CN103674660A - Gram staining two-step method with one-time washing - Google Patents
Gram staining two-step method with one-time washing Download PDFInfo
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- CN103674660A CN103674660A CN201210336839.3A CN201210336839A CN103674660A CN 103674660 A CN103674660 A CN 103674660A CN 201210336839 A CN201210336839 A CN 201210336839A CN 103674660 A CN103674660 A CN 103674660A
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Abstract
The invention discloses a gram staining two-step method with one-time washing. The method comprises the following steps: evenly coating a slide with a to-be-detected material at room temperature; drying and fixing; dropwise adding a primary dye on a smear; dyeing and pouring out the primary dye and then directly dropwise adding a counter-stain; dyeing and washing one time, and carrying out microscopic examination after drying, wherein gram positive bacteria stained in purple and gram negative bacteria stained in red are formed at the center of the smear. Compared with the prior art, the dose of the dye and the dose of water are greatly reduced by the gram staining two-step method, the primary staining and re-staining time is shortened, 50% of dye and 50% of water are saved when the dye efficiency is improved, and the gram staining two-step method is applicable to large-scale checked operation.
Description
Technical field
The present invention relates to a kind of Gram staining, particularly relate to a kind of washing gram stain two-step method once.
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 Gram's stain generally comprises just dyes, mordant dyeing, four steps such as decolours, redyes, 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, be difficult to accomplish standardization, consume a large amount of dyestuffs and water simultaneously, easily cause 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 more 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 for current gram stain two-step method dyestuff and water consumption larger, the problem that dyeing time is longer, provides a kind of washing gram stain two-step method once, and the consumption of the method dyestuff and water is little, just dye and the time of redying short, be applicable to large batch of checked operation.
Object of the present invention can be achieved through the following technical solutions:
A washing gram stain two-step method once, comprising:
At room temperature, material to be checked is evenly coated on slide, dry, fixing, first stain is added drop-wise on smear, dyeing, inclines and just after stain, directly drips counterstain, dyeing again, once, dry rear microscopy, is empurpled gram positive bacterias and dyes red gram-negative bacteria in smear central authorities in washing
Wherein said just stain is the aqueous solution that contains mass percent 0.03125% crystal violet; 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 traditional Grain stain method and improved two step decoration methods, because amount of dye used is more, 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 finds through research, use the dyeing liquor of dilution, reduce the consumption of dyestuff, the water-washing step saving after just dying does not affect Color, and Color is still equally good with known method.
Preferably, the described just dripping quantity of stain is 1～4ml in the present invention, 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.
Preferably, the diameter that is coated in the material to be checked on slide described in is 1.0～2.0cm.
In the present invention, described material to be checked is preferably bacterial cultures or censorship secretion.
Gram stain two-step method of washing of the present invention has the following advantages:
1. compare with twice gram stain two-step method of existing washing, method of the present invention has been omitted washing one step after just dying, simplified operation steps, method is more easy and twice gram stain two-step method of Color and washing is the same, and the method compared with prior art can be saved 50% water.
2. the method has greatly reduced the consumption of dyestuff, and has shortened the time of just dying and redying, and 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, 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 1ml 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, 1 second after washing dyes, 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 the counterstain that 1ml is made into by the ethanolic solution that contains 0.0625% basic fuchsin, 0.005% potassium iodide, 0.001% iodine, 2 seconds after washings dye, microscopy after dry, can see empurpled gram positive bacteria and dye red gram-negative bacteria in smear central authorities, 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 4ml 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 that 2ml is made into by the ethanolic solution that contains 0.0625% basic fuchsin, 0.005% potassium iodide, 0.001% iodine, 5 seconds after washings dye, microscopy after dry, can see empurpled gram positive bacteria and dye red gram-negative bacteria in smear central authorities, 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 (5)
1. a washing gram stain two-step method once, comprising:
At room temperature, material to be checked is evenly coated on slide, dry, fixing, first stain is added drop-wise on smear, dyeing, inclines and just after stain, directly drips counterstain, dyeing again, once, dry rear microscopy, is empurpled gram positive bacterias and dyes red gram-negative bacteria in smear central authorities in washing
Wherein said just stain is the aqueous solution that contains mass percent 0.03125% crystal violet;
Described counterstain is the ethanolic solution that contains following mass percent component, wherein 0.0625% basic fuchsin, 0.005% potassium iodide, 0.001% iodine.
2. washing as claimed in claim 1 gram stain two-step method once, 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.
3. washing as claimed in claim 2 gram stain two-step method once, is characterized in that, the 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.
4. washing as claimed in claim 1 gram stain two-step method once, is characterized in that, described in be coated in the material to be checked on slide diameter be 1.0～2.0cm.
5. washing as claimed in claim 1 gram stain two-step method once, is characterized in that, described material to be checked is bacterial cultures or censorship secretion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210336839.3A CN103674660A (en) | 2012-09-12 | 2012-09-12 | Gram staining two-step method with one-time washing |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201210336839.3A CN103674660A (en) | 2012-09-12 | 2012-09-12 | Gram staining two-step method with one-time washing |
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| CN103674660A true CN103674660A (en) | 2014-03-26 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198470A (en) * | 2016-06-30 | 2016-12-07 | 江苏莱芙时代生物科技有限公司 | A kind of fungal detection fluorescence staining liquid and application |
| 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 |
|---|---|---|---|---|
| CN101126689A (en) * | 2007-08-07 | 2008-02-20 | 叶鹰 | Gram stain two-step method |
| CN102220236A (en) * | 2010-04-15 | 2011-10-19 | 叶元康 | Automatic staining instrument with Gram staining two-step method |
-
2012
- 2012-09-12 CN CN201210336839.3A patent/CN103674660A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101126689A (en) * | 2007-08-07 | 2008-02-20 | 叶鹰 | Gram stain two-step method |
| CN102220236A (en) * | 2010-04-15 | 2011-10-19 | 叶元康 | Automatic staining instrument with Gram staining two-step method |
Non-Patent Citations (1)
| Title |
|---|
| 叶元康: "革兰染色的现况和进展", 《中国医药生物技术》, vol. 6, no. 6, 31 December 2011 (2011-12-31), pages 424 - 425 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198470A (en) * | 2016-06-30 | 2016-12-07 | 江苏莱芙时代生物科技有限公司 | A kind of fungal detection fluorescence staining liquid and application |
| 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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