CN103674661A - Washing-free Gram staining method - Google Patents
Washing-free Gram staining method Download PDFInfo
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- CN103674661A CN103674661A CN201210337417.8A CN201210337417A CN103674661A CN 103674661 A CN103674661 A CN 103674661A CN 201210337417 A CN201210337417 A CN 201210337417A CN 103674661 A CN103674661 A CN 103674661A
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Abstract
The invention provides a washing-free Gram staining method which comprises the following steps: uniformly coating a material to be detected on a slide at room temperature, drying, fixing, dropwise adding a primary stain on a smear, staining, removing the primary stain and dropwise adding an afterstain directly; after staining, removing the afterstain, and performing microscopic examination after drying, wherein Gram positive bacteria stained into purple and Gram-negative bacteria stained into red are positioned in the center of the smear. Compared with a conventional Gram staining method, the washing-free Gram staining method has the advantages that a washing step is saved, the operation steps are simplified, lots of water resources are saved, and the amount of dyes is greatly reduced due to the method. Moreover, the staining time is shortened, the staining efficiency is improved, the dyes are saved by 50 percent, and the environmental pollution is reduced.
Description
Technical field
The present invention relates to a kind of Gram staining, particularly relate to the Grain stain method of not washing.
Background technology
Gram staining is widely used a kind of differential staining in bacteriology, and 1884 Nian You Denmark doctor Gram found.Bacterium is first dyeed through basic-dyeable fibre crystallization, and after the mordant dyeing of iodine liquid, with alcohol, decolours, this look of the bacterium having under certain condition is not divested, and what have is divested, and therefore bacterium can be divided into two large classes, the former is gram-positive bacteria (G+), and the latter is Gram-negative bacteria (G-).In order to observe conveniently, after decolouring, by a kind of orchil red grade as luxuriant in alkalescence, redye again.Positive bacteria is still with purple, and negative bacterium is by red-dyed.The bacillus and the overwhelming majority and the coccus that have gemma, and all actinomyces and fungi are all gram positive reaction; Vibrios, conveyor screw and most of pathogenic bactacins all present negative reaction.
Gram-positive bacteria and Gram-negative bacteria have a lot of difference on chemical composition and physiological property, and staining reaction is different.It is generally acknowledged now that Gram-positive thalline contains special nucleoprotein magnesium salts and the compound of polysaccharide, it is combined very firm with the compound of iodine and crystal violet, is difficult for decolouring, negative bacterium compound combination degree is low, absorbing dye is poor, easily decolouring, and this is the Main Basis of staining reaction.In addition, positive bacteria thalline isoelectric point is low compared with negative bacterium, under identical PH condition, dyes, and positive bacteria absorption basic-dyeable fibre is a lot, is therefore difficult for sloughing, and negative bacterium is contrary.So condition during dyeing will strictly be controlled.For example, under the condition of highly basic, dye, two class bacterium absorption basic-dyeable fibres are all many, all can be positive reaction; When PH is very low, can all be negative reaction.In addition, the cell membrane of two class bacterium etc. are also inconsistent to the permeability of crystal violet-Surgidine, and the saturating property of positive bacteria is little, therefore be difficult for being decoloured, the saturating property of negative bacterium is large, easily decolouring.So bleaching time, discoloration method also should strictly be controlled.
Traditional Grain stain four step rule is general main to be comprised just and dye, mordant dyeing, decolour, redye four steps, and concrete operation method is: 1) smear is fixed, 2) ammonium oxalate crystal violet dyes 1 minute, 3) water rinses, 4) add iodine liquid and cover painting face and dye 1 minute, 5) washing, with thieving paper, absorb moisture, 6) add 95% alcohol number and drip, and shake and decolour gently, 30 seconds after washings, suck moisture, 7) luxuriant red colouring liquid dyed after 10 seconds, and tap water rinses, dry microscopy.Wherein just dying, mordant dyeing, all need washing to remove dyestuff after decolouring, redye each step, avoid the impact on Color of the dyestuff that adheres to.Grain stain four step rule is effective, but operation more complicated, quality control is difficult for carrying out, and is difficult to accomplish standardization, consumes more dyestuff and water simultaneously, easily causes the waste of resource and the pollution of environment.
Developed at present a kind of Grain stain three-step approach, the method mainly comprise just dye, mordant dyeing and redye three steps, wherein just dying, mordant dyeing and redye each step after all need washing to remove dyestuff, avoid the impact on Color of the dyestuff that adheres to, although Grain stain three-step approach is compared four step rule, step reduces by a step, shortened dyeing time, but the method still comprises the step of three washings, the large water gaging of same consumption, traditional Grain stain three-step approach still needs to consume more dyestuff in addition, easily causes the waste of resource and the pollution of environment.
A kind of gram stain two-step method of washing twice is disclosed at present in patent CN101126689B, mainly comprise and just dye and redye two steps, after each step, need equally washing except the dyestuff of attachment removal, the Gram’s staining four step rule that the method is more traditional, operate easier and method is reliable, but the method is same, consume a large amount of dyestuff and water, and just dye and time of redying longer, be unsuitable for large batch of checked operation.
Summary of the invention
The object of the invention is in current Grain stain three-step approach and gram stain two-step method because the step that comprises washing causes consuming a large amount of water, the amount of dye of simultaneously using is large, the problem that dyeing time is long, a kind of Grain stain method of not washing is provided, the method has been saved the step of washing, save water resource, reduced dyestuff use amount simultaneously, reduced the pollution to environment.
Object of the present invention can be achieved through the following technical solutions:
The Grain stain method of not washing, comprising:
At room temperature, material to be checked is evenly coated on slide, dry, fixing, again first stain is added drop-wise on smear, dyeing, inclines and just after stain, directly drips counterstain, after dyeing, counterstain inclines, microscopy after dry, is empurpled gram positive bacterias and dyes red gram-negative bacteria in smear central authorities
Wherein said just stain is the aqueous solution that contains mass percent 0.03125% crystal violet.
In existing Grain stain method, washing is considered to an indispensable step, because can remove by washing the dyestuff being attached on smear, avoid the impact on Color, yet inventor is through research discovery, in Grain stain method, use the dyeing liquor of dilution, reduce the consumption of dyestuff, save water-washing step to not impact of Color, Color is still equally good with known method.
In the present invention, preferably, described counterstain is the ethanolic solution that contains following mass percent component, wherein 0.0625% basic fuchsin, 0.005% potassium iodide, 0.001% iodine.
In the present invention, preferably, the described just dripping quantity of stain is 1～4ml, and first stain dyeing time is 10～50 seconds; The dripping quantity of described counterstain is 0.8～2ml, and counterstain dyeing time is 1～5 second.The present invention more preferably described just dripping quantity of stain is 2ml, and first stain dyeing time is 30 seconds, and the dripping quantity of described counterstain is 1ml, and counterstain dyeing time is 2 seconds.
In the present invention, preferably, described counterstain is divided into counterstain A and counterstain B, and wherein counterstain A is the solution that contains mass percent 0.005% potassium iodide, 0.001% iodine, and counterstain B is the ethanolic solution that contains mass percent 0.0625% basic fuchsin; Described counterstain A and counterstain B drip in two steps.
In the present invention, preferably, the described just dripping quantity of stain is 1～3ml, just stain dyeing time is 10～50 seconds, and the dripping quantity of described counterstain A is 0.8～2ml, and counterstain A dyeing time is 1～5 second, the dripping quantity of described counterstain B is 0.8～2ml, and counterstain B dyeing time is 1～5 second.The present invention more preferably described just dripping quantity of stain is 2ml, and first stain dyeing time is 30 seconds, and the dripping quantity of described counterstain A is 1ml, and counterstain A dyeing time is 2 seconds, and the dripping quantity of described counterstain B is 1ml, and counterstain B dyeing time is 2 seconds.
In the present invention, preferably, described in be coated in the material to be checked on slide diameter be 1.0～2.0cm.
In the present invention, preferably, described material to be checked comprises bacterial cultures, censorship secretion.
The Gram staining of not washing of the present invention has the following advantages:
1. compare with existing Gram staining, method of the present invention has been saved the step of washing, has simplified operation steps, saved a large amount of water resources, and Color is the same with existing Gram staining, and the method compared with prior art can be saved 100% water.
2. the method has greatly reduced the consumption of dyestuff, and has shortened dyeing time, when improving dyeing efficiency, has saved 50% dyestuff, has reduced the pollution to environment.
Embodiment
Below the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, rather than whole embodiment.Embodiment based in the present invention, those of ordinary skills, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
The slide using in the present invention, first stain, mordant, counterstain and all adopt conventional material and facility for the instrument of microscopy, the method for microscopy adopts conventional sense method of the prior art.
Embodiment 1
Get clean slide, at room temperature, bacterial cultures is evenly coated on slide, dry, fixing, wherein the diameter of smear is 1.0cm, drip again 4ml by containing first stain that 0.03125% crystal violet aqueous solution is made into smear, dye 10 seconds, incline and just after stain, do not wash, directly drip 0.8ml by containing 0.0625% basic fuchsin, 0.005% potassium iodide, the counterstain that the ethanolic solution of 0.001% iodine is made into, dye after 5 seconds, counterstain inclines, microscopy after dry, in smear central authorities, can see empurpled gram positive bacteria and dye red gram-negative bacteria, with distinct contrast.
Embodiment 2
Get clean slide, at room temperature, censorship secretion is evenly coated on slide, dry, fixing, wherein the diameter of smear is 1.5cm, drip again 2ml by containing first stain that 0.03125% crystal violet aqueous solution is made into smear, dye 30 seconds, incline and just after stain, do not wash, directly drip 1ml by containing 0.0625% basic fuchsin, 0.005% potassium iodide, the counterstain that the ethanolic solution of 0.001% iodine is made into, dye after 2 seconds, counterstain inclines, microscopy after dry, in smear central authorities, can see empurpled gram positive bacteria and dye red gram-negative bacteria, with distinct contrast.
Embodiment 3
Get clean slide, at room temperature, censorship secretion is evenly coated on slide, dry, fixing, wherein the diameter of smear is 2.0cm, drip again 1ml by containing first stain that 0.03125% crystal violet aqueous solution is made into smear, dye 50 seconds, incline and just after stain, do not wash, directly drip 2ml by containing 0.0625% basic fuchsin, 0.005% potassium iodide, the counterstain that the ethanolic solution of 0.001% iodine is made into, dye after 1 second, counterstain inclines, microscopy after dry, in smear central authorities, can see empurpled gram positive bacteria and dye red gram-negative bacteria, with distinct contrast.
Embodiment 4
Get clean slide, at room temperature, bacterial cultures is evenly coated on slide, dry, fixing, wherein the diameter of smear is 1.0cm, drip again 4ml by containing first stain that 0.03125% crystal violet aqueous solution is made into smear, dye 10 seconds, incline and just after stain, do not wash, directly drip the counterstain A that 0.8ml is made into by the solution that contains 0.005% potassium iodide and 0.001% iodine, dye 5 seconds, incline after counterstain A and do not wash, directly drip the counterstain B that 0.8ml is made into by the ethanolic solution that contains 0.0625% basic fuchsin, dye after 5 seconds, the counterstain B that inclines does not wash, microscopy after dry, in smear central authorities, can see empurpled gram positive bacteria and dye red gram-negative bacteria, with distinct contrast.
Embodiment 5
Get clean slide, at room temperature, censorship secretion is evenly coated on slide, dry, fixing, wherein the diameter of smear is 1.5cm, drip again 2ml by containing first stain that 0.03125% crystal violet aqueous solution is made into smear, dye 30 seconds, incline and just after stain, do not wash, directly drip the counterstain A that 1ml is made into by the solution that contains 0.005% potassium iodide and 0.001% iodine, dye 4 seconds, incline after counterstain A and do not wash, directly drip the counterstain B that 1ml is made into by the ethanolic solution that contains 0.0625% basic fuchsin, dye after 2 seconds, the counterstain B that inclines does not wash, microscopy after dry, in smear central authorities, can see empurpled gram positive bacteria and dye red gram-negative bacteria, with distinct contrast.
Embodiment 6
Get clean slide, at room temperature, bacterial cultures is evenly coated on slide, dry, fixing, wherein the diameter of smear is 2.0cm, drip again 1ml by containing first stain that 0.03125% crystal violet aqueous solution is made into smear, dye 50 seconds, incline and just after stain, do not wash, directly drip the counterstain A that 2ml is made into by the solution that contains 0.005% potassium iodide and 0.001% iodine, dye 2 seconds, incline after counterstain A and do not wash, directly drip the counterstain B that 2ml is made into by the ethanolic solution that contains 0.0625% basic fuchsin, dye after 1 second, the counterstain B that inclines does not wash, microscopy after dry, in smear central authorities, can see empurpled gram positive bacteria and dye red gram-negative bacteria, with distinct contrast.
The above; only for the better embodiment of the present invention, but protection scope of the present invention do not limit to therewith, is anyly familiar with in technical scope that those skilled in the art disclose in the present invention; the variation that can expect easily or replacement, within all should being encompassed in protection scope of the present invention.
Claims (7)
1. the Grain stain method of not washing, comprising:
At room temperature, material to be checked is evenly coated on slide, dry, fixing, again first stain is added drop-wise on smear, dyeing, inclines and just after stain, directly drips counterstain, after dyeing, counterstain inclines, microscopy after dry, is empurpled gram positive bacterias and dyes red gram-negative bacteria in smear central authorities
Wherein said just stain is the aqueous solution that contains mass percent 0.03125% crystal violet.
2. Grain stain method of not washing as claimed in claim 1, is characterized in that, described counterstain is the ethanolic solution that contains following mass percent component, wherein 0.0625% basic fuchsin, 0.005% potassium iodide, 0.001% iodine.
3. Grain stain method of not washing as claimed in claim 1, it is characterized in that, described counterstain is divided into counterstain A and counterstain B, wherein counterstain A is the solution that contains mass percent 0.005% potassium iodide, 0.001% iodine, and counterstain B is the ethanolic solution that contains mass percent 0.0625% basic fuchsin; Described counterstain A and counterstain B drip in two steps.
4. Grain stain method of not washing as claimed in claim 2, is characterized in that, the described just dripping quantity of stain is 1～4ml, and first stain dyeing time is 10～50 seconds; The dripping quantity of described counterstain is 0.8～2ml, and counterstain dyeing time is 1～5 second.
5. Grain stain method of not washing as claimed in claim 3, it is characterized in that, the described just dripping quantity of stain is 1～3ml, just stain dyeing time is 10～50 seconds, the dripping quantity of described counterstain A is 0.8～2ml, counterstain A dyeing time is 1～5 second, and the dripping quantity of described counterstain B is 0.8～2ml, and counterstain B dyeing time is 1～5 second.
6. Grain stain method of not washing as claimed in claim 1, is characterized in that, described in be coated in the material to be checked on slide diameter be 1.0～2.0cm.
7. Grain stain method of not washing as claimed in claim 1, is characterized in that, described material to be checked comprises bacterial cultures, censorship secretion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210337417.8A CN103674661A (en) | 2012-09-12 | 2012-09-12 | Washing-free Gram staining method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210337417.8A CN103674661A (en) | 2012-09-12 | 2012-09-12 | Washing-free Gram staining method |
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| CN103674661A true CN103674661A (en) | 2014-03-26 |
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| CN201210337417.8A Pending CN103674661A (en) | 2012-09-12 | 2012-09-12 | Washing-free Gram staining method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108663253A (en) * | 2018-08-14 | 2018-10-16 | 苏州丰泰医疗用品贸易有限公司 | One kind quickly carrying out Gram-stained method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0286255A2 (en) * | 1987-04-06 | 1988-10-12 | Becton, Dickinson and Company | Improved stain for acid-fast bacilli |
| CN101126689B (en) * | 2007-08-07 | 2012-05-16 | 叶鹰 | Gram stain two-step method |
-
2012
- 2012-09-12 CN CN201210337417.8A patent/CN103674661A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0286255A2 (en) * | 1987-04-06 | 1988-10-12 | Becton, Dickinson and Company | Improved stain for acid-fast bacilli |
| CN101126689B (en) * | 2007-08-07 | 2012-05-16 | 叶鹰 | Gram stain two-step method |
Non-Patent Citations (3)
| Title |
|---|
| 丁红辉: "革兰染色二步法在阴道分泌物涂片中的临床应用", 《检验医学》, vol. 24, no. 10, 31 October 2009 (2009-10-31), pages 755 - 756 * |
| 刘婷芝: "细菌革兰氏染色改良两步法与经典四步法染色结果的临床评价", 《国际医药卫生导报》, vol. 16, no. 4, 28 February 2010 (2010-02-28) * |
| 黄元桐等: "革兰氏染色三步法与质量控制", 《微生物学报》, vol. 36, no. 1, 31 December 1996 (1996-12-31), pages 76 - 78 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108663253A (en) * | 2018-08-14 | 2018-10-16 | 苏州丰泰医疗用品贸易有限公司 | One kind quickly carrying out Gram-stained method |
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Application publication date: 20140326 |