CN1828296A - Maladera sp. spermary chromosome production and observation method - Google Patents
Maladera sp. spermary chromosome production and observation method Download PDFInfo
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- CN1828296A CN1828296A CNA200510048723XA CN200510048723A CN1828296A CN 1828296 A CN1828296 A CN 1828296A CN A200510048723X A CNA200510048723X A CN A200510048723XA CN 200510048723 A CN200510048723 A CN 200510048723A CN 1828296 A CN1828296 A CN 1828296A
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Abstract
Wherein, with adult sperm cell of Maladera sp. As material, pre-processing for 6-14h with colchicine; using 0.4%KCl to hypotonic reaction for 30min; curing with methaol and glacial acetic acid (3:1) for 30-60min; finally, dyeing with Giemsa for 10-20min. this invention obtains clear chromosome with number as n = 9.
Description
Technical field:
The invention belongs to biological technical field, provide a kind of Maladera sp spermary chromosome to make observational technique.
Background technology:
Maladera sp Maladera sp. is subordinate to coleoptera, suede Scarabaeidae, thin,tough silk Serica, be to get the main chafer class pest of eating the crops such as tobacco of causing harm, because this worm and other thin,tough silk Serica chafer morphological feature fairly similar, simple at present dependence identification of morphology method still can not accurately be identified, therefore the classification to Maladera sp still only rests on the genus, still species indeterminata.Yet, the identification of morphology method is for very similar the belonging to chafer together and then can't accurately identify of form, for not possessing molecular biology experiment condition person, the molecular biology identification method also is subjected to certain restriction, and, can't clearly observe chromosome morphology and the numeration of this worm after the at present existing chromosomal method for making of utilization for utilizing the still manque chromosome of karyotyping rule to make observational technique.
Summary of the invention:
The present invention has overcome the deficiencies in the prior art, provide a kind of accurately, the chromosomal making observational technique of Maladera sp easily.
Step of the present invention is:
1, draw materials and pre-service: the Maladera sp adult is fixed on dissects on the cake wax, take out spermary, put into 0.04～0.06% colchicine solution and handle 6～14h, the stoste of colchicine 0.1～0.3% is stored in the refrigerator.
2, solution preparation: Giemsa reagent 1g, glycerine 50ml, methyl alcohol 50ml puts into mortar with Giemsa reagent 1g earlier, adds 5ml glycerine again and grinds and it is dissolved be the homogenate shape, add glycerine again, shake up the back in the bottle of packing into after the mixing and add methyl alcohol;
3, the preparation of slide sample: spermary is taken out from the colchicine immersion liquid, use distilled water flushing, under anatomical lens, separate every spermary tubule again, reject spermary tubule impurity such as protein on every side, the hypotonic processing 30min in 0.4%KCl solution and two kinds of solution of distilled water of elder generation, move into immobile liquid (methyl alcohol: glacial acetic acid=3: 1) interior behind 30～60min, move in 70% alcohol, place refrigerator, from immobile liquid, take out the spermary tubule, on slide glass, put a spermary tubule, blot with thieving paper, with distillation washing 3 times, blot then, drip one of last 0.05% colchicum liquid, covered is knocked gently and is made the break meiotic cell of the inside of spermary tubule flow out with dyeing liquor and contact, and microslide is placed directly on the open and flat desktop, get two-layer clean thieving paper lid in the above, on the cover glass position, vertically exert pressure;
4, dyeing: with 10 times of Giemsa (PH6.7～7.0) stoste dilutions, 10～20min dyes with phosphate buffer (PH6.8);
5, microscopy: the slide sample that makes is examined under a microscope, counted, and the Maladera sp spermary chromosome is rod-short;
Wherein: Maladera sp adult spermary is put into 0.04～0.06% colchicine solution handled 10～14 hours, the set time is 46～60min in immobile liquid, and dyeing time is 15～20min.
Is material according to this inventive method with Maladera sp adult spermary, through the colchicine pre-service, through the hypotonic processing of KCl solution, fixing, after Giemsa dyeing, to make and observe Maladera sp chromosome, metaphase in cell division is many mutually, and it is clear, so chromosome clear as seen, and the chromosomal quantity that can accurately, intuitively count can be used for Maladera sp chromosome karyotype analysis and kind and identifies.
Embodiment
Embodiment one
Code fetch thin,tough silk cockchafer adult spermary is put into 0.05% colchicine solution and handled 6 hours.
Colchicine solution concentration is 0.1%; The preparation of Giemsa solution is by Giemsa reagent 1g, glycerine 50ml, and methyl alcohol 50ml puts into mortar with Giemsa reagent 1g earlier, adds 5ml glycerine again and grinds and it is dissolved be the homogenate shape, add glycerine again, shake up the back in the bottle of packing into after the mixing and add methyl alcohol.
The preparation of slide sample: spermary is taken out from the colchicine immersion liquid, the flushing back is divided into the spermary tubule under anatomical lens, the hypotonic processing 30min in 0.4%KCl solution and two kinds of solution of distilled water of elder generation, move into immobile liquid (methyl alcohol: glacial acetic acid=3: 1) interior behind 30min, take out the spermary tubule, on microslide, put a spermary tubule, drip one of last 0.05% colchicum liquid, cover clean cover glass, knock and make the break meiotic cell of the inside of seminiferous tubule flow out with dyeing liquor and contact.
Dyeing: with 10 times of Giemsa (PH6.7～7.0) stoste dilutions, dyeing 10min vertically exerts pressure on the cover glass position with phosphate buffer (PH6.8).
Microscopy: slide sample is examined under a microscope, is counted.
The result: Maladera sp spermary cell chromosome cell metaphase, n=9 person accounts for 85%, and chromosome all is rod-short.
Embodiment two
Code fetch thin,tough silk cockchafer adult spermary is put into 0.05% colchicine solution and handled 14 hours.
Colchicine solution concentration is 0.3%, and the preparation of Giemsa solution is by Giemsa reagent 1g, glycerine 50ml, methyl alcohol 50ml earlier puts into mortar with Giemsa reagent 1g, adds 5ml glycerine again and grinds and it is dissolved be the homogenate shape, add glycerine again, shake up the back in the bottle of packing into after the mixing and add methyl alcohol.
The preparation of slide sample: spermary is taken out from the colchicine immersion liquid, the flushing back is divided into the spermary tubule under anatomical lens, the hypotonic processing 30min in 0.4%KCl solution and two kinds of solution of distilled water of elder generation, move into immobile liquid (methyl alcohol: glacial acetic acid=3: 1) interior behind 60min, take out the spermary tubule, on microslide, put a spermary tubule, drip one of last 0.05% colchicum liquid, cover clean cover glass, knock and make the break meiotic cell of the inside of seminiferous tubule flow out with dyeing liquor and contact.
Dyeing: with 10 times of Giemsa (PH6.7～7.0) stoste dilutions, dyeing 20min vertically exerts pressure on the cover glass position with phosphate buffer (PH6.8).
Microscopy: slide sample is examined under a microscope, is counted.
The result: Maladera sp spermary cell chromosome cell metaphase, n=9 person accounts for 85%, and chromosome all is rod-short.
Embodiment three
Code fetch thin,tough silk cockchafer adult spermary is put into 0.05% colchicine solution and handled 10 hours.
Colchicine solution concentration is 0.2%, and the preparation of Giemsa solution is by Giemsa reagent 1g, glycerine 50ml, methyl alcohol 50ml earlier puts into mortar with Giemsa reagent 1g, adds 5ml glycerine again and grinds and it is dissolved be the homogenate shape, add glycerine again, shake up the back in the bottle of packing into after the mixing and add methyl alcohol.
The preparation of slide sample: spermary is taken out from the colchicine immersion liquid, the flushing back is divided into the spermary tubule under anatomical lens, the hypotonic processing 30min in 0.4%KCl solution and two kinds of solution of distilled water of elder generation, move into immobile liquid (methyl alcohol: glacial acetic acid=3: 1) interior behind 45min, take out the spermary tubule, on microslide, put a spermary tubule, drip one of last 0.05% colchicum liquid, cover clean cover glass, knock and make the break meiotic cell of the inside of seminiferous tubule flow out with dyeing liquor and contact.
Dyeing: with 10 times of Giemsa (PH6.7～7.0) stoste dilutions, dyeing 15min vertically exerts pressure on the cover glass position with phosphate buffer (PH6.8).
Microscopy: slide sample is examined under a microscope, is counted.
The result; Maladera sp spermary cell chromosome cell metaphase, n=9 person accounts for 85%, and chromosome all is rod-short.
Claims (4)
1. a Maladera sp spermary chromosome is made observational technique, the steps include:
(1) draw materials and pre-service: the Maladera sp adult is fixed on dissects on the cake wax, take out spermary, put into 0.04～0.06% colchicine solution and handle 6～14h, the stoste of colchicine 0.1～0.3% is stored in the refrigerator;
(2) solution preparation: Giemsa reagent 1g, glycerine 50ml, methyl alcohol 50ml puts into mortar with Giemsa reagent 1g earlier, adds 5ml glycerine again and grinds and it is dissolved be the homogenate shape, add glycerine again, shake up the back in the bottle of packing into after the mixing and add methyl alcohol;
(3) preparation of slide sample: spermary is taken out from the colchicine immersion liquid, use distilled water flushing, under anatomical lens, separate every spermary tubule again, reject spermary tubule impurity such as protein on every side, the hypotonic processing 30min in 0.4%KCl solution and two kinds of solution of distilled water of elder generation, move into immobile liquid (methyl alcohol: glacial acetic acid=3: 1) interior behind 30～60min, move in 70% alcohol, place refrigerator, from immobile liquid, take out the spermary tubule, on slide glass, put a spermary tubule, blot with thieving paper, with distillation washing 3 times, blot then, drip one of last 0.05% colchicum liquid, covered is knocked gently and is made the break meiotic cell of the inside of spermary tubule flow out with dyeing liquor and contact, and microslide is placed directly on the open and flat desktop, get two-layer clean thieving paper lid in the above, on the cover glass position, vertically exert pressure;
(4) dyeing: with 10 times of Giemsa (PH 6.7～7.0) stoste dilutions, 10～20min dyes with phosphate buffer (PH 6.8);
(5) microscopy: the slide sample that makes is examined under a microscope, counted, and the Maladera sp spermary chromosome is rod-short.
2, Maladera sp spermary chromosome according to claim 1 is made observational technique, and it is characterized in that: the Maladera sp adult spermary in the step (1) is put into 0.04～0.06% colchicine solution and handled 10～14 hours.
3, Maladera sp spermary chromosome according to claim 1 is made observational technique, and it is characterized in that: the regular time in immobile liquid in the step (3) is 46～60min.
4, Maladera sp spermary chromosome according to claim 1 is made observational technique, and it is characterized in that: the dyeing time in the step (4) is 15～20min.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA200510048723XA CN1828296A (en) | 2005-12-22 | 2005-12-22 | Maladera sp. spermary chromosome production and observation method |
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| CNA200510048723XA CN1828296A (en) | 2005-12-22 | 2005-12-22 | Maladera sp. spermary chromosome production and observation method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102914466A (en) * | 2011-08-01 | 2013-02-06 | 马铁军 | Method for pretreating to-be-detected cells and reagents used by same |
| CN103983497A (en) * | 2014-06-12 | 2014-08-13 | 周口师范学院 | Preparation method of turbellarian worm chromosome specimen |
| CN105616029A (en) * | 2015-12-30 | 2016-06-01 | 河南农业大学 | Cotton plant bug adult brain dissection method |
| CN109540633A (en) * | 2019-01-23 | 2019-03-29 | 北京仁基源医学研究院有限公司 | Karyotyping is dedicated to prepare high resolution chromosome dye liquor |
| CN113551960A (en) * | 2021-07-21 | 2021-10-26 | 山东省花生研究所 | Preparation method of scarab chromosome karyotype |
-
2005
- 2005-12-22 CN CNA200510048723XA patent/CN1828296A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102914466A (en) * | 2011-08-01 | 2013-02-06 | 马铁军 | Method for pretreating to-be-detected cells and reagents used by same |
| CN102914466B (en) * | 2011-08-01 | 2015-08-12 | 马铁军 | A kind of method to cell pretreatment to be checked and agents useful for same thereof |
| CN103983497A (en) * | 2014-06-12 | 2014-08-13 | 周口师范学院 | Preparation method of turbellarian worm chromosome specimen |
| CN105616029A (en) * | 2015-12-30 | 2016-06-01 | 河南农业大学 | Cotton plant bug adult brain dissection method |
| CN109540633A (en) * | 2019-01-23 | 2019-03-29 | 北京仁基源医学研究院有限公司 | Karyotyping is dedicated to prepare high resolution chromosome dye liquor |
| CN109540633B (en) * | 2019-01-23 | 2021-10-29 | 北京仁基源医学研究院有限公司 | Special preparation of high-resolution chromosome dye solution for karyotype analysis |
| CN113551960A (en) * | 2021-07-21 | 2021-10-26 | 山东省花生研究所 | Preparation method of scarab chromosome karyotype |
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