CN102703599B - Improved preparation method of kelp chromosome - Google Patents
Improved preparation method of kelp chromosome Download PDFInfo
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Abstract
The invention discloses an improved preparation method of a kelp chromosome. The steps are absorbing surplus moisture after kelp gamobiums are centrifuged, keeping the kelp gamobiums to be deposited, and adding colchicines solution for evenly shaking and stewing; conducting hypotension on the kelp gamobiums by using potassium chloride solution, fixing the kelp gamobiums by sing kano stationary liquid after hypotension, conducting enzymolysis and wall removal through mixed enzyme liquid of cellulose R-10 solution, pectinase solution and liquation enzyme R-10 solution, absorbing supernate by using a liquor relief gun after the kelp gamobiums are centrifuged, adding glacial acetic acid for evenly mixing to form cell suspension, absorbing the cell suspension by using a dropper to an upper dropping sheet of a centrifuging and pre-cooling glass slide, burning the bottom face of the glass slide by using an alcohol burner for one time, and naturally drying or stoving, dying the kelp gamobium chromosome adhered to the glass slide by using giemsa staining fluid, and observing and photographing under a microscope. The chromosomes obtained by using the improved preparation method are good in segregation, differentiable in shape and denumerable in chromosome split pahse, and the improved preparation method is simple and rapid in method, strong in repeatability and capable of opening in a general laboratory.
Description
Technical field
The present invention relates to chromosomal preparation method, relate in particular to the chromosomal preparation method of improved sea-tangle.
Background technology
The sporophyte of sea-tangle is individual large, is tame object.Kelp gametophyte is small filament, is the object of artificial culture seed, is also the basis of kelp germplasm resource conservation.Kelp gametophyte has dividing of male and female, and megagametophyte is comprised of one or several cell, and microgametophyte is generally comprised of a plurality of cells, and microgametophyte cell is little compared with megagametophyte cell individual.In developmental process, megagametophyte increases the volume of cell, and microgametophyte carries out the division of cell, increases the number of cell, forms cellulous branch body or group's spherule together.
Sea-tangle karyomit(e) is less, due to the difference of method of chromosome preparation, causes sea-tangle chromosome number to have dispute.Mainly with section method and iron haematoxylin dyeing in early days.Section method is more original method, for calculating the time-consuming and out of true of karyomit(e).Multiplex pressed disc method and aceto-camine, Chloral Hydrate iron acetate phenodin or aceto-orcein dyeing subsequently.Twentieth century six the seventies Chinese scholars are observed chromosome number to kelp gametophyte cell by pressed disc method, and most scholars think to only have 22, and minority scholar thinks 31.To nineteen nineties, Japanese scholars Hiroshi Yabu etc. thinks that sea-tangle gametid [cell approximately has 32 karyomit(e)s (to see paper " chromosome numbers of four main laminarias (brown alga) ", " Japanese phycology magazine ", the 39th the 2nd phase of volume in 1991,185-187 page).Chinese scholar Zhou Liran in 2004 etc. utilize compressing tablet and brazilwood extract dyeing to observe kelp gametophyte to have 31 karyomit(e)s.The karyomit(e) that pressed disc method obtains is point-like more, cannot differential staining volume morphing, can not do karyotyping and (see paper " a kind of improved sea-tangle microgametophyte (heterokontae) method of chromosome preparation ", " hydrobiology ", the 512nd phase in 2004,141-144 page).The scholars such as Liu Yu (see paper " the chromosomal DAPI dyeing of sea-tangle and caryogram initial analysis ", publication is in " aquatic product journal " the 01st phase in 2012,50-54 page) utilize the method for dripping sheet method and DAPI fluorescent dyeing, observe kelp gametophyte cell and there are 31 karyomit(e)s, Chromosome spread is better, and number form can be distinguished.But make fluorochromine can only pass through fluorescent microscope ability observations, fluorescent microscope market sale price is higher, can not popularize use.
Summary of the invention
The invention provides a kind of Chromosome spread good, form can be distinguished and the denumerable chromosomal preparation method of improved sea-tangle.
Technical solution of the present invention is:
The chromosomal preparation method of sea-tangle, the steps include:
(1) kelp gametophyte pre-treatment: the centrifugal rear absorption excessive moisture of kelp gametophyte retains kelp gametophyte precipitation, and add colchicine solution vibration to shake up standing;
(2) kelp gametophyte is hypotonic: kelp gametophyte is hypotonic with Klorvess Liquid;
(3) kelp gametophyte is fixed: the kelp gametophyte after hypotonic is fixed with Kano stationary liquid;
(4) enzymolysis removes wall: with the mixed enzyme solution of cellulase R-10 solution, polygalacturonase solution, macerozyme R-10 solution, carry out enzymolysis and remove wall.
(5) cold sheet: enzymolysis goes that the kelp gametophyte after wall is centrifugal absorbs supernatant liquor afterwards with liquid-transfering gun, add glacial acetic acid, vibration is mixed into cell suspension, with dropper, draw cell suspension and on the slide glass from precooling, drip sheet, slide glass bottom surface is naturally dried or dries after spirit lamp flame burns once;
(6) Ji's nurse Sa dyeing: adopt Ji's nurse Sa staining fluid to dye to the kelp gametophyte karyomit(e) being attached on slide glass.
(7) micro-Microscopic observation and taking pictures.
The colchicine solution of above-mentioned steps (1) is that weightmeasurement ratio is 0.2% colchicine solution, and kelp gametophyte adds after 0.2% colchicine solution at 4 ℃ standing 7～8 hours.
The concentration of the Klorvess Liquid of above-mentioned steps (2) is 0.075mol/L, and it is after the Klorvess Liquid of 0.075mol/L, to vibrate and shake up, at room temperature standing 30～40 minutes that kelp gametophyte precipitation adds concentration.
The Kano stationary liquid of above-mentioned steps (3) is the volume ratio configuration by 3: 1 by methyl alcohol and glacial acetic acid, at 4 ℃ of Kano stationary liquids, fixes three times, each 20 minutes, at 4 ℃ of the Kano stationary liquids that finally renew again, fixes 24 hours.
In above-mentioned steps (4), cellulase R-10 solution, polygalacturonase solution, macerozyme R-10 solution mix after shaking up and become mixed enzyme solution by the volume ratio of 2: 1: 1, now with the current, place at 4 ℃.
In above-mentioned steps (5), from the height of 30～40 centimetres of the slide glasss of precooling, drip sheet.
Technique effect of the present invention is: the present invention adopts the method for cold sheet and Giemsa staining, has obtained Chromosome spread better, and form can be distinguished and denumerable karyomit(e) division phase.Ji's nurse Sa dyeing is simultaneously to whole chromosome dyeing, there will not be olistherozone territory, and uses simple microscope to get final product observations.The inventive method adopts the method for cold sheet and the dyeing of Ji's nurse Sa, has obtained Chromosome spread better, and form can be distinguished and denumerable karyomit(e) division phase.In addition, the inventive method is easy fast, and repeatable strong, common laboratory all can operate.
Accompanying drawing explanation
1, Fig. 1 is No. 2, east hybridization kelp gametophyte karyomit(e) photo prepared by the inventive method.
2, Fig. 2 utilizes the kelp gametophyte karyomit(e) photo of pressed disc method and brazilwood extract dyeing for Chinese scholar Zhou Liran.
3, Fig. 3 is Ji's nurse Sa dyeing slide glass bridging vertical view.
4, Fig. 4 is Ji's nurse Sa dyeing slide glass bridging side-view.
In figure, 1, sheet glass, 2, have chromosome specimen slide glass, 3, slide glass, 4, have chromosome specimen slide glass and the hole between sheet glass.
Specific embodiments
Below in conjunction with drawings and Examples, describe in detail:
One, the present embodiment kelp gametophyte is taken from " No. 2, east " hybridization kelp gametophyte that country of Shandong Oriental Ocean Sci-tech Co., Ltd., marine alga Engineering Technical Research Centre germplasm is preserved storehouse preservation.
Kelp gametophyte also can gather as follows: in the sea-tangle mature period, select healthy and strong sporophyte, taken off young sporangial main laminaria piece, with sterilizing seawater (raw seawater boil obtain sterilizing seawater for 2 minutes) and sterilizing cotton, (degreasing cotton is with entering pressure steam sterilizer 120 ℃ of temperature after kraft paper parcel, pressure 0.165MPa sterilizing obtains sterilizing cotton for 15 minutes) scouring multipass, remove dirt settling and assorted algae, after main laminaria piece dries in the shade, be placed in the sterilizing seawater nutrient solution that has added SODIUMNITRATE and potassium primary phosphate, kelp spore is concentrated to be diffused 1～3 hour, then kelp spore water is poured in the staining jar that is placed with slide glass, kelp spore is attached to and on slide glass, forms sea-tangle sporoblast, under suitable temperature (10 ± 2 ℃) and illumination (1000～1500Lx) condition, cultivate and form female in about one week, the male gametophyte that can distinguish.At this moment, adopt under the microscope microcapillary partition method that single female, microgametophyte are taken out respectively, female, microgametophyte is placed in respectively different sterilizing seawater nutrient solutions and carries out single culture, female, microgametophyte continues under 1500Lx illumination condition, to nourish and grow for 24 hours in sterilizing seawater nutrient solution, just can form clone, i.e. gametophyte clone.Then enter kelp gametophyte and preserve the stage, kelp gametophyte is continuous light (1500Lx) in sterilizing seawater nutrient solution, within every 7 days, changes sterilizing seawater nutrient solution one time, is about to former sterilizing seawater nutrient solution and pours out, retain kelp gametophyte, then add the sterilizing seawater nutrient solution of equivalent.
Two, the present embodiment adopts following laboratory apparatus:
A, illumination box: SPX-300B-G type, Shanghai Medical Equipment Plant of Bo Xun Industrial Co., Ltd.
B, desk centrifuge: TGL-16C Xing， Anting Scientific Instrument Factory, Shanghai
C, refrigerator: BCD-216TX Xing， Qingdao HaiEr Co., Ltd
D, microscope: XSP-C204 type, Shanghai Medical Equipment Plant of Bo Xun Industrial Co., Ltd.
E, digital camera: the IXUS 310HS of Canon
F, full temperature are cultivated shaking table: QYC-200 type, Shanghai new talent medicine equipment Manufacturing Co., Ltd
Three, the present embodiment adopts following experiment reagent
1) configuration sodium nitrate solution: take 121 grams of SODIUMNITRATE (molecular formula NaNO3, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, analytical pure), after dissolving with sterilizing seawater, be settled to 500ml with sterilizing seawater.The sterilizing seawater of the present embodiment seawater of all making a living boils 2 minutes, and naturally cools to the sterilizing seawater of room temperature.
2) configuration potassium dihydrogen phosphate: take 17.5 grams of potassium primary phosphates (molecular formula KH2PO4, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, analytical pure), after dissolving with sterilizing seawater, be settled to 1000ml with sterilizing seawater.
3) configuration sterilizing seawater nutrient solution: measure respectively above-mentioned 1ml sodium nitrate solution and 1ml potassium dihydrogen phosphate, be added to 4000ml through boiling 2 minutes and naturally cooling in the sterilizing seawater of room temperature, shake up standby.
4) configure 0.2% colchicine solution: take 0.2 gram of colchicine powder (upper sea blue season development in science and technology company limited, import packing), first use after 1ml anhydrous alcohol solution, then be settled to 100ml with sterilizing seawater, with brown bottle splendid attire with cover and in 4 ℃ of refrigerators, preserve.
5) configuration 0.075mol/L Klorvess Liquid: take 0.5588 gram, Repone K (chemical formula KCl， Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, analytical pure), after dissolving with sterilizing seawater, be settled to 100ml with sterilizing seawater.
6) configuration Kano stationary liquid: by anhydrous methanol and the glacial acetic acid proportional arrangement of 3: 1 by volume, now with the current, 4 ℃ of refrigerators are placed.
7) configure cellulase R-10 (Cellulase " Onozuka " R-10) solution of 1% concentration: take 0.1 gram of cellulase R-10 powder (Japanese Yakult company produce), in the 0.067mol/L phosphate buffered saline buffer that the pH that is dissolved in 10ml is 7.0, shake up standby.
8) configure macerozyme R-10 (Macerozyme R-10) solution of 1% concentration: take 0.05 gram of macerozyme R-10 powder (production of Beijing Suo Laibao Science and Technology Ltd.), in the 0.067mol/L phosphate buffered saline buffer that the pH that is dissolved in 5ml is 7.0, shake up standby.
9) configure 1% polygalacturonase solution: take 0.05 gram of polygalacturonase powder (upper sea blue season development in science and technology company limited produce), in the 0.067mol/L phosphate buffered saline buffer that the pH that is dissolved in 5ml is 7.0, shake up standby.
10) the 0.067mol/L phosphate buffered saline buffer that configuration pH is 7.0: configuration A liquid: 0.067mol/L potassium dihydrogen phosphate, take potassium primary phosphate (molecular formula KH2PO4, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, analytical pure) 9.112 grams, after dissolving at 60 ℃ of 50～100ml distilled water, with distilled water, be settled to 1000ml.Configuration B liquid: 0.067mol/L disodium hydrogen phosphate solution, take disodium hydrogen phosphate (molecular formula Na2HPO412H2O, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, analytical pure) 23.986 grams, after dissolving at 60 ℃ of 50～100ml distilled water, with distilled water, be settled to 1000ml.Measure the A liquid of 38.8ml and the B liquid of 61.2ml and mix, both can obtain pH and be the phosphate buffered saline buffer of 7.0 0.067mol/L.
11) configuration Ji's nurse Sa (Giemsa) staining fluid: Ji's nurse Sa concentrated solution and the Ji's nurse Sa diluent ratio of 1: 9 is by volume mixed, and matching while using, preserves with brown bottle with cover.Ji's nurse Sa concentrated solution and Ji's nurse Sa diluent are Beijing Suo Laibao Science and Technology Ltd. and produce.
12) methyl alcohol, dehydrated alcohol, glacial acetic acid: be Shanghai Chemical Reagent Co., Ltd., Sinopharm Group and produce, analytical pure.
Four, the present embodiment kelp gametophyte method of chromosome preparation
(1) colchicine pre-treatment
The good kelp gametophyte of cultivation conditions means that micro-Microscopic observation kelp gametophyte cell size is even, the kelp gametophyte that pigment is evenly distributed (female, microgametophyte all can), it is 1.5ml plastic centrifuge tube that the good kelp gametophyte of cultivation conditions of getting volume and have large grain of rice size is put into volume, rotating speed 1000rpm (rev/min), centrifugal 5min (minute) after with liquid-transfering gun, absorb excessive moisture and retain kelp gametophyte precipitation, add above-mentioned 0.2% colchicine solution 1ml, vibration shakes up, in 4 ℃ of refrigerators standing 7～8 hours.
Here adopt colchicine pre-treatment object to be: 1. to stop or destroy the formation of Spindle microtubule in plant mitotic division process, owing to there is no the effect of Spindle, cell mitogen process was prevented in the metaphase in cell division stage, thus can cumulative comparison many chromosome images in metaphase in cell division; 2. promote karyomit(e) to concentrate and shorten, between minimizing karyomit(e), winding is overlapping mutually, favourable chromosomal high dispersing; 3. change kytoplasm viscosity, promote chromosome clear, promote tenuigenin clean.
(2) kelp gametophyte is hypotonic
At rotating speed 1000rpm, after centrifugal 5min, with liquid-transfering gun, absorb supernatant, retain gametophyte precipitation, adding concentration is that Repone K (KCl) the solution 1ml of 0.075mol/L is hypotonic, vibration shakes up, under room temperature standing 30 minutes.
Hypotonic Main Function has two: 1. promote cell plasmolysis, be conducive to enzymolysis process and remove cell walls; 2. promote tenuigenin viscosity-modifying, be conducive to chromosome image clear.
(3) kelp gametophyte is fixed
At rotating speed 1000rpm, after centrifugal 5min, with liquid-transfering gun, absorb supernatant, retain kelp gametophyte precipitation.The Kano stationary liquid 1ml that adds 4 ℃ of placements of new configuration, inhales gently and beats (suck and release) solution 10 times with liquid-transfering gun, and standing 20min in 4 ℃ of refrigerators, at rotating speed 1000rpm, absorbs supernatant with liquid-transfering gun after centrifugal 5min, retains gametophyte precipitation.The Kano stationary liquid 1ml that adds again 4 ℃ of placements of new configuration, inhales gently and beats solution 10 times with liquid-transfering gun, and standing 20min in 4 ℃ of refrigerators, at rotating speed 1000rpm, absorbs supernatant with liquid-transfering gun after centrifugal 5min, retains gametophyte precipitation.The Kano stationary liquid 1ml that again adds 4 ℃ of placements of new configuration, inhales gently and beats solution 10 times with liquid-transfering gun, and standing 20min in 4 ℃ of refrigerators, at rotating speed 1000rpm, absorbs supernatant with liquid-transfering gun after centrifugal 5min, retains gametophyte precipitation.Finally add 1ml Kano stationary liquid, standing more than 24 hours in 4 ℃ of refrigerators.
Fixing Main Function: 1. rapid cell killing, retains split image.2. make the sex change of karyomit(e) nucleoprotein, precipitation, present karyomit(e) true form and structure.3. make tenuigenin protoplastis protein denaturation, precipitation, be conducive to karyomit(e) background clear.
(4) enzymolysis removes wall
At rotating speed 1000rpm, after centrifugal 5min, with liquid-transfering gun, absorb supernatant, retain kelp gametophyte precipitation.Adding distil water 1ml enters centrifuge tube, with liquid-transfering gun, inhales gently and beats 10 flushing kelp gametophyte precipitations of solution, at rotating speed 1000rpm, after centrifugal 5min, absorbs the distilled water in centrifuge tube again with liquid-transfering gun, notes not siphoning away kelp gametophyte, retains kelp gametophyte.Add new distilled water 1ml to enter centrifuge tube, with liquid-transfering gun, inhale gently and beat 10 flushing kelp gametophyte precipitations of solution, at rotating speed 1000rpm, after centrifugal 5min, with liquid-transfering gun, absorb the distilled water in centrifuge tube again, retain kelp gametophyte.Above-mentioned circulation step repeats to rinse kelp gametophyte precipitation and amounts to 3～5 times.At rotating speed 1000rpm, after centrifugal 5min, with liquid-transfering gun, absorb supernatant, retain kelp gametophyte precipitation, add the mixed enzyme solution of newly joining (after cellulase R-10 solution, polygalacturonase solution, macerozyme R-10 solution are mixed by the volume ratio of 2: 1: 1, to shake up, now with the current, 4 ℃ of refrigerators are placed) 1ml, cultivates on shaking tables in 200rpm enzymolysis 18 hours 37 ℃ of full temperature.At rotating speed 1000rpm, 5min is centrifugal absorbs upper clear enzyme solution gently afterwards with liquid-transfering gun, with liquid-transfering gun, get 1ml distilled water again and inhale gently and beat 10 flushing gametophyte precipitations of solution, again at rotating speed 1000rpm, after centrifugal 5min, with liquid-transfering gun, absorb distilled water, add new 1ml distilled water to inhale gently and beat solution 10 times, gametophyte precipitation is rinsed.
Enzymolysis goes the effect of wall: 1. biology dissociates and process to substitute physics and chemistry processings of dissociating, and has reduced karyomit(e) distortion, distortion, presents really karyomit(e) firmly.2. remove cell walls fiber, pectin and kytoplasm to the impact of karyomit(e) quantity, make Chromosome spread space larger.3. the image such as favourable kinetochore satellite is complete clear.
(5) cold sheet
At rotating speed 1000rpm, after centrifugal 5min, with liquid-transfering gun, absorb supernatant liquor, add 100～200 μ L (microlitre) glacial acetic acids, vibration is mixed into cell suspension.Pick and place the clean slide soaking in 4 ℃ of refrigerator distilled water, control control water on filter paper, with dropper, draw cell suspension and drip sheet at the height of 30～40 centimetres of the slide glasss from precooling, every slide glass drips 2～3, then immediately slide glass one end is lifted, and blow gently, cell is disperseed rapidly, and the slide glass one side of then not dripping cell suspension is crossed once on spirit lamp flame, makes Chromosome spread and expansion, at slide glass, do not have one end of chromosomal one side to mark, under room temperature, dry.
The effect of cold sheet: 1. utilize the poor cell that allows of certain altitude break on slide glass, karyomit(e) is discharged in cell.2. the cell not breaking drops on the slide glass of precooling after spirit lamp flame, chromosomal loss while utilizing temperature head that cell rupture, this cell rupture mode can be avoided dripping sheet.
(6) Ji's nurse Sa dyeing
As shown in Figure 3 and Figure 4, get a sheet glass and lie on experiment table, separately get two clean new slide glasss according to long parallel the sequencing of one side, distance is less than the length of a slide glass.Room temperature is dried and had the slide glass of chromosome specimen pendulum on two new slide glasss, and have chromosomal one to face down, as putting up a bridge, there is the slide glass of chromosome specimen to set successively other, between the slide glass that two have a chromosome specimen, there is not space, between the slide glass that has a chromosome specimen and sheet glass, just form the hole that two ends communicate, with dropper, draw Ji's nurse Sa staining fluid, one end by hole, slowly inject Ji's nurse Sa staining fluid, note firmly evenly not producing bubble, Ji's nurse Sa staining fluid is all full of after hole, standing 30～40min under room temperature.The tap of fetching boiling water, washes down staining fluid with very slow current along hole one end, notices that flow velocity is too not fast, prevents that the slide glass of chromosome specimen is flushed away, and then will have the slide glass of chromosome specimen to take out, and room temperature is dried.
(7) micro-Microscopic observation and taking pictures
Eyepiece (WF16 */10 of amplifying 16 times are selected in this experiment, amplify 16 times, field number is 10 millimeters) constant, rotate objective changement, first under 10 times of object lens, find the karyomit(e) division phase that Chromosome spread is good and denumerable, then cedar oil is dropped on karyomit(e), then forward under 100 times of object lens and take pictures, under oily mirror, take pictures.
Fig. 1 is the chromosomal photo of kelp gametophyte that utilizes the inventive method to prepare, and as seen from the figure, dyes scattered, and length scale can distinguish, number is 31.
Fig. 2 is the kelp gametophyte karyomit(e) that Chinese scholar Zhou Liran in 2004 utilizes pressed disc method and brazilwood extract dyeing, and karyomit(e) is point-like, and shape size can not be recognized.
Claims (2)
1. the chromosomal preparation method of improved sea-tangle, the steps include:
(1) kelp gametophyte pre-treatment: the centrifugal rear absorption excessive moisture of kelp gametophyte retains kelp gametophyte precipitation, and after adding 0.2% colchicine solution vibration to shake up at 4 ℃ standing 7 hours;
(2) kelp gametophyte is hypotonic: it is after the Klorvess Liquid of 0.075mol/L, to vibrate and shake up, at room temperature standing 30～40 minutes that kelp gametophyte adds concentration;
(3) kelp gametophyte is fixed: the kelp gametophyte after hypotonic is fixed with Kano stationary liquid, described Kano stationary liquid is to be configured by the volume ratio of 3:1 by methyl alcohol and glacial acetic acid, at 4 ℃ of Kano stationary liquids, fix three times, each 20 minutes, at 4 ℃ of the Kano stationary liquids that finally renew again, fix 24 hours;
(4) enzymolysis removes wall: with the mixed enzyme solution of cellulase R-10 solution, polygalacturonase solution, macerozyme R-10 solution, carry out enzymolysis and remove wall, described cellulase R-10 solution, polygalacturonase solution, macerozyme R-10 solution become mixed enzyme solution after shaking up by the volume ratio mixing of 2:1:1, now with the current, place at 4 ℃;
(5) cold sheet: enzymolysis goes that the kelp gametophyte after wall is centrifugal absorbs supernatant liquor afterwards with liquid-transfering gun, add glacial acetic acid, vibration is mixed into cell suspension, with dropper, draw cell suspension and drip sheet at the height of 30～40 centimetres of the slide glasss from precooling, slide glass bottom surface is naturally dried or dries after spirit lamp flame burns once;
(6) Ji's nurse Sa dyeing: adopt Ji's nurse Sa staining fluid to dye to the kelp gametophyte karyomit(e) being attached on slide glass;
(7) micro-Microscopic observation and taking pictures.
2. the chromosomal preparation method of a kind of improved sea-tangle according to claim 1, it is characterized in that: the dyeing of described step (6) Ji's nurse Sa is that a sheet glass is lain on experiment table, separately get two clean new slide glasss according to long parallel the sequencing of one side, distance is less than the length of a slide glass, room temperature is dried and had the slide glass of chromosome specimen pendulum on two new slide glasss, and have chromosomal one to face down, between the slide glass that has a chromosome specimen and sheet glass, just form the hole that two ends communicate, with dropper, draw Ji's nurse Sa staining fluid, by one end of hole, inject Ji's nurse Sa staining fluid, Ji's nurse Sa staining fluid is all full of after hole, standing 30～40min under room temperature, then with current, along hole one end, wash down staining fluid, finally will there is the slide glass of chromosome specimen to take out, room temperature is dried.
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| CN108007753A (en) * | 2018-01-22 | 2018-05-08 | 上海海洋大学 | A kind of method of chromosome preparation of Enteromorpha sporinite |
| CN110055209B (en) * | 2019-03-13 | 2020-04-21 | 海南省海洋与渔业科学院 | Preparation method of Talaromyces protoplast |
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