CN110907254A - Wright-Giemsa dyeing reagent and preparation method thereof - Google Patents
Wright-Giemsa dyeing reagent and preparation method thereof Download PDFInfo
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- CN110907254A CN110907254A CN201911067690.1A CN201911067690A CN110907254A CN 110907254 A CN110907254 A CN 110907254A CN 201911067690 A CN201911067690 A CN 201911067690A CN 110907254 A CN110907254 A CN 110907254A
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- 238000004043 dyeing Methods 0.000 title claims abstract description 29
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title abstract description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 105
- 239000000975 dye Substances 0.000 claims description 128
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 78
- 239000000243 solution Substances 0.000 claims description 44
- 235000011187 glycerol Nutrition 0.000 claims description 27
- 238000010817 Wright-Giemsa staining Methods 0.000 claims description 25
- 239000012128 staining reagent Substances 0.000 claims description 17
- 239000011259 mixed solution Substances 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 14
- 239000000843 powder Substances 0.000 claims description 14
- 239000002245 particle Substances 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 9
- 238000004040 coloring Methods 0.000 abstract description 5
- 238000010186 staining Methods 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 9
- 239000012192 staining solution Substances 0.000 description 6
- 230000003203 everyday effect Effects 0.000 description 5
- 210000000601 blood cell Anatomy 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- KFZNPGQYVZZSNV-UHFFFAOYSA-M azure B Chemical compound [Cl-].C1=CC(N(C)C)=CC2=[S+]C3=CC(NC)=CC=C3N=C21 KFZNPGQYVZZSNV-UHFFFAOYSA-M 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 238000007447 staining method Methods 0.000 description 2
- 230000008719 thickening Effects 0.000 description 2
- 239000012905 visible particle Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000002738 Giemsa staining Methods 0.000 description 1
- 241001212699 Pinctada martensii Species 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000003756 cervix mucus Anatomy 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- SEACYXSIPDVVMV-UHFFFAOYSA-L eosin Y Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 SEACYXSIPDVVMV-UHFFFAOYSA-L 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
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- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The embodiment of the invention discloses a Wright-Giemsa dyeing reagent and a preparation method thereof, wherein the Wright-Giemsa dyeing reagent is formed by mixing a first Wright-Giemsa dye solution and a second Wright-Giemsa dye solution, wherein the second Wright-Giemsa dye solution comprises the following components in parts by mass, 10-20 parts of Wright dye, 1-5 parts of Giemsa dye and 5000 parts of methanol 4000-. The embodiment of the invention mixes the finished product BasoWright-Giemsa dye solution with the BasoWright-Giemsa dye solution of the embodiment of the invention to obtain the new Wright-Giemsa dye solution, which has the advantages of obvious coloring, good dyeing effect, good dyed cell resolution, simple preparation and low cost.
Description
Technical Field
The invention relates to the technical field of chemical reagent preparation, in particular to a Wright-Giemsa dyeing reagent and a preparation method thereof.
Background
The staining of blood smear is widely used in medical laboratory, and is an important technique for observing the morphology of blood cells, and the quality of staining of blood smear directly affects the identification of the morphology of blood cells and the diagnosis of diseases. The blood cell staining solution has many kinds, and the international committee for standardization in hematology (ICSH) recommends that romanofsky (Romanowsky) staining be a standard staining method, the main components of the staining solution are azure B and eosin Y, and the content of the azure B is required to be more than 80%, but the Romanowsky staining method is difficult to popularize in various countries due to the high price of the azure B.
Wright-Giemsa staining method is mostly adopted in domestic medical examination laboratories and is mainly used for staining blood cells, and can also be used for staining bone marrow smears, vaginal secretion cell smears, exfoliated cell smears and the like. Generally, commercial dyeing solution is purchased or prepared by self in clinic, the quality of the reagent is different, and the dyeing effect is difficult to satisfy. The commercial reagent is most common in Wright-Giemsa dye liquor produced by the Zhuhai Baso company, and the main dyeing defects of the dye liquor produced by the Zhuhai Baso company in clinical examination work are as follows: the coloring of the particles is more obvious, such as thickening of neutral particles, thickening and increasing of A particles, and easy particles of lymphocytes and monocytes; the cytoplasm is blue, and some cell edges are easy to appear red.
In conclusion, the dyeing reagents in the prior art also have the defects of high preparation cost, alkaline dyeing, long dyeing time, unobvious particle coloring or no visible particles, and further improvement is needed.
Disclosure of Invention
The embodiment of the invention aims to provide a Wright-Giemsa dyeing reagent and a preparation method thereof, which are used for solving the problems of alkaline dyeing, long dyeing time, unobvious particle coloring or no visible particle and high cost of the existing dye solution.
In order to achieve the above object, an embodiment of the present invention provides a Wright-Giemsa dyeing reagent, which is formed by mixing a first Wright-Giemsa dye solution and a second Wright-Giemsa dye solution, wherein the second Wright-Giemsa dye solution includes, by mass, 10-20 parts of Wright dye, 1-5 parts of Giemsa dye, and 5000 parts of methanol 4000-.
Preferably, the second Wright-Giemsa stain further comprises glycerin.
Preferably, the second Wright-Giemsa dye solution comprises the following components in parts by mass, 12-15 parts of Wright dye, 2-4 parts of Giemsa dye, 1-5 parts of glycerol and 4800 parts of methanol 4500-.
Preferably, the second Wright-Giemsa dye solution comprises the following components, by mass, 10 parts of Wright dye, 2 parts of Giemsa dye, 3 parts of glycerol and 5000 parts of methanol.
Preferably, the first Wright-Giemsa dye liquor is a finished Wright-Giemsa dye liquor.
Preferably, the mixing ratio of the first Wright-Giemsa dye liquor and the second Wright-Giemsa dye liquor is 1: 2.
the embodiment of the invention also provides a method for preparing the Wright-Giemsa staining reagent, which comprises the following steps of firstly preparing a second Wright-Giemsa staining solution:
taking Wright dye, Giemsa dye, methanol and glycerol according to the mixture ratio;
crushing the Wright dye and the Giemsa dye to form mixture powder, adding glycerol until no powder particles precipitate to form a mixed solution, and standing for 12-16 hours;
adding methanol into the mixed solution, and grinding for several times until the methanol is used up to form a second Wright-Giemsa dye solution;
and mixing the first Wright-Giemsa dye solution and the second Wright-Giemsa dye solution to form the Wright-Giemsa dye reagent.
The second Wright-Giemsa stain was shaken daily for 1-3 min.
Preferably, the mixed solution is left standing for 12 to 16 hours and then ground for 20 to 80 minutes.
The embodiment of the invention has the following advantages:
the embodiment of the invention mixes the finished product BasoWright-Giemsa dye solution with the BasoWright-Giemsa dye solution of the embodiment of the invention to obtain the new Wright-Giemsa dye solution, which has the advantages of obvious coloring, good dyeing effect, good dyed cell resolution, simple preparation and low cost.
Drawings
FIG. 1 is a photomicrograph showing staining of smears in comparative examples provided in examples of the present invention.
FIG. 2 is a photomicrograph showing the staining of a smear of Wright-Giemsa staining reagent according to an embodiment of the present invention.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
In the embodiment of the invention, the first Wright-Giemsa dye liquor is a finished product Wright-Giemsa dye liquor. Specifically, the first Wright-Giemsa dye solution is a Wright-Giemsa dye solution produced by Beisuo Biotechnology Co., Ltd (Baso).
The embodiment of the invention provides a Wright-Giemsa dyeing reagent, which is formed by mixing a first Wright-Giemsa dye solution and a second Wright-Giemsa dye solution, wherein the second Wright-Giemsa dye solution comprises the following components in parts by mass, 10-20 parts of Wright dye, 1-5 parts of Giemsa dye and 5000 parts of methanol 4000-. The second Wright-Giemsa stain also included glycerin. Preferably, the second Wright-Giemsa dye solution comprises the following components, by mass, 12-15 parts of Wright dye, 2-4 parts of Giemsa dye, 1-5 parts of glycerol and 4500-4800 parts of methanol.
The Wright-Giemsa dye solution produced by the Pinctada martensii Soloo Baso is mixed with the Wright-Giemsa dye solution provided by the embodiment of the invention according to a certain proportion, so that the advantages of good smear dyeing effect, easy identification of each structure and low cost are achieved.
Example 1
In the embodiment of the invention, the second Wright-Giemsa dye solution comprises the following components, by mass, 10 parts of Wright dye, 1 part of Giemsa dye, 1 part of glycerol and 4000 parts of methanol.
In the embodiment of the invention, the second Wright-Giemsa dyeing reagent is prepared by taking Wright dye, Giemsa dye, methanol and glycerol according to the mixture ratio;
crushing the Wright dye and the Giemsa dye to form mixture powder, adding glycerol until no powder particles precipitate to form a mixed solution, standing for 12-16 hours, and grinding for 20 minutes;
adding methanol into the mixed solution, grinding for several times until the methanol is used up, and oscillating the second Wright-Giemsa dye solution for 1-3min every day to form a second Wright-Giemsa dye solution;
the first Wright-Giemsa dye liquor and the second Wright-Giemsa dye liquor prepared in the embodiment of the invention are fully mixed according to the volume ratio of 1:2 to form the Wright-Giemsa dye reagent.
Example 2
In the embodiment of the invention, the second Wright-Giemsa dye solution comprises the following components, by mass, 20 parts of Wright dye, 5 parts of Giemsa dye, 5 parts of glycerol and 5000 parts of methanol.
In the embodiment of the invention, the second Wright-Giemsa dyeing reagent is prepared by taking Wright dye, Giemsa dye, methanol and glycerol according to the mixture ratio;
crushing the Wright dye and the Giemsa dye to form mixture powder, adding glycerol until no powder particles precipitate to form a mixed solution, standing for 12-16 hours, and grinding for 30 minutes;
adding methanol into the mixed solution, grinding for several times until the methanol is used up, and oscillating the second Wright-Giemsa dye solution for 1min every day to form a second Wright-Giemsa dye solution;
the first Wright-Giemsa dye liquor and the second Wright-Giemsa dye liquor prepared in the embodiment of the invention are fully mixed according to the volume ratio of 1:2 to form the Wright-Giemsa dye reagent.
Example 3
In the embodiment of the invention, the second Wright-Giemsa dye solution comprises the following components, by mass, 15 parts of Wright dye, 2 parts of Giemsa dye, 3 parts of glycerol and 4500 parts of methanol.
In the embodiment of the invention, the second Wright-Giemsa dyeing reagent is prepared by taking Wright dye, Giemsa dye, methanol and glycerol according to the mixture ratio;
crushing the Wright dye and the Giemsa dye to form mixture powder, adding glycerol until no powder particles are precipitated to form a mixed solution, standing for 10 hours, and grinding for 40 minutes;
adding methanol into the mixed solution, grinding for several times until the methanol is used up, and oscillating the second Wright-Giemsa dye solution for 1min every day to form a second Wright-Giemsa dye solution;
the first Wright-Giemsa dye liquor and the second Wright-Giemsa dye liquor prepared in the embodiment of the invention are fully mixed according to the volume ratio of 1:2 to form the Wright-Giemsa dye reagent.
Example 4
In the embodiment of the invention, the second Wright-Giemsa dye solution comprises the following components in parts by mass, namely 16 parts of Wright dye, 3 parts of Giemsa dye, 4 parts of glycerol and 4600 parts of methanol.
In the embodiment of the invention, the second Wright-Giemsa dyeing reagent is prepared by taking Wright dye, Giemsa dye, methanol and glycerol according to the mixture ratio;
crushing the Wright dye and the Giemsa dye to form mixture powder, adding glycerol until no powder particles precipitate to form a mixed solution, standing for 11 hours, and grinding for 40 minutes;
adding methanol into the mixed solution, grinding for several times until the methanol is used up, and oscillating the second Wright-Giemsa dye solution for 2min every day to form a second Wright-Giemsa dye solution;
the first Wright-Giemsa dye liquor and the second Wright-Giemsa dye liquor prepared in the embodiment of the invention are fully mixed according to the volume ratio of 1:2 to form the Wright-Giemsa dye reagent.
Example 5
In the embodiment of the invention, the second Wright-Giemsa dye solution comprises the following components, by mass, 19 parts of Wright dye, 4 parts of Giemsa dye, 4 parts of glycerol and 4800 parts of methanol.
In the embodiment of the invention, the second Wright-Giemsa dyeing reagent is prepared by taking Wright dye, Giemsa dye, methanol and glycerol according to the mixture ratio;
crushing the Wright dye and the Giemsa dye to form mixture powder, adding glycerol until no powder particles are precipitated to form a mixed solution, standing for 12 hours, and grinding for 50 minutes;
adding methanol into the mixed solution, grinding for several times until the methanol is used up, and oscillating the second Wright-Giemsa dye solution for 3min every day to form a second Wright-Giemsa dye solution;
the first Wright-Giemsa dye liquor and the second Wright-Giemsa dye liquor prepared in the embodiment of the invention are fully mixed according to the volume ratio of 1:2 to form the Wright-Giemsa dye reagent.
The Wright-Giemsa staining reagents prepared in examples 1 to 5 were stored under sealed conditions and protected from light, and were generally stored under sealed conditions for 3 months or more.
The second Wright-Giemsa dye liquor in the embodiments 1-5 of the present invention was prepared by purchasing the components, and the first Wright-Giemsa dye liquor was the finished Wright-Giemsa dye liquor.
Comparative example
1. Preparing a smear;
2. adding Wright-Giemsa staining reagent produced by the product of the Zhuhai Baso, dripping 1 drop of the Wright-Giemsa staining reagent on the smear to allow the human color liquid to cover the whole smear film, and standing for 0.5-1 min;
3. adding phosphate buffer solution, adding 2-3 times of Wright-Giemsa staining reagent, blowing and uniformly mixing by using an aurilave, staining and standing for 5-10min, judging the staining degree of nucleated cells observed under a low power microscope, directly washing by using water flow after good staining is observed under the low power microscope, and paying attention not to pour out human color solution firstly and then washing;
4. the staining effect was observed under a microscope, as shown in FIG. 1.
Test examples
1. Preparing a smear;
2. adding the Wright-Giemsa staining reagent of the embodiment of the invention, dripping 1 drop of the Wright-Giemsa staining reagent on the smear, allowing the human color liquid to cover the whole smear film, and standing for 0.5-1 min;
3. adding phosphate buffer solution, adding 2-3 times of Wright-Giemsa staining reagent, blowing and uniformly mixing by using an aurilave, staining and standing for 5-10min, judging the staining degree of nucleated cells observed under a low power microscope, directly washing by using water flow after good staining is observed under the low power microscope, and paying attention not to pour out human color solution firstly and then washing;
4. the staining effect was observed under a microscope, as shown in FIG. 2.
As shown in FIG. 1 and FIG. 2, in the test results of the smear stained by the BasoWright-Giemsa staining solution in the comparative example and the smear stained by the staining solution in the embodiment of the present invention, the obtained Wright-Giemsa staining solution in the embodiment of the present invention can show clear cell staining effect, the cytoplasm, the nucleus, the granule, etc. of the cell can obtain satisfactory staining effect, and the stained cell has good resolution; the invention has simple formula and low cost, and is very favorable for the popularization of clinical examination laboratories. The Wright-Giemsa dye liquor provided by the embodiment of the invention has short dyeing time.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. The Wright-Giemsa dyeing reagent is characterized by being formed by mixing a first Wright-Giemsa dyeing liquid and a second Wright-Giemsa dyeing liquid, wherein the second Wright-Giemsa dyeing liquid comprises the following components in parts by mass, 10-20 parts of Wright dye, 1-5 parts of Giemsa dye and 5000 parts of methanol 4000-.
2. The Wright-Giemsa staining reagent of claim 1,
the second Wright-Giemsa stain also includes glycerin.
3. The Wright-Giemsa staining reagent of claim 2,
the second Wright-Giemsa dye solution comprises the following components, by mass, 12-15 parts of Wright dye, 2-4 parts of Giemsa dye, 1-5 parts of glycerol and 4800 parts of methanol 4500-.
4. The Wright-Giemsa staining reagent of claim 3,
the second Wright-Giemsa dye solution comprises the following components, by mass, 10 parts of Wright dye, 2 parts of Giemsa dye, 3 parts of glycerol and 5000 parts of methanol.
5. The Wright-Giemsa staining reagent of claim 1,
the first Wright-Giemsa dye liquor is a finished product Wright-Giemsa dye liquor.
6. The Wright-Giemsa staining reagent of claim 1,
the mixing ratio of the first Wright-Giemsa dye liquor to the second Wright-Giemsa dye liquor is 1: 2.
7. a method of preparing the Wright-Giemsa staining reagent of claim 1,
first, preparing a second Wright-Giemsa dye solution:
taking Wright dye, Giemsa dye, methanol and glycerol according to the mixture ratio;
crushing the Wright dye and the Giemsa dye to form mixture powder, adding glycerol until no powder particles precipitate to form a mixed solution, and standing for 12-16 hours;
adding methanol into the mixed solution, and grinding for several times until the methanol is used up to form a second Wright-Giemsa dye solution;
and mixing the first Wright-Giemsa dye solution and the second Wright-Giemsa dye solution to form the Wright-Giemsa dye reagent.
8. The Wright-Giemsa staining reagent of claim 7,
the second Wright-Giemsa stain was shaken daily for 1-3 min.
9. The Wright-Giemsa staining reagent of claim 7,
and after the mixed solution is kept stand for 12 to 16 hours, grinding for 20 to 80 minutes.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050003471A1 (en) * | 2003-07-03 | 2005-01-06 | Wang Fu-Sheng | Methods of detecting megakaryocytes |
| CN105300772A (en) * | 2015-09-30 | 2016-02-03 | 成都华西海圻医药科技有限公司 | Wright-Giemsa compound staining solution and preparation method thereof |
| CN108414327A (en) * | 2017-10-31 | 2018-08-17 | 天津协和华美医学诊断技术有限公司 | A kind of Wright-Giemsa staining reagents and its application 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 |
-
2019
- 2019-11-04 CN CN201911067690.1A patent/CN110907254B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050003471A1 (en) * | 2003-07-03 | 2005-01-06 | Wang Fu-Sheng | Methods of detecting megakaryocytes |
| CN105300772A (en) * | 2015-09-30 | 2016-02-03 | 成都华西海圻医药科技有限公司 | Wright-Giemsa compound staining solution and preparation method thereof |
| CN108414327A (en) * | 2017-10-31 | 2018-08-17 | 天津协和华美医学诊断技术有限公司 | A kind of Wright-Giemsa staining reagents and its application 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 |
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| Title |
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