CN104089807A - Germ staining method - Google Patents
Germ staining method Download PDFInfo
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- CN104089807A CN104089807A CN201410321905.9A CN201410321905A CN104089807A CN 104089807 A CN104089807 A CN 104089807A CN 201410321905 A CN201410321905 A CN 201410321905A CN 104089807 A CN104089807 A CN 104089807A
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- paraffin section
- bacterium
- toluidine blue
- dimethylbenzene
- ethyl alcohol
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Abstract
The invention discloses a germ staining method. Paraffin sections are quickly stained by the germ staining method, so that germs in the paraffin sections can be well positioned; furthermore, the method is also applicable to germ smears, and the steps are simple and are easy to control. According to the germ staining method disclosed by the invention, staining is implemented by a toluidine blue solution. The method disclosed by the invention has the following advantages that the method can be used for staining the germ smears and also displaying the germs in paraffin section tissues; compared with a conventional germ staining method, the method disclosed by the invention has the advantages of convenience and quickness.
Description
Technical field
The present invention relates to a kind of Bacterial stain method, belong to microorganism field.
Background technology
Bacterium is a class prokaryotic microorganism, because thalline is little, translucent, wants its size and geometric of clearer observation, needs just can see after dyed and microscope amplification.Conventional bacterial stain has the method for singly dying, staining counter and differential staining, mainly contains the methods such as Gram’s staining, methylene blue, acid-fast stain, Wright's staining and the dyeing of Ji's nurse Sa.Before carrying out above-mentioned dyeing, need to prepare bacterium smear, but also rare to the Bacterial stain report in the tissue of paraffin section.Developing a kind of Bacterial stain method tool that had not only been applicable to paraffin section but also had been applicable to bacterium smear is of great significance.
Summary of the invention
The object of this invention is to provide a kind of Bacterial stain method---toluidine blue laws, utilize the method to carry out rapid dyeing to paraffin section, can well locate bacterium wherein, and, the method is applicable too to bacterium smear, and step is simple, easily controls.
The invention provides a kind of Bacterial stain method, is to dye with toluidine blue solution;
Described toluidine blue solution is prepared as follows:
A liquid: 0.8g toluidine blue is dissolved in 80 ml distilled waters;
B liquid: 0.6g potassium permanganate is dissolved in 20 ml distilled waters;
A liquid is boiled, then B drop is entered in A liquid, then boil, with distilled water, complement to 100 milliliters, natural cooled and filtered and get final product;
The described time of boiling is 10min.
In said method, the time of described dyeing is 5-15s, is specially 10s.
In above-mentioned arbitrary described method, described bacterium is the bacterium in paraffin section or bacterium smear.
In above-mentioned arbitrary described method, after described dyeing, also comprise the steps: that the ethanol water of the paraffin section after toluidine blue solution-dyed or bacterium smear being put into volumn concentration 95% washes away loose colour;
The bacterium that the tissue color that the described standard that washes away loose colour is paraffin section becomes in light blue or bacterium smear demonstrates obvious blue form.
In above-mentioned arbitrary described method, described paraffin section also comprises the step dewaxing to water before dyeing in toluidine blue solution;
Described dewaxing to the water ethanol water that is specifically 95% through mixed liquor, absolute ethyl alcohol, the volumn concentration of dimethylbenzene, equal-volume dimethylbenzene and absolute ethyl alcohol successively by paraffin section, the ethanol water that volumn concentration is 90%, the ethanol water that volumn concentration is 80%, ethanol water, the distilled water that volumn concentration is 70%, the time of paraffin section in each reagent is 3-5min;
Described bacterium smear also comprises the step that flame is fixing before dyeing in toluidine blue solution;
Described flame is fixedly that natural drying bacterium smear is smeared towards upper, with its back side, on flame flame envelope, heats, and the non-scald on hand of take is fixed as degree.
In above-mentioned arbitrary described method, described paraffin section, after washing away loose colour, also comprises it is passed through to absolute ethyl alcohol successively, the mixed liquor of isopyknic dimethylbenzene and absolute ethyl alcohol, dimethylbenzene, the finally step with neutral gum mounting by section;
Described paraffin section is at described absolute ethyl alcohol, and mixed liquor, the time in dimethylbenzene of isopyknic dimethylbenzene and absolute ethyl alcohol are 5-10min, are specially 5min;
Described bacterium smear, after washing away loose colour, also comprises the step that nature dries.
In above-mentioned arbitrary described method, described paraffin section is the paraffin section of pigeon crop tissue or the paraffin section of bangladesh tiger animal intestinal tissue;
The smear that described bacterium smear is bacillus cereus.
Method tool provided by the invention has the following advantages:
1, method of the present invention not only can dye to bacterium smear, and can show the bacterium in paraffin section tissue.
2, method of the present invention is compared with existing Bacterial stain method (such as Gram's stain and Wright Stain), has advantage easily and efficiently.
Accompanying drawing explanation
Fig. 1 is that Toluidine blue staining shows the bacterium Fig. 1 in crop tissue paraffin section de.
Fig. 2 is that Toluidine blue staining shows the bacterium Fig. 2 in crop tissue paraffin section de.
Fig. 3 is that Toluidine blue staining shows the bacterium Fig. 1 in intestinal tissue paraffin section.
Fig. 4 is that Toluidine blue staining shows the bacterium Fig. 2 in intestinal tissue paraffin section.
Fig. 5 is that Toluidine blue staining shows bacillus cereus pure culture Fig. 1.
Fig. 6 is that Toluidine blue staining shows bacillus cereus pure culture Fig. 2.
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The compound method of toluidine blue solution:
A liquid: toluidine blue 0.8g is dissolved in 80 ml distilled waters;
B liquid: potassium permanganate 0.6g is dissolved in 20 ml distilled waters;
The A liquid having dissolved is boiled 10 minutes, then the B liquid having dissolved is dropwise added in A liquid, then boil 10 minutes, with distilled water, complement to 100 milliliters, treat that nature cooled and filtered is standby.
Bacillus cereus (Bacillus cereus) disclosed in document " Mizuka Yamaguchi; Takao Kawai.A new method for rapid and quantitative detection of the Bacillus cereus emetic toxin cereulide in food products by liquid chromatography-tandem mass spectrometry analysis.Food Microbiology34 (2013) 29-37 ", and public Ke Cong China Agricultural University obtains.
The paraffin section Ke Cong China Agricultural University of pigeon crop tissue obtains.
The paraffin section Ke Cong China Agricultural University of bangladesh tiger intestinal tissue obtains.
The toluidine blue laws of bacterium dyeing in embodiment 1, paraffin section
One, get the paraffin section of pigeon crop tissue, dewax to water (successively through dimethylbenzene, dimethylbenzene: absolute ethyl alcohol (volume ratio 1:1), absolute ethyl alcohol, volumn concentration is 95% ethanol water, volumn concentration is 90% ethanol water, volumn concentration is 80% ethanol water, volumn concentration is 70% ethanol water, distilled water (each step time 3-5min)), section is put into the toluidine blue solution 5-15s preparing, put it in the ethanol water of volumn concentration 95% and wash away loose colour, microscope is controlled (tissue color becomes light blue), section is passed through to absolute ethyl alcohol (dehydration) successively, dimethylbenzene: absolute ethyl alcohol (volume ratio 1:1), dimethylbenzene (de-ethanol) (each 5min, 5-10min all can), finally will cut into slices and use neutral gum mounting.
The section of sealing under normal temperature state is dried naturally, and with microscopic examination (it is blue that bacterium is), the Bacterial stain result in the paraffin section of the pigeon crop tissue of the 10s that dyes in toluidine blue solution as depicted in figs. 1 and 2.
Two, get the paraffin section of bangladesh tiger intestinal tissue, according to the method for step 1, carry out the dyeing of toluidine blue laws, the Bacterial stain result in the paraffin section of the 10s that dyes in toluidine blue solution as shown in Figure 3 and Figure 4.
The toluidine blue laws dyeing of embodiment 2, bacterium smear
(the cultivation that bacillus cereus is rule on plain agar nutrient culture media of bacillus cereus pure culture, 37 ℃, cultivate 24 hours), (bacillus cereus bacterium colony of picking is dissolved in 1 ml distilled water to carry out bacterium smear, then with disinfection inoculation ring, get a ring bacterium liquid, central authorities in slide are coated with into suitable big or small thin layer equably), obtain the smear of bacillus cereus pure culture, natural drying.Flame is fixed (by dried smear, make to smear towards upper, with its back side on spirit lamp flame envelope as pendulum sample dragged for several times back and forth, heating slightly, can not be too hot, take non-scald on hand as degree, be fixed), in smear position, drip toluidine blue solution-dyed 5-15s, with the ethanol water of volumn concentration 95%, rinse 30s (15s-30s all can) and wash away loose colour, microscope is controlled (till seeing blue bacterium), naturally dries microscopic examination, and result as shown in Figure 5 and Figure 6.
Claims (6)
1. a Bacterial stain method, is to dye with toluidine blue solution;
Described toluidine blue solution is prepared as follows:
A liquid: 0.8g toluidine blue is dissolved in 80 ml distilled waters;
B liquid: 0.6g potassium permanganate is dissolved in 20 ml distilled waters;
A liquid is boiled, then B drop is entered in A liquid, then boil, with distilled water, complement to 100 milliliters, natural cooled and filtered and get final product.
2. method according to claim 1, is characterized in that: the time of described dyeing is 5-15s, is specially 10s.
3. method according to claim 1 and 2, is characterized in that: described bacterium is the bacterium in paraffin section or bacterium smear.
4. according to the arbitrary described method of claim 1-3, it is characterized in that: after described dyeing, also comprise the steps: that the ethanol water of the paraffin section after toluidine blue solution-dyed or bacterium smear being put into volumn concentration 95% washes away loose colour.
5. according to the arbitrary described method of claim 1-4, it is characterized in that: described paraffin section also comprises the step dewaxing to water before dyeing in toluidine blue solution;
Described dewaxing to the water ethanol water that is specifically 95% through mixed liquor, absolute ethyl alcohol, the volumn concentration of dimethylbenzene, equal-volume dimethylbenzene and absolute ethyl alcohol successively by paraffin section, the ethanol water that volumn concentration is 90%, the ethanol water that volumn concentration is 80%, ethanol water, the distilled water that volumn concentration is 70%, the time of paraffin section in each reagent is 3-5min;
Described bacterium smear also comprises the step that flame is fixing before dyeing in toluidine blue solution;
Described flame is fixedly that natural drying bacterium smear is smeared towards upper, with its back side, on flame flame envelope, heats, and the non-scald on hand of take is fixed as degree.
6. according to the arbitrary described method of claim 1-5, it is characterized in that: described paraffin section is after washing away loose colour, also comprise it is passed through to absolute ethyl alcohol successively, the mixed liquor of isopyknic dimethylbenzene and absolute ethyl alcohol, dimethylbenzene, the finally step with neutral gum mounting by section;
Described paraffin section is at described absolute ethyl alcohol, and the mixed liquor of isopyknic dimethylbenzene and absolute ethyl alcohol, the time in dimethylbenzene are 5-10min, are specially 5min;
Described bacterium smear, after washing away loose colour, also comprises the step that nature dries.
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| CN201410321905.9A CN104089807A (en) | 2014-07-07 | 2014-07-07 | Germ staining method |
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| CN201410321905.9A CN104089807A (en) | 2014-07-07 | 2014-07-07 | Germ staining method |
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Citations (4)
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|---|---|---|---|---|
| CN101915693A (en) * | 2010-07-07 | 2010-12-15 | 新疆医科大学 | Human embryonic trigeminus based three-dimensional reconstruction method by using histotomy staining |
| CN102221526A (en) * | 2011-03-09 | 2011-10-19 | 叶欣 | Composite reagent for screening early-stage oral intima cell disease and application method thereof |
| CN102243154A (en) * | 2011-04-02 | 2011-11-16 | 中山大学附属第一医院 | Method for staining peripheral nerve sections |
| US20130337441A1 (en) * | 2010-12-06 | 2013-12-19 | Dako Denmark A/S | Combined histological stain |
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2014
- 2014-07-07 CN CN201410321905.9A patent/CN104089807A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101915693A (en) * | 2010-07-07 | 2010-12-15 | 新疆医科大学 | Human embryonic trigeminus based three-dimensional reconstruction method by using histotomy staining |
| US20130337441A1 (en) * | 2010-12-06 | 2013-12-19 | Dako Denmark A/S | Combined histological stain |
| CN102221526A (en) * | 2011-03-09 | 2011-10-19 | 叶欣 | Composite reagent for screening early-stage oral intima cell disease and application method thereof |
| CN102243154A (en) * | 2011-04-02 | 2011-11-16 | 中山大学附属第一医院 | Method for staining peripheral nerve sections |
Non-Patent Citations (6)
| Title |
|---|
| 刘雨田，邹艳丽，王志亮: "免疫组化法检测我国疯牛病结果初报", 《中国动物检疫》, vol. 19, no. 4, 31 December 2002 (2002-12-31), pages 26 - 27 * |
| 吴洪娟等: "肥大细胞电镜半薄切片染色方法", 《电子显微学报》, vol. 31, no. 5, 31 October 2012 (2012-10-31), pages 452 - 454 * |
| 张晓梅: "用免疫组织化学染色法检测猪生殖- 呼吸道综合征病毒(PRRSV)抗原的研究", 《动物医学进展》, vol. 20, no. 3, 31 December 1999 (1999-12-31), pages 175 * |
| 田海燕，韩德平，胡艳欣: "两种固定液对甲苯胺蓝染色效果的影响", 《中国兽医杂志》, vol. 46, no. 9, 31 December 2010 (2010-12-31) * |
| 邓传远，郭素枝等: "茉莉叶石蜡制片技术的改进", 《三明学院学报》, vol. 25, no. 2, 30 June 2008 (2008-06-30) * |
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