CN101135616A - Humoral biological cell test-tube internally dyeing method - Google Patents
Humoral biological cell test-tube internally dyeing method Download PDFInfo
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- CN101135616A CN101135616A CNA2007100468967A CN200710046896A CN101135616A CN 101135616 A CN101135616 A CN 101135616A CN A2007100468967 A CNA2007100468967 A CN A2007100468967A CN 200710046896 A CN200710046896 A CN 200710046896A CN 101135616 A CN101135616 A CN 101135616A
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- methylene blue
- dyeing
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Abstract
The method features the following: 1) preparing the Wright's staining liquid; 2) preparing the phosphate buffer solution with pH6.4-6.8; 3) preparing alkali methylene blue liquid; 4) preparing the test tube staining count liquid; 5) staining count; 6) sort check.
Description
[technical field]
The present invention relates to field of biomedicine technology, a kind of specifically humoral biological cell test-tube internally dyeing method.
[background technology]
Body fluid biological cell dyeing, tradition is used slide method, and is cumbersome, and is subjected to dyeing time, is coated with the influence of tablet quality, usually is not that to dye be exactly to dye shallowly deeply, causes the difficulty in the eucaryotic cell structure identification, can not be in time correct obtain the result.
[summary of the invention]
Purpose of the present invention overcomes the deficiencies in the prior art exactly, and a kind of humoral biological cell test-tube internally dyeing method that provides.
For achieving the above object, design a kind of humoral biological cell test-tube internally dyeing method, it is characterized in that: (1) preparation Wright's staining liquid: get methylene blue Yihong dyestuff 1 gram, after adding 3-5 ml methanol or glycerine and in mortar, grinding evenly, pour in the graduated cylinder, pour in the lump in the graduated cylinder with the washed with methanol mortar, adding methyl alcohol to the amount of graduated cylinder at last is 600 milliliters, behind the mixing, airtight preservation is standby; (2) phosphate buffer of preparation PH6.4-6.8; (3) the alkaline methylene blue dyeing liquor of preparation: get 30 milliliters of methylene blue ethanol saturated solutions, 0.1 milliliter of mixing of 100 milliliters of adding distil waters and 10% potassium hydroxide aqueous solution; (4) preparation test tube dyeing counting liquid: get 10 milliliters of Wright's staining liquid 20ml and alkaline methylene blue dyeing liquors, mix, filter, be sub-packed in the Kang Shi pipe, 0.1 milliliter of every pipe is standby; (5) dyeing counting: get the Kang Shi that contains 0.1 milliliter of test tube dyeing counting liquid and manage, 0.1 milliliter of the phosphate buffer of adding PH6.4-6.8, add 0.2 milliliter of detected body fluid again, mixing dyeing 5-10 minute, charge into after counting chamber treats that cell sinks, red, the leucocyte 5 big lattice sums of counting multiply by 4 and obtain every cubic millimeter of cell number under low power lens; (6) sort check: adopt the high power lens pair cell to carry out Direct Classification.The phosphate buffer of described preparation PH6.4-6.8 is for getting 20 milliliter of 1% sodium hydrogen phosphate Na
2HPO
4With 30 milliliter of 1% potassium dihydrogen phosphate KH
2PO
4, add water to 1000 milliliters after the mixing.Described methylene blue ethanol saturated solution adds 50 milliliters of mixings of 95% ethanol for getting methylene blue 3-5 gram, adds the dissolving of 2-3 gram methylene blue mixing again, and after adding methylene blue, methylene blue no longer dissolves, have methylene blue to be deposited in bottle at the bottom of, promptly make methylene blue ethanol saturated solution.Described humoral biological cell test-tube internally dyeing method is applied to the cytoscopy of clinical ascites pleural fluid or joint fluid or irrigating solution or cerebrospinal fluid.
The present invention compared with prior art, removed the step of the centrifugal smear staining of classic method from, and can make suit, join a pipe dye liquor, empty test tube, one towards the little glass rod of liquid, several the soft paper of wiping the counting version, allow and use promptly totally again soon, have fast, advantage accurately, and reagent is simple, can be shared with normal dyeing reagent, do not need valuable specific apparatus, be fit to use under hospitals at different levels and the combat condition out of office.
[specific embodiment]
The present invention is further illustrated below in conjunction with example.
Embodiment:
One, preparation Wright's staining liquid, i.e. methylene blue Yihong dyeing liquor:
Get methylene blue Yihong dyestuff 1 gram, add 3-5 ml methanol or glycerine and grinds in mortar evenly afterwards to going in 1000 milliliters of graduated cylinders, in the lump to going in the graduated cylinder, add methyl alcohol to 600 milliliter with the washed with methanol mortar at last, behind the mixing, airtight preservation is standby.
Two, preparation PH6.4～6.8 phosphate buffers:
Get 1% sodium hydrogen phosphate Na
2HPO
420 milliliters, 1% potassium dihydrogen phosphate KH
2PO
430 milliliters, after two liquid mix, adding distil water to 1000 milliliter.
Three, the alkaline methylene blue dyeing liquor of preparation
Get methylene blue 3-5 gram, add 50 milliliters of mixings of 95% ethanol, add the dissolving of 2-3 gram methylene blue mixing again, after adding methylene blue, methylene blue no longer dissolves, have methylene blue to be deposited in bottle at the bottom of, promptly make methylene blue ethanol saturated solution, get 30 milliliters of methylene blue ethanol saturated solutions, 0.1 milliliter of 100 milliliters of adding distil waters and 10% potassium hydroxide aqueous solution, mixing is an alkalize methylene blue dyeing liquor.
Four, prepare dyeing counting liquid in vitro
Get 20 milliliters of Wright's staining liquid, 10 milliliters of alkaline methylene blue dyeing liquors mix, filter, and are sub-packed in the Kang Shi pipe, and 0.1 milliliter of every pipe is standby.
Five, dyeing counting:
Get and contain 0.1 milliliter of one of Kang Shi pipe of dyeing counting liquid in vitro, 0.1 milliliter of the phosphate buffer of adding PH6.4-6.8,0.2 milliliter of detected body fluid, mixing dyeing 5-10 minute, charge into after counting chamber treats that cell sinks, red, the leucocyte 5 big lattice sums of counting take advantage of 4 to obtain every cubic millimeter of cell number under low power lens, and sum is taken advantage of diluted 2 times of 4 expression samples, count 5 big lattice and are converted into 1 cubic metre and should take advantage of 2.
Six, sort check:
Since in the peritoneal effluent when coelomic fluid and CAPD mainly based on neutrophil leucocyte, Yihong cell, lymphocyte, the mesothelial cell is rare or lose, therefore can in counting chamber, directly do classification, promptly counting finishes, conversion high power lens Direct Classification, eucaryotic cell structure is clear, differentiates easily, adopt this colouring method, the features of shape of cell under high power lens is:
Red blood cell: significantly yellow positive circle or the slightly light plate-like that is in center;
Lymphocyte: nuclear is circular, dyes dark blue purple, and slurry dyes pale blue less, and caryoplasm circle is obvious;
Neutrophil cell: nuclear is band shape or leaflet, dyes dark blue purple, and caryoplasm circle is obvious, and it is faint yellow that endochylema is, and fine particle as seen;
Eosinophil: nuclear dyes mulberry with leaflet neutrality, endochylema and particle;
Mesothelial cell's (regular pattern composite): nuclear circle or oval, off normal is dyed deep blue purple, and slurry enriches pale blue;
When seeing that a large amount of mutants or cellular morphology are irregular, can be with the residue specimen centrifuge, the taking precipitate smear that in vitro dye, under oily mirror, classify and observe, still efficient and convenient than classic method.
As courage and uprightness or the too high sample of leukocyte count, can dilute behind the sample dyeing counting as stated above as one sees fit, the result should take advantage of extension rate, and the degenerative cell is subjected to look bad, but the proper extension dyeing time.
Claims (4)
1. humoral biological cell test-tube internally dyeing method, it is characterized in that: (1) preparation Wright's staining liquid: get methylene blue Yihong dyestuff 1 gram, after adding 3-5 ml methanol or glycerine and in mortar, grinding evenly, pour in the graduated cylinder, pour in the graduated cylinder in the lump with the washed with methanol mortar again, adding methyl alcohol to the amount of graduated cylinder at last is 600 milliliters, and behind the mixing, airtight preservation is standby; (2) phosphate buffer of preparation PH6.4-6.8; (3) the alkaline methylene blue dyeing liquor of preparation: get 30 milliliters of methylene blue ethanol saturated solutions, 0.1 milliliter of mixing of 100 milliliters of adding distil waters and 10% potassium hydroxide aqueous solution; (4) dyeing counting liquid in the preparation examination: get 10 milliliters of Wright's staining liquid 20ml and alkaline methylene blue dyeing liquors, mix, filter, be sub-packed in the Kang Shi pipe, 0.1 milliliter of every pipe is standby; (5) dyeing counting: get and contain 0.1 milliliter of one of Kang Shi pipe of dyeing counting liquid in vitro, 0.1 milliliter of the phosphate buffer of adding PH6.4-6.8, add 0.2 milliliter of detected body fluid again, mixing dyeing 5-10 minute, charge into after counting chamber treats that cell sinks, red, the leucocyte 5 big lattice sums of counting take advantage of 4 to obtain every cubic millimeter of cell number under low power lens; (6) sort check: adopt the high power lens pair cell to carry out Direct Classification.
2. a kind of humoral biological cell test-tube internally dyeing method as claimed in claim 1 is characterized in that: the phosphate buffer of described preparation PH6.4-6.8 is for getting 20 milliliter of 1% sodium hydrogen phosphate Na
2HPO
4With 30 milliliter of 1% potassium dihydrogen phosphate KH
2PO
4, mix back adding distil water to 1000 milliliter.
3. the described a kind of humoral biological cell test-tube internally dyeing method of claim 1, it is characterized in that: described methylene blue ethanol saturated solution is for getting methylene blue 3-5 gram, add 50 milliliters of mixings of 95% ethanol, add the dissolving of 2-3 gram methylene blue mixing again, after adding methylene blue, methylene blue no longer dissolves, have methylene blue to be deposited in bottle at the bottom of, promptly make methylene blue ethanol saturated solution.
4. a kind of humoral biological cell test-tube internally dyeing method as claimed in claim 1 is characterized in that: described humoral biological cell test-tube internally dyeing method is applied to the cytoscopy of clinical ascites pleural fluid or joint fluid or irrigating solution or cerebrospinal fluid.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100468967A CN101135616A (en) | 2007-10-10 | 2007-10-10 | Humoral biological cell test-tube internally dyeing method |
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| CNA2007100468967A CN101135616A (en) | 2007-10-10 | 2007-10-10 | Humoral biological cell test-tube internally dyeing method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103323313A (en) * | 2012-03-23 | 2013-09-25 | 黄伏生 | Liquid-based cell analyzer staining method of cells exfoliated from serous cavity by using Wright staining method |
-
2007
- 2007-10-10 CN CNA2007100468967A patent/CN101135616A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103323313A (en) * | 2012-03-23 | 2013-09-25 | 黄伏生 | Liquid-based cell analyzer staining method of cells exfoliated from serous cavity by using Wright staining method |
| CN103323313B (en) * | 2012-03-23 | 2016-06-29 | 黄伏生 | Rui Shi method is for the serous cavity exfoliative cyte colouring method of liquid basal cell instrument |
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Open date: 20080305 |