CN104568551A - Acid resistant staining method - Google Patents
Acid resistant staining method Download PDFInfo
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- CN104568551A CN104568551A CN201310495973.2A CN201310495973A CN104568551A CN 104568551 A CN104568551 A CN 104568551A CN 201310495973 A CN201310495973 A CN 201310495973A CN 104568551 A CN104568551 A CN 104568551A
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Abstract
The invention relates to an acid resistant staining method. The method is characterized by comprising the following steps: (1), putting a material smear to be detected on a slide, and drying and fixing the material smear to be detected so as to obtain a sample; (2), dripping primary stain to cover the sample, and after staining for 1-2 minutes, removing the primary stain; (3), dripping decolorizing counterstain to cover the sample, after staining for 25-35 seconds, washing the sample by water, and performing microscopic examination after drying, wherein the primary stain is a solution comprising 1.0-2.0 weight percent of basic fuchsin and 5-7 weight percent of phenol, and the decolorizing counterstain is a methylene blue solution of 0.6-0.8 weight percent. Compared with the prior art, the method disclosed by the invention has the advantages that heating is selectable in staining; the method can be used for manual and automatic staining, and is beneficial to the standardization and the automation of staining; the method disclosed by the invention is reliable, and the result is distinct and is easy to observe and judge.
Description
Technical field
The present invention relates to a kind of Bacterial stain method, especially relate to a kind of Ziehi-Neelsen stain.
Background technology
Ziehi-Neelsen stain (acid-fast staining method) is initiated and the bacterial stain improving through F. Qi Er (Ziehl) and create by Paul Ehrlich (F.Ehrlich) for 1882.Wherein most is representational is Qi Er-Nelson (Ziehl-Neelsen) decoration method to tulase and Qi Er-Jia Beite (Ziehl-Gabbet) decoration method.After carbolfuchsin dyeing, with acidic alcohol decolouring, then carry out counterstain with methylene blue.
The principle of acid-fast stain is as follows: containing a large amount of lipids in the cell membrane of mycobacterium (containing tulase), be enclosed in the outside of peptide glycan, so the general not easy coloring of mycobacterium, it be impelled painted through heating and extend dyeing time.But after the mycolic acid in mycobacterium is combined with dyestuff, be just difficult to by acid bleaching agent bleaching, therefore named acid-fast stain.
Qi Er-Nelson's Ziehi-Neelsen stain makes mycolic acid become compound with carbolfuchsin strong bonded in a heated condition, with hydrochloride alcohol process also nondiscoloration.When adding after alkaline methylene blue redyes again, mycobacterium is still red, and the material in other bacteriums and background be blueness.
Acid-fast stain general step is as follows:
1) just contaminate: clamp smear preparation with glass slide clamp, drip carbolfuchsin 2-3 and drip, in the heating slowly of flame eminence, be sure not boiling, there is steam and away from keyboard, if dye liquor evaporation reduces, dye liquor should be added again, in order to avoid dry, heat 5 minutes, rinse with water after sample cooling.
2) decolour: 3% hydrochloride alcohol decolours 3 minutes; Rinse with water.
3) redye: redye 30 seconds with alkaline methylene blue solution, washing, blot with thieving paper and use oily sem observation afterwards.
Being formulated as follows of dye liquor in above-mentioned acid-fast stain process:
1, carbolfuchsin: basic fuchsin alcoholization solution 10mL and 5% carbolic acid 90mL mixes;
2,3% hydrochloride alcohol: concentrated hydrochloric acid 3mL mixes with 95% alcohol 97mL;
3, Lv Shi methylene blue liquid: methylene blue alcoholization liquid 30mL mixes with potassium hydroxide (1:10000) 100mL.
Being more than conventional acid-fast stain step, is three steps dyeing and three washings, it operates more complicated, waste water and the time long.Modern Laboratory is busy with one's work high with cost, every technical method is had to the requirement made every effort to easy and save, to complete more task within limited time and cost.
Chinese patent CN101126689A discloses a kind of gram stain two-step method, the method gets clean microslide, get from kit 10 ~ 30ML (1 droplet) just stain (A agent) be added drop-wise on microslide, get again about with the homogenize material smear to be checked of dye liquor equivalent, after 15 seconds, smear need not be done, the counterstain (B agent) dripping 30 ~ 60ML (2 droplet) immediately covers sample, about 15 seconds, washing, microscopy after dry.At smear place, the gram positive bacteria dyed ruddy gram-negative bacteria He dye darkviolet can be seen, with distinct contrast.Compared with prior art, the present invention have easy and simple to handle, method is reliable, cost performance good, result is easy to observe and judge and the advantage such as free from environmental pollution.But Grain stain and acid-fast stain are two kinds of different decoration methods on bacteriology, its purposes and practical significance different, Gram staining is little for the reference significance of antibacterial decoration method.
Summary of the invention
Object of the present invention be exactly provide that a kind of staining procedure is simple, dyeing time is short to overcome defect that above-mentioned prior art exists, dye the Ziehi-Neelsen stain that can not heat.
Object of the present invention can be achieved through the following technical solutions:
A kind of Ziehi-Neelsen stain, comprises the following steps:
(1) clean slide is got, by material smear to be checked on slide, and dry, fixing, obtain sample;
(2) drip just stain and cover sample, dye 1 ~ 2 minute, can heat and not heat, incline just stain, do not wash;
(3) drip decolouring counterstain, cover sample, dye 25 ~ 35 seconds, washing, microscopy after dry.
Further, described first stain is the solution containing the basic fuchsin of 1.0 ~ 2.0wt% and the phenol of 5 ~ 7wt%.
Further, described first stain is the solution containing the basic fuchsin of 1.6wt% and the phenol of 6.4wt%.
Further, described solution to be distilled water, acetone or volume fraction be 95% ethanol.
Further, described decolouring counterstain is the methylene blue solution of 0.6 ~ 0.8wt%.
Closer, described decolouring counterstain is the methylene blue solution of 0.8wt%.
Further, described material to be checked comprises bacterial cultures, censorship secretion or body fluid.
Further, the dripping quantity of described first stain is 1 ~ 3ml, and the dripping quantity of described decolouring counterstain is 1 ~ 3ml.
Further, in step (3) after microscopy, dying red material to be checked is antiacid positive bacteria, and dying blue material to be checked is antiacid negative bacterium, with distinct contrast.
Compared with prior art, the present invention has the following advantages and beneficial effect:
(1) Ziehi-Neelsen stain of the present invention, is only with two kinds of dyeing liquors with only need two steps dye and once wash, can accomplishes the dyeing of fast and convenient water saving.And the acid-fast stain three-step approach of classics, dye liquor has first dye liquor, destainer and redyes liquid three kinds, and dyeing needs three step dyeing, three washings and heats, and dyeing time 8 minutes 30 seconds, whole process wants about 10 minutes.Therefore method of the present invention is than Qi Er-Nelson's decoration method of often returning use, and dyeing time saves 60%, and water is saved more than 60% and dye liquor production and packaging logistics cost and respectively reduced by 25%.Method of the present invention heats when dyeing and does not heat; Can be used for artificial and robotization dyeing, be conducive to standards for dyeing and robotization.Method of the present invention is reliable, distinct being easy to of result is observed and judged.
(2) Ziehi-Neelsen stain of the present invention or kit will have important actual application value and good market outlook, be conducive to biology or medical practice, be conducive to commercialization transport, and safe and reliable, there is very large potential market, significantly society and economic benefit can be produced.
(3) method of the present invention be quicker, more convenient, more save, more humane acid-fast stain method.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
Get clean slide and material to be checked (body fluid) requires smear routinely, dry and flame or acetone are fixed.First drip the first stain of 3ml (distilled water solution for containing the basic fuchsin of 1.6wt% and the phenol of 6.4wt%) and cover sample, dye 2 minutes, just tiltedly dye liquor is removed, do not wash, drip 3ml decolouring counterstain (methylene blue solution of 0.8wt%) again and cover sample, dye 30 seconds, washing, microscopy after dry.Can see in smear, dye red for antiacid positive bacteria with what dye blue look is antiacid negative bacterium, with distinct contrast.
Embodiment 2
Get clean slide and material to be checked (censorship secretion) requires smear routinely, dry and flame or acetone are fixed.First drip the first stain of 2ml (acetone soln for containing the basic fuchsin of 1.0wt% and the phenol of 5.4wt%) and cover sample, dye 1 minute, just tiltedly dye liquor is removed, do not wash, drip 2ml decolouring counterstain (methylene blue solution of 0.8wt%) again and cover sample, dye 30 seconds, washing, microscopy after dry.Can see in smear, dye red antiacid positive bacteria and the antiacid negative bacterium dying blue look, with distinct contrast.
Embodiment 3
Get clean slide and material to be checked (bacterial cultures) requires smear routinely, dry and flame or acetone are fixed.First drip the first stain of 1ml (ethanolic solution for containing the basic fuchsin of 2.0wt% and the phenol of 7.0wt%) and cover sample, dye 1.5 minutes, just tiltedly dye liquor is removed, do not wash, drip 1ml decolouring counterstain (methylene blue solution of 0.8wt%) again and cover sample, dye 30 seconds, washing, microscopy after dry.Can see in smear, dye red for antiacid positive bacteria with what dye blue look is antiacid negative bacterium, with distinct contrast.
Embodiment 4
A kind of Ziehi-Neelsen stain, comprises the following steps:
(1) clean slide is got, by material to be checked (bacterial cultures) smear on slide, and dry, fixing, obtain sample;
(2) drip the first stain of 1ml (acetone soln for containing the basic fuchsin of 1.0wt% and the phenol of 5wt%) and cover sample, dye 1 minute, incline just stain, do not wash;
(3) drip 1ml decolouring counterstain (methylene blue solution of 0.6wt%), cover sample, dye 25 seconds, washing, microscopy after dry, after microscopy, dying red material to be checked is antiacid positive bacteria, and dying blue material to be checked is antiacid negative bacterium, with distinct contrast.
Embodiment 5
A kind of Ziehi-Neelsen stain, comprises the following steps:
(1) clean slide is got, by material to be checked (bacterial cultures) smear on slide, and dry, fixing, obtain sample;
(2) drip the first stain of 3ml (aqueous solution for containing the basic fuchsin of 2.0wt% and the phenol of 7wt%) and cover sample, dye 2 minutes, incline just stain, do not wash;
(3) drip 3ml decolouring counterstain (methylene blue solution of 0.7wt%), cover sample, dye 35 seconds, washing, microscopy after dry, after microscopy, dying red material to be checked is antiacid positive bacteria, and dying blue material to be checked is antiacid negative bacterium, with distinct contrast.
Claims (9)
1. a Ziehi-Neelsen stain, is characterized in that, comprises the following steps:
(1) by material smear to be checked on slide, and dry, fixing, obtain sample;
(2) drip just stain and cover sample, dye 1 ~ 2 minute, incline just stain;
(3) drip decolouring counterstain, cover sample, dye 25 ~ 35 seconds, washing, microscopy after dry.
2. a kind of Ziehi-Neelsen stain according to claim 1, is characterized in that, described first stain is the solution containing the basic fuchsin of 1.0 ~ 2.0wt% and the phenol of 5 ~ 7wt%.
3. a kind of Ziehi-Neelsen stain according to claim 2, is characterized in that, described first stain is the solution containing the basic fuchsin of 1.6wt% and the phenol of 6.4wt%.
4. a kind of Ziehi-Neelsen stain according to Claims 2 or 3, is characterized in that, described solution to be distilled water, acetone or volume fraction be 95% ethanol.
5. a kind of Ziehi-Neelsen stain according to claim 1, is characterized in that, described decolouring counterstain is the methylene blue solution of 0.6 ~ 0.8wt%.
6. a kind of Ziehi-Neelsen stain according to claim 5, is characterized in that, described decolouring counterstain is the methylene blue solution of 0.8wt%.
7. a kind of Ziehi-Neelsen stain according to claim 1, is characterized in that, described material to be checked comprises bacterial cultures, censorship secretion or body fluid.
8. a kind of Ziehi-Neelsen stain according to claim 1, is characterized in that, the dripping quantity of described first stain is 1 ~ 3ml, and the dripping quantity of described decolouring counterstain is 1 ~ 3ml.
9. a kind of Ziehi-Neelsen stain according to claim 1, is characterized in that, in step (3) after microscopy, dying red material to be checked is antiacid positive bacteria, and dying blue material to be checked is antiacid negative bacterium, with distinct contrast.
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| CN201310495973.2A CN104568551A (en) | 2013-10-18 | 2013-10-18 | Acid resistant staining method |
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| CN201310495973.2A CN104568551A (en) | 2013-10-18 | 2013-10-18 | Acid resistant staining method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593396A (en) * | 2018-05-04 | 2018-09-28 | 江苏中济万泰生物医药有限公司 | The preparation method and application method of malachite green solution |
| CN108931413A (en) * | 2018-07-23 | 2018-12-04 | 江苏诺鬲生物科技有限公司 | A kind of quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis |
| CN112945680A (en) * | 2021-02-26 | 2021-06-11 | 珠海贝索生物技术有限公司 | Phenol-free acid-fast bacterium staining solution and staining method thereof |
| CN115791346A (en) * | 2022-12-24 | 2023-03-14 | 上饶市广丰区疾病预防控制中心 | Mycobacterium tuberculosis acid-fast staining solution and preparation and staining method thereof |
-
2013
- 2013-10-18 CN CN201310495973.2A patent/CN104568551A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593396A (en) * | 2018-05-04 | 2018-09-28 | 江苏中济万泰生物医药有限公司 | The preparation method and application method of malachite green solution |
| CN108931413A (en) * | 2018-07-23 | 2018-12-04 | 江苏诺鬲生物科技有限公司 | A kind of quick antiacid fluorescent dyeing reagent of modified form mycobacterium tuberculosis |
| CN112945680A (en) * | 2021-02-26 | 2021-06-11 | 珠海贝索生物技术有限公司 | Phenol-free acid-fast bacterium staining solution and staining method thereof |
| CN115791346A (en) * | 2022-12-24 | 2023-03-14 | 上饶市广丰区疾病预防控制中心 | Mycobacterium tuberculosis acid-fast staining solution and preparation and staining method thereof |
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