 8.0) and 10 ul Proteinase Ks, it is put into 56 DEG C of water-bath, overnight；
3. the solution after overnight is sopped up, retain Aphytis sample, it is rear that 200ul100% alcohol is added；
4. taking a spill glass slide, Aphytis sample is drawn onto glass slide recess together with a small amount of alcohol, simultaneously to alcohol volatilization
10ul pure water is added, sample back is upward；
5. sample is placed in Nikon microscope (Eclipse when the volatilization of Aphytis sample back skin pure water is clean
Ni-U microscopic photography fan-shaped lug structure under)；
6. after shooting, sopping up extra pure water, drop 20ul caryophyllus oil floods Aphytis sample；
7. taking a common glass slide, Aphytis sample is moved on on common glass slide together together with caryophyllus oil, is dissected, carefully
Remove head, feeler and wing, the remaining whole appearance of polypide；
8. sopping up extra caryophyllus oil with filter paper, add a thin layer canada balsam, fixes head, feeler, wing and polypide；
9. being dried 24 hours using drying (40 DEG C) of piece machine；
10. taking the sample of above-mentioned drying, successively add canada balsam 2-3 times, finally makes head, feeler, wing and polypide difference
It is buried in canada balsam.Since wing and feeler thickness are small, a small amount of canada balsam of addition, 2 times；Head and
Polypide thickness is larger, to be successively added canada balsam 3 times, is buried in it completely clear when observing in natural gum to improve
Degree.
11. taking round coverslip (diameter 0.6cm) mounting before canada balsam is not completely dried；
12. being dried 2 weeks using drying (40 DEG C) of piece machine until drying, to effectively remove the bubble in glue；
It observes, shoot 13. the sample after drying is placed under Nikon microscope (Eclipse Ni-U).
Result of taking pictures is as shown in attached drawing 1-2, it can be seen that Aphytis sample of the present invention can be obviously observed and chest
The prominent microstructure of uromere rear fan-shaped, and identification of taking pictures is carried out, and can guarantee the slide sample persistence of production.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (2)
1. a kind of flaking method of microexamination Aphytis, it is characterised in that: described method includes following steps:
(1) 100% alcohol impregnates, cleans fresh Aphytis sample；
(2) Aphytis sample is put into 1.5ml centrifuge tube, 90ul Tissue lysates and 10 ul Proteinase Ks is added, are put into
In 56 DEG C of water-bath, overnight；
(3) solution after staying overnight is sopped up, retains Aphytis sample, it is rear that 100% alcohol of 200ul is added；
(4) a spill glass slide is taken, Aphytis sample is drawn onto glass slide recess together with alcohol, it is clean simultaneously to alcohol volatilization
10ul pure water is added, sample back is upward；
(5) when the volatilization of Aphytis sample back skin pure water is clean, sample is placed in microscopic photography under microscope and is fanned
Shape lug structure；
(6) after shooting, extra pure water is sopped up, drop 20ul caryophyllus oil floods Aphytis sample；
(7) a common glass slide is taken, Aphytis sample is moved on on common glass slide together together with caryophyllus oil, is dissected, carefully
Head, feeler and wing are removed, the whole appearance of polypide is left；
(8) extra caryophyllus oil is sopped up with filter paper, adds a thin layer canada balsam, fix head, feeler, wing and polypide；
(9) it is dried 24 hours using 40 DEG C of baking piece machines；
(10) sample of above-mentioned drying is taken, is successively added canada balsam 2-3 times, head, feeler, wing and polypide difference are finally made
It is buried in canada balsam；
(11) before canada balsam does not solidify completely, round coverslip mounting is taken；
(12) it is dried 2 weeks using 40 DEG C of piece machine of baking until drying, to effectively remove the bubble in glue；
(13) sample after drying is placed in microscopically observation, shooting.
2. a kind of flaking method of microexamination Aphytis according to claim 1, it is characterised in that: the tissue is split
Solution liquid is 10 mM Tris-HCl, 100mM EDTA, 1wt.% SDS, pH 8.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810874528.XA CN109141997A (en) | 2018-08-03 | 2018-08-03 | A kind of flaking method of microexamination Aphytis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810874528.XA CN109141997A (en) | 2018-08-03 | 2018-08-03 | A kind of flaking method of microexamination Aphytis |
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| Publication Number | Publication Date |
|---|---|
| CN109141997A true CN109141997A (en) | 2019-01-04 |
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| CN201810874528.XA Pending CN109141997A (en) | 2018-08-03 | 2018-08-03 | A kind of flaking method of microexamination Aphytis |
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| Country | Link |
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| CN (1) | CN109141997A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110286016A (en) * | 2019-07-11 | 2019-09-27 | 云南大学 | A kind of living insects feeler directed fixing device in irregular shape and preparation method thereof |
| CN110338153A (en) * | 2019-07-15 | 2019-10-18 | 杭州森康林业科技有限公司 | A immobilization technique of pine xylophilus worms for microinjection |
| CN116264891A (en) * | 2021-12-16 | 2023-06-20 | 天津农学院 | A kind of glass slide preparation method of small bee specimen |
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|---|---|---|---|---|
| JP2000010017A (en) * | 1998-06-17 | 2000-01-14 | Nec Corp | Cover glass for microscopic observation and observation method |
| CN103759994A (en) * | 2014-01-02 | 2014-04-30 | 河南科技大学 | Larva paraffin section forming method |
| CN104020030A (en) * | 2014-06-09 | 2014-09-03 | 北京农学院 | Preparation method of insect sample for scanning electron microscope |
| CN105699145A (en) * | 2016-02-25 | 2016-06-22 | 广西医科大学 | Method for preparing transmission electron microscope sample by embedding drosophila melanogaster in agar |
| CN105928752A (en) * | 2016-04-13 | 2016-09-07 | 沈阳大学 | Method for staining paraffin section of adult insect |
-
2018
- 2018-08-03 CN CN201810874528.XA patent/CN109141997A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000010017A (en) * | 1998-06-17 | 2000-01-14 | Nec Corp | Cover glass for microscopic observation and observation method |
| CN103759994A (en) * | 2014-01-02 | 2014-04-30 | 河南科技大学 | Larva paraffin section forming method |
| CN104020030A (en) * | 2014-06-09 | 2014-09-03 | 北京农学院 | Preparation method of insect sample for scanning electron microscope |
| CN105699145A (en) * | 2016-02-25 | 2016-06-22 | 广西医科大学 | Method for preparing transmission electron microscope sample by embedding drosophila melanogaster in agar |
| CN105928752A (en) * | 2016-04-13 | 2016-09-07 | 沈阳大学 | Method for staining paraffin section of adult insect |
Non-Patent Citations (1)
| Title |
|---|
| 张慧: "黄蚜小蜂属部分种类的形态鉴别及分子系统发育研究", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110286016A (en) * | 2019-07-11 | 2019-09-27 | 云南大学 | A kind of living insects feeler directed fixing device in irregular shape and preparation method thereof |
| CN110286016B (en) * | 2019-07-11 | 2023-08-04 | 云南大学 | Irregular antenna directional fixing device for living fruit fly insects and manufacturing method thereof |
| CN110338153A (en) * | 2019-07-15 | 2019-10-18 | 杭州森康林业科技有限公司 | A immobilization technique of pine xylophilus worms for microinjection |
| CN116264891A (en) * | 2021-12-16 | 2023-06-20 | 天津农学院 | A kind of glass slide preparation method of small bee specimen |
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