CN108414327A - A kind of Wright-Giemsa staining reagents and its application method - Google Patents
A kind of Wright-Giemsa staining reagents and its application method Download PDFInfo
- Publication number
- CN108414327A CN108414327A CN201711049078.2A CN201711049078A CN108414327A CN 108414327 A CN108414327 A CN 108414327A CN 201711049078 A CN201711049078 A CN 201711049078A CN 108414327 A CN108414327 A CN 108414327A
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- CN
- China
- Prior art keywords
- mother liquor
- wright
- application method
- giemsa
- dyestuffs
- 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 32
- 239000012128 staining reagent Substances 0.000 title claims abstract description 25
- 238000010817 Wright-Giemsa staining Methods 0.000 title claims abstract description 17
- 210000004027 cell Anatomy 0.000 claims abstract description 17
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 14
- 239000012530 fluid Substances 0.000 claims abstract description 10
- 239000012620 biological material Substances 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000004040 coloring Methods 0.000 claims abstract description 6
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims abstract description 5
- 210000001185 bone marrow Anatomy 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 51
- 239000012452 mother liquor Substances 0.000 claims description 36
- 239000003153 chemical reaction reagent Substances 0.000 claims description 12
- 239000012224 working solution Substances 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000004570 mortar (masonry) Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 238000007664 blowing Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000010422 painting Methods 0.000 claims description 2
- 238000002738 Giemsa staining Methods 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 210000005259 peripheral blood Anatomy 0.000 claims 1
- 239000011886 peripheral blood Substances 0.000 claims 1
- 239000003086 colorant Substances 0.000 abstract description 9
- 238000004043 dyeing Methods 0.000 abstract description 7
- 238000002360 preparation method Methods 0.000 abstract description 6
- 210000004976 peripheral blood cell Anatomy 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 238000010186 staining Methods 0.000 abstract description 2
- 239000000975 dye Substances 0.000 description 10
- 239000000835 fiber Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- SJEYSFABYSGQBG-UHFFFAOYSA-M Patent blue Chemical compound [Na+].C1=CC(N(CC)CC)=CC=C1C(C=1C(=CC(=CC=1)S([O-])(=O)=O)S([O-])(=O)=O)=C1C=CC(=[N+](CC)CC)C=C1 SJEYSFABYSGQBG-UHFFFAOYSA-M 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000000981 basic dye Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000004885 white matter Anatomy 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
- 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
Landscapes
- 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 present invention provides a kind of Wright Giemsa staining reagents and its application methods, the technical solution first characterizes staining reagent from preparation method angle, the preparation method is simple and convenient, can be completed with the short period, and gained coloring agent has the longer term of validity under specific Conservation environment.On this basis, the present invention has carried out brand-new design around it to the colouring method of biological material smear, this method first has the coloring agent of 0.25mL to smear staining 30s, and 1mL phosphate buffers are then added dropwise again and continue to keep 20min, is most dried after clear water is cleaned afterwards and completes dyeing.Coloring agent preparation of the present invention is simple, dyeing time is short, excellent effect, various types of cells are easy to differentiate, is primarily adapted for use in the chromoscopy of the samples smears such as bone marrow fluid cell, peripheral blood cells, Pleural-peritoneal fluid cell and cerebrospinal fluid cell.
Description
Technical field
The present invention relates to biological reagent technical fields, further to the preparation and application of biomaterial coloring agent, tool
Body is related to a kind of Wright-Giemsa staining reagents and its application method.
Background technology
Since commercial reagent coloring is not good enough instantly, so as to cause to make a fault to cell classification or interpretation, because
This is formulated it and dyeing time makes corresponding adjustment.Wright dyestuffs are by acid dyes Yihong and basic dye methylenum careuleum group
At, it is good to colorings such as cytoplasmic components and neutrophil granules, but Wright dyestuffs to nuclear targeting not as good as Giemsa contaminate
Material.Giemsa dyestuffs are made of reddish black and Yihong, are worked well to nuclear targeting.On the one hand the effect of methanol can make Yihong
It is dissociated into the E- of ionic condition, methylenum careuleum is made to be dissociated into the M+ of ionic condition；On the other hand it can play the role of fixed cell.
Wright-Giemsa dye liquors have chemical affinity interaction, intracellular Multiple components to belong to egg the existing physical absorption of cell dyeing again
White matter class and nucleic acid material are ampholytes, depending on the electrically charged pH value with solution, phosphate buffer is used for
PH value necessary to cell color is adjusted, ideal observing effect only can be just dyed under most suitable pH condition.
Invention content
The present invention is directed to the technological deficiency for the prior art, a kind of Wright-Giemsa staining reagents are provided and its are made
With method, it is used for the samples such as marrow liquid cell, peripheral blood cells, Pleural-peritoneal fluid cell, cerebrospinal fluid cell in the prior art with solution and applies
The ineffective technical problem of the staining reagent of piece.
Another technical problem to be solved by the present invention is that in the prior art related coloring agent when in use needed for dyeing when
Between it is longer.
The invention solves another technical problem be related coloring agent in the prior art preparation method it is cumbersome.
To realize that the above technical purpose, the present invention use following technical scheme：
A kind of Wright-Giemsa staining reagents, the staining reagent are prepared by the following method：
1) with weight ratio for 5:1 ratio mixes Wright dyestuffs and Giemsa dyestuffs, with Wright dyestuffs and methanol
Amount ratio is 1:500(g:ML ratio) takes methanol for use；
2) mixture of Wright dyestuffs and Giemsa dyestuffs is ground to powdery in mortar；
3) it takes the 1/10 of methanol total volume to be added into powder to continue to grind 20min, the dye liquor dissolved is poured into palm fibre
In color reagent bottle, undissolved solid content is obtained；
4) step 3) is repeated until solid content all dissolvings, the methanol of surplus is all added to brown reagent bottle；
5) sealing is kept in dark place 1 month under room temperature.
Preferably, the term of validity of the Wright-Giemsa staining reagents is 5 years.
A kind of application method of above-mentioned Wright-Giemsa staining reagents, includes the following steps：To the smear of biological material
Phosphate buffer working solution 1mL is added dropwise after 30s, blows mixing for Wright-Giemsa dye liquors described in upper dropwise addition 0.25mL,
Smear is rinsed with circulating water after 20min, is dried.
Preferably, the biological material is bone marrow fluid cell, peripheral blood cells, Pleural-peritoneal fluid cell or cerebrospinal fluid cell.
Preferably, in the colouring method, smear is completed on dye horse.
Preferably, the mixing of blowing is completed with ear washing bulb.
Preferably, the phosphate buffer working solution is mixed to get by mother liquor A below and mother liquor B, it is described
Mother liquor A is the KH of a concentration of 1% (g/mL)2PO4Aqueous solution；The mother liquor B is the Na of a concentration of 1% (g/mL)2HPO4It is water-soluble
Liquid.
Preferably, the mother liquor A and mother liquor B using it is preceding respectively in brown reagent bottle, 4 DEG C under the conditions of seal and keep away
Light preserves.
Preferably, the term of validity of the mother liquor A and mother liquor B is 6 months.
Preferably, the phosphate buffer working solution obtains by the following method：By the mother liquor A and mother liquor
B is with 3:2(mL:ML ratio mixing), choose A volumes 100/3 is used as final volume, with water by the mixture of mother liquor A and mother liquor B
It is settled to the final volume.
Preferably, the pH of the phosphate buffer working solution is 6.8.
Preferably, the phosphate buffer working solution is valid for three months.
The present invention provides a kind of Wright-Giemsa staining reagents and its application method, the technical solution first from
Method angle processed characterizes staining reagent, and the preparation method is simple and convenient, can be completed with the short period, gained coloring agent
There is the longer term of validity under specific Conservation environment.On this basis, the present invention surrounds its dye to biological material smear
Color method has carried out brand-new design, and this method first has the coloring agent of 0.25mL to smear staining 30s, 1mL phosphoric acid is then added dropwise again
Buffer solution continues to keep 20min, is most dried after clear water is cleaned afterwards and completes dyeing.Coloring agent of the present invention prepares simple, dyeing
Time is short, excellent effect, various types of cells are easy to differentiate, be primarily adapted for use in bone marrow fluid cell, peripheral blood cells, Pleural-peritoneal fluid cell and
The chromoscopy of the samples smear such as cerebrospinal fluid cell.
Specific implementation mode
The specific implementation mode of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.In addition to being defined, institute in following embodiment
Technical and scientific term has the identical meanings being commonly understood by with those skilled in the art of the invention.
Embodiment 1
A kind of Wright-Giemsa staining reagents, the staining reagent are prepared by the following method：
1) with weight ratio for 5:1 ratio mixes Wright dyestuffs and Giemsa dyestuffs, with Wright dyestuffs and methanol
Amount ratio is 1:500(g:ML ratio) takes methanol for use；
2) mixture of Wright dyestuffs and Giemsa dyestuffs is ground to powdery in mortar；
3) it takes the 1/10 of methanol total volume to be added into powder to continue to grind 20min, the dye liquor dissolved is poured into palm fibre
In color reagent bottle, undissolved solid content is obtained；
4) step 3) is repeated until solid content all dissolvings, the methanol of surplus is all added to brown reagent bottle；
5) sealing is kept in dark place 1 month under room temperature.
Wherein, the term of validity of the Wright-Giemsa staining reagents is 5 years.
A kind of application method for stating Wright-Giemsa staining reagents, includes the following steps：To the painting on piece of biological material
Wright-Giemsa dye liquors described in 0.25mL are added dropwise, phosphate buffer working solution 1mL is added dropwise after 30s, blows mixing, 20min
Smear is rinsed with circulating water afterwards, is dried.
Wherein, the biological material is peripheral blood cells.
In the colouring method, smear is completed on dye horse.
The mixing of blowing is completed with ear washing bulb.
The phosphate buffer working solution is mixed to get by mother liquor A below and mother liquor B, and the mother liquor A is dense
Degree is the KH of 1% (g/mL)2PO4Aqueous solution；The mother liquor B is the Na of a concentration of 1% (g/mL)2HPO4Aqueous solution.
The mother liquor A and mother liquor B using it is preceding respectively in brown reagent bottle, 4 DEG C under the conditions of seal and be kept in dark place.
The term of validity of the mother liquor A and mother liquor B is 6 months.
The phosphate buffer working solution obtains by the following method：By the mother liquor A and mother liquor B with 3:2
(mL:ML ratio mixing), choose A volumes 100/3 are used as final volume, are settled to the mixture of mother liquor A and mother liquor B with water
The final volume.
Embodiment 2
A kind of Wright-Giemsa staining reagents, the staining reagent are prepared by the following method：
1) with weight ratio for 5:1 ratio mixes Wright dyestuffs and Giemsa dyestuffs, with Wright dyestuffs and methanol
Amount ratio is 1:500(g:ML ratio) takes methanol for use；
2) mixture of Wright dyestuffs and Giemsa dyestuffs is ground to powdery in mortar；
3) it takes the 1/10 of methanol total volume to be added into powder to continue to grind 20min, the dye liquor dissolved is poured into palm fibre
In color reagent bottle, undissolved solid content is obtained；
4) step 3) is repeated until solid content all dissolvings, the methanol of surplus is all added to brown reagent bottle；
5) sealing is kept in dark place 1 month under room temperature.
A kind of application method for stating Wright-Giemsa staining reagents, includes the following steps：To the smear of Pleural-peritoneal fluid cell
Phosphate buffer working solution 1mL is added dropwise after 30s, blows mixing for Wright-Giemsa dye liquors described in upper dropwise addition 0.25mL,
Smear is rinsed with circulating water after 20min, is dried.
The embodiments of the present invention have been described in detail above, but content is only the preferred embodiment of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement etc. done in the application range of the present invention, should all
It is included within protection scope of the present invention.
Claims (10)
1. a kind of Wright-Giemsa staining reagents, it is characterised in that the staining reagent is prepared by the following method：
1) with weight ratio for 5:1 ratio mixes Wright dyestuffs and Giemsa dyestuffs, with Wright dyestuffs and methanol usage
Than being 1:500(g:ML ratio) takes methanol for use；
2) mixture of Wright dyestuffs and Giemsa dyestuffs is ground to powdery in mortar；
3) it takes the 1/10 of methanol total volume to be added into powder to continue to grind 20min, the dye liquor dissolved is poured into brown examination
In agent bottle, undissolved solid content is obtained；
4) step 3) is repeated until solid content all dissolvings, the methanol of surplus is all added to brown reagent bottle；
5) sealing is kept in dark place 1 month under room temperature.
2. a kind of Wright-Giemsa staining reagents according to claim 1, it is characterised in that the Wright-
The term of validity of Giemsa staining reagents is 5 years.
3. the application method of Wright-Giemsa staining reagents described in a kind of claim 1, it is characterised in that including following step
Suddenly：Wright-Giemsa dye liquors described in 0.25mL are added dropwise to the painting on piece of biological material, phosphate buffer work is added dropwise after 30s
Make liquid 1mL, blow mixing, rinses smear with circulating water after 20min, dry.
4. application method according to claim 3, it is characterised in that the biological material is that bone marrow fluid cell, peripheral blood are thin
Born of the same parents, Pleural-peritoneal fluid cell or cerebrospinal fluid cell.
5. application method according to claim 3, it is characterised in that in the colouring method, smear is complete on dye horse
At.
6. application method according to claim 3, it is characterised in that the mixing of blowing is completed with ear washing bulb.
7. application method according to claim 3, it is characterised in that the phosphate buffer working solution is by below
What mother liquor A and mother liquor B was mixed to get, the mother liquor A is the KH of a concentration of 1% (g/mL)2PO4Aqueous solution；The mother liquor B is dense
Degree is the Na of 1% (g/mL)2HPO4Aqueous solution.
8. application method according to claim 7, it is characterised in that the mother liquor A and mother liquor B using before respectively in
In brown reagent bottle, seals and be kept in dark place under the conditions of 4 DEG C.
9. application method according to claim 8, it is characterised in that the term of validity of the mother liquor A and mother liquor B is 6
Month.
10. application method according to claim 7, it is characterised in that the phosphate buffer working solution is by following
What method obtained：By the mother liquor A and mother liquor B with 3:2(mL:ML ratio mixing) chooses the 100/3 of A volumes and is used as whole body
Product, the final volume is settled to water by the mixture of mother liquor A and mother liquor B.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711049078.2A CN108414327A (en) | 2017-10-31 | 2017-10-31 | A kind of Wright-Giemsa staining reagents and its application method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711049078.2A CN108414327A (en) | 2017-10-31 | 2017-10-31 | A kind of Wright-Giemsa staining reagents and its application method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108414327A true CN108414327A (en) | 2018-08-17 |
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ID=63125270
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711049078.2A Pending CN108414327A (en) | 2017-10-31 | 2017-10-31 | A kind of Wright-Giemsa staining reagents and its application method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110907254A (en) * | 2019-11-04 | 2020-03-24 | 成都市第六人民医院 | Wright-Giemsa dyeing reagent and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105842037A (en) * | 2016-03-21 | 2016-08-10 | 山东农业大学 | Staining method for simultaneously displaying mast cells and acidophilic cells |
| CN107300495A (en) * | 2017-06-15 | 2017-10-27 | 托克逊县夏乡畜牧业服务中心 | A kind of colouring method of pyriform worm blood film compound stain solution and blood film |
-
2017
- 2017-10-31 CN CN201711049078.2A patent/CN108414327A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN105842037A (en) * | 2016-03-21 | 2016-08-10 | 山东农业大学 | Staining method for simultaneously displaying mast cells and acidophilic cells |
| CN107300495A (en) * | 2017-06-15 | 2017-10-27 | 托克逊县夏乡畜牧业服务中心 | A kind of colouring method of pyriform worm blood film compound stain solution and blood film |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110907254A (en) * | 2019-11-04 | 2020-03-24 | 成都市第六人民医院 | Wright-Giemsa dyeing reagent and preparation method thereof |
| CN110907254B (en) * | 2019-11-04 | 2022-10-11 | 成都市第六人民医院 | Wright-Giemsa dyeing reagent and preparation method thereof |
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Application publication date: 20180817 |