CN1392266A - Quick gram staining liquid and method - Google Patents
Quick gram staining liquid and method Download PDFInfo
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- CN1392266A CN1392266A CN 01114296 CN01114296A CN1392266A CN 1392266 A CN1392266 A CN 1392266A CN 01114296 CN01114296 CN 01114296 CN 01114296 A CN01114296 A CN 01114296A CN 1392266 A CN1392266 A CN 1392266A
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Abstract
The quick Gram staining liquid is prepared with the following material: Crystal Violet solution comprising Crystal Violet, absolute alcohol, glycol methyl ether, ammonium oxalate and distilled water; iodine liquid comprising potassium iodide, iodine, sodium chloride and distilled water; decoloring agent comprising acetone and ethanol; and counterstain liquid comprising fuchsin, absolute alcohol, phenylic acid, Tween and distilled water. The staining method includes the flame fixing of smear, adding Crystal Violet, water washing and sucking to eliminate water; adding iodine liquid, water washing and sucking to eliminate water; adding decoloring agent while stirring and water washing; and adding counterstain liquid, water washing, drying and microscope inspection. The present invention can detect bacteria fast and accurately, and is especially suitable for use in countryside health organization.
Description
The present invention relates to gram-positive microorganism and Gram-negative bacteria quick gram staining liquid, the invention still further relates to the working method of quick gram staining liquid.
Gram staining liquid at first adopts 20 milligrams of crystal violet solution or aldrich mixtures (100 milligrams of 95% alcohol, 400 milligrams of Viola crystallinas), 8 milligrams of 1% ammonium oxalate solutions, cooperates respectively, mixes then; Next adopts Lugol's iodine solution, and 200 milligrams of potassiumiodides, 100 milligrams of iodine, 300 milligrams of distilled water are earlier with 200 milligrams of distilled water, with 200 milligrams of potassiumiodide dissolvings, and then add 100 milligrams of iodine thorough mixing, iodine is dissolved fully, then adding distil water to 300 milligram; Another is a discoloring agent, adopts 95% alcohol; At last with redying liquid, 10 milligrams of basic fuchsins, 10 milligrams of 95% alcohol, 90 milligrams of 5% PHENOL 99.8 MIN ((CARBOLIC ACID)).Its operation steps is: at first smear is fixing on flame, drips crystal violet solution and dyes 1 minute, and secondly washing is to drip Lugol's iodine solution, dyes 1 minute after washing; Be to drip the decolouring of 95% alcohol once more, should shake slide frequently this moment, till no purple comes off, 30 second after washing; The 4th is to drip to redye liquid and dyed 1 minute, and washing waits to do microscopy.The problem that aforesaid method mainly exists is: the one, and Viola crystallina is with after ammonium oxalate solution mixes, and the shelf time is short, if there is precipitation to occur, should prepare use again, the cost height; The 2nd, Lugol's iodine solution is put perishable long afterwards, and is careless slightly in the operation, just produces Different Results, and whole dyeing course needs 210 seconds, more time-consuming, increased labor intensity of operating personnel.
The quick gram staining liquid that the object of the present invention is to provide a kind of easy preparation, never degenerate, cost is low, the shelf time is long.
Another object of the present invention is to provide a kind of easy to operate, fast and accurately working method.
The object of the present invention is achieved like this:
Staining fluid, by following several preparation of raw material:
A, crystal violet solution
Viola crystallina 600-1000 milligram dehydrated alcohol 8-12 milligram
Propylene glycol monomethyl ether 8-12 milligram ammonium oxalate 700-900 milligram
Distilled water 70-90 milligram
Mix, do not produce precipitation, preserve 1-2.
B, iodine liquid
Potassiumiodide 200-500 milligram iodine 50-300 milligram
Sodium-chlor 150-350 milligram distilled water 80-120 milligram
Mix, be brown and deposit, never degenerate, preserve 1-2.
C, discoloring agent
Acetone 35-45 milligram 95% ethanol 55-65 milligram
Mix.
D, redye liquid
Magenta 450-550 milligram dehydrated alcohol 8-12 milligram
5% PHENOL 99.8 MIN ((CARBOLIC ACID)) 8-12 milligram tween-80 200-500 milligram
Distilled water 70-90 milligram
Its working method is:
1, smear is fixed through flame, adds crystal violet solution, dyes 3-7 and washes second, uses the filter paper wipe dry;
2, add iodine liquid and dye 3-7 second, the filter paper wipe dry is used in washing;
3, add discoloring agent and constantly shake, 10-30 second till do not have purple and come off, washing;
4, add and redye liquid and dye 3-7 second, the back microscopy is done in washing.Just thalline to be purple be gram-positive microorganism, taking on a red color is Gram-negative bacteria.
After the iodine liquid of quick gram liquid enters thalline, there are enough amounts to combine with Viola crystallina rapidly, this binding substances combines with the endobacillary Yeast Nucleic Acid magnesium salts of Gram-positive protein complex again, therefore, gram-positive microorganism picked-up Viola crystallina is many and firm, Gram-negative bacteria lacks the Yeast Nucleic Acid magnesium salts, so, do not combine with Viola crystallina, and by the combination of PHENOL 99.8 MIN ((CARBOLIC ACID)) azaleine.The iso-electric point of gram-positive microorganism (PH2-3) is low than negative bacterium (PH4-5), and is electronegative more at certain following of PH, and therefore easy and positively charged basic dyestuff combination is difficult for by bleaching agent bleaching.Gram-positive microorganism is different with the permeability of Gram-negative bacteria mycoderm, the mixed solution of corresponding employing 95% alcohol of discoloring agent and acetone, gram-positive microorganism is difficult for penetrating, so be difficult for decolouring, because acetone solution is strong, make the dissolving of dyestuff and Surgidine thoroughly, so, can not produce false gram-positive microorganism.
The present invention compared with prior art, have the following advantages and effect: proportioning is easy, is easy to be grasped by clinical worker, be particularly suitable for the tyro, the dyeing whole process only needs 30 seconds, and is easy to operate, do not cause false positive and false negative, do not precipitate, never degenerate, with low cost, long 1-2 of shelf-time, portable, easily grasp, quick, convenient, the accurate bacterial detection of energy especially is fit to the rural basic level health unit.
Embodiment 1:
Staining fluid has following raw material system to join
A, crystal violet solution
10 milligrams of 800 milligrams of dehydrated alcohols of Viola crystallina
800 milligrams of 10 milligrams of ammonium oxalate of propylene glycol monomethyl ether
80 milligrams of distilled water
B, iodine liquid
150 milligrams of 300 milligrams of iodine of potassiumiodide
100 milligrams of 270 milligrams of distilled water of sodium-chlor
C, discoloring agent
60 milligrams of 40 milligram of 95% ethanol of acetone
D, redye liquid
10 milligrams of 500 milligrams of dehydrated alcohols of magenta
500 milligrams of 10 milligrams of tween-80s of 5% PHENOL 99.8 MIN ((CARBOLIC ACID))
80 milligrams of distilled water
Its operation steps is as follows:
1, smear is fixed through flame, adds crystal violet solution, dyes washing in 5 seconds, uses the filter paper wipe dry;
2, add iodine liquid and dyed 5 seconds, the filter paper wipe dry is used in washing;
3, add discoloring agent and constantly shake, till do not have purple and come off 20 seconds, washing;
4, add and redye liquid and dyed 5 seconds, the back microscopy is done in washing, just thalline to be purple be gram-positive microorganism, taking on a red color is Gram-negative bacteria.
Embodiment 2:
A, crystal violet solution
8 milligrams of 1000 milligrams of dehydrated alcohols of Viola crystallina
700 milligrams of 12 milligrams of ammonium oxalate of propylene glycol monomethyl ether
90 milligrams of distilled water
B, iodine liquid
50 milligrams of 200 milligrams of iodine of potassiumiodide
80 milligrams of 350 milligrams of distilled water of sodium-chlor
C, discoloring agent
55 milligrams of 45 milligram of 95% ethanol of acetone
D, redye liquid
8 milligrams of 450 milligrams of dehydrated alcohols of magenta
500 milligrams of 12 milligrams of tween-80s of 5% PHENOL 99.8 MIN ((CARBOLIC ACID))
90 milligrams of distilled water
Implementation step is identical with embodiment 1.Embodiment 3:A, crystal violet solution
12 milligrams of 600 milligrams of dehydrated alcohols of Viola crystallina
900 milligrams of 8 milligrams of ammonium oxalate of propylene glycol monomethyl ether
70 milligrams of B of distilled water, iodine liquid
300 milligrams of 500 milligrams of iodine of potassiumiodide
120 milligrams of C of 150 milligrams of distilled water of sodium-chlor, discoloring agent
65 milligrams of D of 35 milligram of 95% ethanol of acetone, redye liquid
12 milligrams of 550 milligrams of dehydrated alcohols of magenta
200 milligrams of 8 milligrams of tween-80s of 5% PHENOL 99.8 MIN ((CARBOLIC ACID))
70 milligrams of implementation steps of distilled water are identical with embodiment 1.
Claims (5)
1, a kind of quick gram staining liquid, the staining fluid that it is prepared by the following weight proportion raw material:
A, crystal violet solution
10 milligrams of 800 milligrams of dehydrated alcohols of Viola crystallina
800 milligrams of 10 milligrams of ammonium oxalate of propylene glycol monomethyl ether
80 milligrams of distilled water
B, iodine liquid
150 milligrams of 300 milligrams of iodine of potassiumiodide
100 milligrams of 270 milligrams of distilled water of sodium-chlor
C, discoloring agent
60 milligrams of 40 milligram of 95% ethanol of acetone
D, redye liquid
10 milligrams of 500 milligrams of dehydrated alcohols of magenta
500 milligrams of 10 milligrams of tween-80s of 5% carbonic acid
80 milligrams of distilled water.
2, a kind of quick gram staining liquid according to claim 1, wherein the weight proportion of each raw material is:
A, crystal violet solution
10 milligrams of 800 milligrams of dehydrated alcohols of Viola crystallina
800 milligrams of 10 milligrams of ammonium oxalate of propylene glycol monomethyl ether
80 milligrams of distilled water
B, iodine liquid
150 milligrams of 300 milligrams of iodine of potassiumiodide
100 milligrams of 270 milligrams of distilled water of sodium-chlor
C, discoloring agent
60 milligrams of 40 milligram of 95% ethanol of acetone
D, redye liquid
10 milligrams of 500 milligrams of dehydrated alcohols of magenta
500 milligrams of 10 milligrams of tween-80s of 5% PHENOL 99.8 MIN ((CARBOLIC ACID))
80 milligrams of distilled water.
3, a kind of quick gram staining liquid according to claim 1, wherein the weight proportion of each raw material is:
A, crystal violet solution
8 milligrams of 1000 milligrams of dehydrated alcohols of Viola crystallina
700 milligrams of 12 milligrams of ammonium oxalate of propylene glycol monomethyl ether
90 milligrams of distilled water
B, iodine liquid
50 milligrams of 200 milligrams of iodine of potassiumiodide
80 milligrams of 350 milligrams of distilled water of sodium-chlor
C, discoloring agent
55 milligrams of 45 milligram of 95% ethanol of acetone
D, redye liquid
8 milligrams of 450 milligrams of dehydrated alcohols of magenta
500 milligrams of 12 milligrams of tween-80s of 5% PHENOL 99.8 MIN ((CARBOLIC ACID))
90 milligrams of distilled water.
4, a kind of quick gram staining liquid according to claim 1, wherein the weight proportion of each raw material is:
A, crystal violet solution
12 milligrams of 600 milligrams of dehydrated alcohols of Viola crystallina
900 milligrams of 8 milligrams of ammonium oxalate of propylene glycol monomethyl ether
70 milligrams of distilled water
B, iodine liquid
300 milligrams of 500 milligrams of iodine of potassiumiodide
120 milligrams of 150 milligrams of distilled water of sodium-chlor
C, discoloring agent
65 milligrams of 35 milligram of 95% ethanol of acetone
D, redye liquid
12 milligrams of 550 milligrams of dehydrated alcohols of alkalescence product
200 milligrams of 8 milligrams of tween-80s of 5% PHENOL 99.8 MIN ((CARBOLIC ACID))
70 milligrams of distilled water.
5, a kind of method that realizes the described a kind of quick gram staining liquid of claim 1 comprises the following steps:
A, smear are fixed through flame, add crystal violet solution, dye washing in 5 seconds, use the filter paper wipe dry;
B, add iodine liquid and dyed 5 seconds, the filter paper wipe dry is used in washing;
C, add discoloring agent and constantly shake, till do not have purple and come off 20 seconds, washing;
D, add and redye liquid and dyed 5 seconds, the back microscopy is done in washing, just thalline to be purple be gram-positive microorganism, taking on a red color is Gram-negative bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01114296 CN1392266A (en) | 2001-06-19 | 2001-06-19 | Quick gram staining liquid and method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01114296 CN1392266A (en) | 2001-06-19 | 2001-06-19 | Quick gram staining liquid and method |
Publications (1)
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| CN1392266A true CN1392266A (en) | 2003-01-22 |
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| CN 01114296 Pending CN1392266A (en) | 2001-06-19 | 2001-06-19 | Quick gram staining liquid and method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134583A (en) * | 2010-12-31 | 2011-07-27 | 清远海贝生物技术有限公司 | Photosynthetic bacteria counting method |
| WO2013139554A1 (en) * | 2012-03-19 | 2013-09-26 | Ventana Medical Systems, Inc. | Gram staining method with improved decolorization of the crystal violet-iodine complex from gram negative bacteria |
| CN103451260A (en) * | 2013-09-09 | 2013-12-18 | 上海兰卫临床检验有限公司 | Preparation method of anti-acid staining solution quality control product |
| CN107576552A (en) * | 2017-08-29 | 2018-01-12 | 北京林业大学 | A kind of paraffin section colouring method for observing Chinese Rose infection processs |
| CN108593392A (en) * | 2018-01-26 | 2018-09-28 | 广州江元医疗科技有限公司 | A kind of vaginal fluid dyeing liquor and preparation method thereof |
| 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 |
-
2001
- 2001-06-19 CN CN 01114296 patent/CN1392266A/en active Pending
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134583A (en) * | 2010-12-31 | 2011-07-27 | 清远海贝生物技术有限公司 | Photosynthetic bacteria counting method |
| CN108181155B (en) * | 2012-03-19 | 2020-11-27 | 文塔纳医疗系统公司 | Gram staining method with improved decolorization of crystal violet-iodine complex of gram negative bacteria |
| WO2013139554A1 (en) * | 2012-03-19 | 2013-09-26 | Ventana Medical Systems, Inc. | Gram staining method with improved decolorization of the crystal violet-iodine complex from gram negative bacteria |
| CN104245955A (en) * | 2012-03-19 | 2014-12-24 | 文塔纳医疗系统公司 | Gram staining method with improved decolorization of the crystal violet-iodine complex from gram negative bacteria |
| US9023615B2 (en) | 2012-03-19 | 2015-05-05 | Ventana Medical Systems, Inc. | Gram staining method with improved decolorization of the crystal violet-iodine complex from gram negative bacteria |
| JP2015516149A (en) * | 2012-03-19 | 2015-06-11 | ヴェンタナ メディカル システムズ， インク． | Gram staining method with improved decolorization of crystal violet-iodine complex from gram negative bacteria |
| CN108181155A (en) * | 2012-03-19 | 2018-06-19 | 文塔纳医疗系统公司 | Improve the Gram-staining process of the decoloration to crystal violet-Surgidine of gramnegative bacterium |
| CN103451260A (en) * | 2013-09-09 | 2013-12-18 | 上海兰卫临床检验有限公司 | Preparation method of anti-acid staining solution quality control product |
| CN107576552A (en) * | 2017-08-29 | 2018-01-12 | 北京林业大学 | A kind of paraffin section colouring method for observing Chinese Rose infection processs |
| CN107576552B (en) * | 2017-08-29 | 2020-04-07 | 北京林业大学 | Paraffin section dyeing method for observing infection process of China rose black spot |
| CN108593392B (en) * | 2018-01-26 | 2020-09-04 | 广州江元医疗科技有限公司 | Vaginal secretion staining solution and preparation method thereof |
| CN108593392A (en) * | 2018-01-26 | 2018-09-28 | 广州江元医疗科技有限公司 | A kind of vaginal fluid dyeing liquor and preparation method thereof |
| 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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