CN102509504B - Method for preparing chromosome specimen with sepia esculenta embryonic cell - Google Patents
Method for preparing chromosome specimen with sepia esculenta embryonic cell Download PDFInfo
- Publication number
- CN102509504B CN102509504B CN2011102841169A CN201110284116A CN102509504B CN 102509504 B CN102509504 B CN 102509504B CN 2011102841169 A CN2011102841169 A CN 2011102841169A CN 201110284116 A CN201110284116 A CN 201110284116A CN 102509504 B CN102509504 B CN 102509504B
- Authority
- CN
- China
- Prior art keywords
- chromosome
- dripping
- sheet
- embryonic cell
- hypotonic
- 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.)
- Expired - Fee Related
Links
Abstract
A method for preparing a chromosome specimen with a sepia esculenta embryonic cell, which includes the operating procedures of stripping, pre-treating, carrying out hypotonic treatment, fixing, dissociating, dripping, dyeing and washing, and is characterized in that the dripping adopts thermal dripping at the temperature ranging from 40 DEG C to 60 DEG C, the height in the dripping ranges from 1.8mto 2m, and absorbent paper is used for absorbing redundant liquid at a middle position of each liquid drop after each liquid drop is dripped. By means of the thermal dripping, the temperature of the dripping and the height in dripping are reasonably controlled, the chromosome production time is shortened, the chromosome production effect is improved, and the problems of difficulty in mastering of cell rupture degree, chromosome loss, poor chromosome morphology and the like when the chromosome specimen is prepared are solved. The method is used for the chromosome production with the sepia esculenta embryonic cell, so that the good chromosome specimen is obtained, and the chromosome is multiple in phases in metakinesis, clear in morphology and fine in dispersion. Specific effects are indicated in a figure 1.
Description
Technical field:
The invention belongs to the cell biology field, be specifically related to a kind of method of utilizing the golden cuttlefish embryonic cell to prepare chromosome specimen.
Background technology:
The preparation manipulation of chromosome specimen is simple, cheap, the low characteristics of experiment site requirements, it is traditional biogenetics investigative technique always, chromosome specimen preparation comprises: pre-treatment, hypotonic processing, fix, dissociate, drip sheet, step such as dye and develop a film, obtained widespread use in many fields of biogenetics.But owing to the ripe individuality of golden cuttlefish obtain difficulty, cost higher, make reason such as chromosome specimen weak effect, utilize ripe individual preparation chromosome to seem unsatisfactory.The embryo is as one of material for preparing chromosome specimen, obtain relatively easy, cost is low, be widely used in the preparation of chromosome specimen, prepare the normal cold sheet method that adopts routine in the chromosome specimen process the golden cuttlefish embryo, the dry required time of slide is long, easily repeat a sheet, the cell difficulty is broken, and influences chromosome observation.
Summary of the invention:
The technical problem to be solved in the present invention provides and a kind ofly can be good at utilizing the golden cuttlefish embryo to prepare the method for chromosome specimen, this method is dripped the sheet method by heat, height when control is rationally dripped the temperature of sheet and dripped sheet, shortened the film-making time, improved the chromosome sectioning effect, the cell rupture degree is grasped aspect problems such as difficulty, chromosome diminution, chromosome morphology difference when having solved chromosome specimen and preparing.
The present invention realizes by following technical scheme:
A kind of method of utilizing the golden cuttlefish embryonic cell to prepare chromosome specimen, its running program are stripping, pre-treatment, hypotonic processing, fix, dissociate, drip sheet, dye and develop a film; Described sheet: adopt heat to drip the sheet method, 40 ℃-60 ℃ of temperature are dripped sheet height 1.8m-2m, absorb excess liquid in the drop centre position with thieving paper after whenever dripping off one.
As a kind of optimized technical scheme of the present invention, the method that above-mentioned golden cuttlefish embryonic cell prepares chromosome specimen may further comprise the steps:
Stripping: use fixedly ovum of tweezers, needle egg membrane with dissecting needle; Both hands are respectively held tweezers from the rupture of membranes opening part egg membrane that turns up then, peel off egg membrane gradually, expose the embryo;
Pre-treatment: the embryo behind the stripping adopts colchicine to carry out pre-treatment, handles 30min in the colchicine solution with embryonic cell immersion 0.04%;
Hypotonic processing: adopt concentration be 5% KCl as hypotonic medium, handle 45min, remove clean colchicine before the hypotonic processing;
Fixing: adopt Ka Nuoshi liquid (methyl alcohol: glacial acetic acid=3: 1, volume ratio) fixing, 15min changes immobile liquid once at interval, changes immobile liquid altogether 3 times, need remove half hypotonic medium before adding immobile liquid for the first time;
Dissociate: adopting concentration is the 50% glacial acetic acid 45min that dissociates, and removes clean immobile liquid before dissociating;
Drip sheet: adopt heat to drip the sheet method, 40 ℃-60 ℃ of temperature are dripped sheet height 1.8m-2m, absorb excess liquid in the drop centre position with thieving paper after whenever dripping off one;
Dyeing: adopt the dyeing of 10%Giemsa dye liquor, i.e. Giemsa stoste 1ml, the phosphate buffer 9ml of PH 6.8, dyeing time 30min;
Develop a film: wash slide with tap water, facing up of drop arranged during flushing, slide is downward-sloping, and the slide lower end becomes 30 ° with surface level, and the slide frosting contacts with current, and current just become wire to drip, slowly flushing, and the water to flushing no longer has till the color.
The beneficial effect that the present invention is compared with the prior art is:
The present invention adopts heat to drip the sheet method, drips 40 ℃-60 ℃ of sheet temperature, and in this temperature range, cell can finely break, and guarantees that simultaneously drop can be too high and dry not too fast because of temperature, is conducive to drop and extends; Drop is through the height of drop of 1.8m-2m, and cell rupture when contacting with slide can fully discharge chromosome; After drop drips on the slide, absorb excess liquid with thieving paper in the drop centre position immediately, division is concentrated mutually be distributed in drop edge to be conducive to observe, and droplet drying is very fast after absorbing unnecessary liquid, clear-cut is conducive to hold the direction of droplets fall, avoids repeating dripping a sheet.
Description of drawings:
Fig. 1, golden cuttlefish chromosome metacinesis phase
Embodiment:
Be described in detail technical method of the present invention by reference to the accompanying drawings below by concrete instance:
The golden cuttlefish embryonic cell is made the chromosome specimen step and is comprised: stripping, pre-treatment, hypotonic processing, fix, dissociate, drip sheet and dyeing.The present invention has carried out the determining and the improvement of the step of developing a film of the improvement of determining, drip the sheet method, dyeing time of the time of determining, dissociate of the determining of the selection of the determining of the determining of stripping method, pre-treatment time, hypotonic medium and hypotonic time, fixed routine in the chromosome specimen preparation process.
Determining of stripping method: embryonic cell prepares chromosome specimen and generally is used for fish, because the ovum of fish is generally all less, can't carries out stripping and handle to obtain the embryo, so all adopt the mode of direct pre-treatment to carry out.But the golden cuttlefish egg membrane is thick, and the colchicine difficulty is soaked into, and ovum is conducive to stripping acquisition embryo greatly.Use fixedly ovum of tweezers during stripping, needle egg membrane with dissecting needle, both hands are respectively held tweezers from the rupture of membranes opening part egg membrane that turns up then, peel off egg membrane gradually, expose the embryo, have shortened the pre-treatment time that time goes on foot greatly.
The pre-treatment time: sample adopts colchicine to carry out pre-treatment, and the pre-treatment time, the too short or long chromosome morphology that all can make was unfavorable for observing, and handled 30min in the colchicine solution of present embodiment with embryonic cell immersion 0.04%.
Determining of the selection of hypotonic medium and hypotonic time: conventional method adopts filtering sea as hypotonic medium, because concentration of seawater determines to cause the hypotonic time uncertain than difficulty, we adopt KCl as hypotonic medium, and concentration is 5%, handles 45min.Remove clean colchicine before the hypotonic processing.
Determining of fixed routine: after finding that in experimentation immobile liquid is fixed 3 times, can be at-4 ℃ of long preservation samples.We adopt Ka Nuoshi liquid (methyl alcohol: glacial acetic acid=3: 1, volume ratio) fixing, and 15min changes immobile liquid once at interval, changes immobile liquid altogether 3 times.Owing to have glacial acetic acid certain dissociation to be arranged to sample in the immobile liquid, can influence fixed effect, therefore before adding immobile liquid for the first time, only remove half hypotonic medium as buffering, second and third time adds new immobile liquid after then old liquid need being removed fully again.
Dissociate the time: adopt glacial acetic acid to dissociate, concentration is 50%, and the time of dissociating, too short or long all can the influence dripped a sheet effect, and our time set that will dissociate is 45min, and it is respond well to drip sheet, does not have and organizes piece to disturb greatly.Remove clean immobile liquid before dissociating, otherwise influence dissociation solution concentration.
Drip the sheet method: cold conventional sheet method repeats to drip sheet easily, influences chromosome observation, and needs dried overnight, and required time is longer.We adopt heat to drip a sheet method to drip sheet, and 40 ℃-60 ℃ of temperature are dripped sheet height 1.8m-2m, absorb excess liquid in the drop centre position with thieving paper after whenever dripping off one.
Dyeing time: adopt the dyeing of 10%Giemsa dye liquor, be Giemsa stoste (production of GIBCO company) 1ml, the phosphate buffer of PH 6.8 (PBS) 9ml, dyeing time has finally influenced the quality of chromosome specimen, the too short chromosome morphology of dyeing time is unintelligible, the long background colour of dyeing time is deepened, and the dyeing time that we adopt is 30min, cleans slide with tap water then.
The step of developing a film: wash slide with tap water, facing up of drop arranged during flushing, slide is downward-sloping, the slide lower end becomes 30 ° with surface level, the slide frosting contacts with current, and current just become wire to drip, slowly flushing, the water to flushing no longer has till the color.
Adopt said method, we have carried out practical application in Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, the golden cuttlefish embryo chromosome is carried out film-making, the result shows, adopt method of the present invention can obtain good chromosome specimen, chromosome metacinesis mutually many, form is clear, good dispersion, concrete effect is seen Fig. 1.
Claims (2)
1. method of utilizing the golden cuttlefish embryonic cell to prepare chromosome specimen, its running program is stripping, pre-treatment, hypotonic processing, fixes, dissociates, drips sheet, dyes and develop a film, it is characterized in that described sheet adopts heat to drip the sheet method, 40 ℃-60 ℃ of temperature, drip sheet height 1.8m-2m, absorb excess liquid in the drop centre position with thieving paper after whenever dripping off one.
2. method according to claim 1 is characterized in that described stripping: use fixedly ovum of tweezers, needle egg membrane with dissecting needle; Both hands are respectively held tweezers from the rupture of membranes opening part egg membrane that turns up then, peel off egg membrane gradually, expose the embryo;
Described pre-treatment: the embryo behind the stripping adopts colchicine to carry out pre-treatment, handles 30min in the colchicine solution with embryonic cell immersion 0.04%;
Described hypotonic processing: adopt KCl as hypotonic medium, concentration is 5%, handles 45min, removes clean colchicine before the hypotonic processing;
Described fixing: as to adopt Ka Nuoshi liquid, i.e. methyl alcohol: glacial acetic acid=3:1(volume ratio) fixing, 15min changes immobile liquid once at interval, changes immobile liquid altogether 3 times, need remove half hypotonic medium before adding immobile liquid for the first time;
Described dissociating: adopting concentration is the 50% glacial acetic acid 45min that dissociates, and removes clean immobile liquid before dissociating;
Described dyeing: adopt the dyeing of 10%Giemsa dye liquor, i.e. Giemsa stoste 1ml, the phosphate buffer 9ml of PH6.8, dyeing time 30min;
Described developing a film: wash slide with tap water, facing up of drop arranged during flushing, slide is downward-sloping, the slide lower end becomes 30 ° with surface level, the slide frosting contacts with current, and current just become wire to drip, slowly flushing, the water to flushing no longer has till the color.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102841169A CN102509504B (en) | 2011-09-22 | 2011-09-22 | Method for preparing chromosome specimen with sepia esculenta embryonic cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102841169A CN102509504B (en) | 2011-09-22 | 2011-09-22 | Method for preparing chromosome specimen with sepia esculenta embryonic cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102509504A CN102509504A (en) | 2012-06-20 |
| CN102509504B true CN102509504B (en) | 2013-07-10 |
Family
ID=46221580
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011102841169A Expired - Fee Related CN102509504B (en) | 2011-09-22 | 2011-09-22 | Method for preparing chromosome specimen with sepia esculenta embryonic cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102509504B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703599B (en) * | 2012-06-26 | 2014-01-22 | 山东东方海洋科技股份有限公司 | Improved preparation method of kelp chromosome |
| CN104483178B (en) * | 2014-11-24 | 2018-03-30 | 中国水产科学研究院黄海水产研究所 | The preparation method of epinephelus akaara adult fish chromosome |
| CN106680056A (en) * | 2016-12-05 | 2017-05-17 | 浙江海洋大学 | Preparation method for gill chromosome of sepiella maindroni |
| CN112772554A (en) * | 2020-12-30 | 2021-05-11 | 浙江大学 | Method for improving rate of emergence of giant salamanders in breeding |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4031635A (en) * | 1975-04-14 | 1977-06-28 | Brandt Edward E | Manipulative chromosomal model |
| CN201780226U (en) * | 2010-08-30 | 2011-03-30 | 深圳华大基因科技有限公司 | Chromosome specimen dropping device |
-
2011
- 2011-09-22 CN CN2011102841169A patent/CN102509504B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4031635A (en) * | 1975-04-14 | 1977-06-28 | Brandt Edward E | Manipulative chromosomal model |
| CN201780226U (en) * | 2010-08-30 | 2011-03-30 | 深圳华大基因科技有限公司 | Chromosome specimen dropping device |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102509504A (en) | 2012-06-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102509504B (en) | Method for preparing chromosome specimen with sepia esculenta embryonic cell | |
| Severs | Freeze-fracture electron microscopy | |
| CN100552414C (en) | Paraffin section method for seawater fish egg | |
| CN102147337B (en) | Paraffin sectioning method for ocean shellfish D-shaped larva | |
| CN107490511A (en) | A kind of haematoxylin dyeing liquid and HE colouring methods | |
| CN103900883B (en) | Cashmere goat skin is for the preparation method of the ultrathin section of transmission electron microscope observing | |
| CN103940648A (en) | Preparation method for gill tissue paraffin section | |
| La Cour | Improvements in plant cytological technique | |
| CN106047812B (en) | Tumor tissues freeze, recovery kit and its processing method | |
| CN103881968A (en) | Isolated culture method of primary granular cells of goose ovarian follicle | |
| CN112014192A (en) | HE staining method for paraffin section of northern Guizhou goat ovary tissue | |
| CN107702955B (en) | Preparation method of paraffin section | |
| CN104634626A (en) | Improved frozen slicing method and applications thereof | |
| CN110132673B (en) | Method for preparing needle mushroom fruiting body tissue slices | |
| CN108287097A (en) | A kind of thionine eosin stains liquid and preparation method and application | |
| Friedman | A standardized procedure for serum protein electrophoresis on cellulose acetate membrane strips | |
| Sathawane et al. | Nuances of the Papanicolaou stain | |
| Exbrayat | Classical methods of visualization | |
| AU2020103868A4 (en) | Preparation method of apoplast antifreeze protein standard sample of winter rapeseed leaves and protein lysis buffer | |
| Chemes | Ultrastructural analysis of testicular tissue and sperm by transmission and scanning electron microscopy | |
| JP2008239487A (en) | Liquid for rapidly fixing hardly permeable tissue | |
| Carrara et al. | Histological examination of the diabetic kidney | |
| CN105548569A (en) | Detection method for peripheral blood VEGF of renal cancer patient | |
| RU2593343C1 (en) | Method for staining the histological cuts in diagnosing trichinosis | |
| CN104390834A (en) | Sarranine and methyl violet mixed staining method for resin slices and staining solution thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130710 Termination date: 20150922 |
|
| EXPY | Termination of patent right or utility model |