CN115791346A - Mycobacterium tuberculosis acid-fast staining solution and preparation and staining method thereof - Google Patents
Mycobacterium tuberculosis acid-fast staining solution and preparation and staining method thereof Download PDFInfo
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Abstract
The invention provides mycobacterium tuberculosis acid-fast staining solution and a preparation method and a staining method thereof, and relates to the technical field of disease screening. An acid-fast staining solution for mycobacterium tuberculosis, comprising: basic fuchsin, carbolic acid, methylene blue, 95% ethanol and concentrated hydrochloric acid. The mycobacterium tuberculosis acid-fast staining solution disclosed by the invention is easy to stain, and the stained specimen is bright in color and convenient to observe, so that the detection rate of mycobacterium tuberculosis can be improved. The preparation method is simple and convenient to operate, unnecessary workload can be reduced, laboratory pollution is reduced, and working efficiency is improved. The staining method can improve the staining effect of the stained specimen, deepen the color depth of the specimen, facilitate the identification of acid-fast bacilli by inspectors, improve the working efficiency, reduce the staining cost of the specimen and achieve better economic benefits.
Description
Technical Field
The invention relates to the technical field of disease screening, and particularly relates to a mycobacterium tuberculosis acid-fast staining solution and a preparation and staining method thereof.
Background
Tuberculosis (TB) is a common and fatal infectious disease caused by mycobacteria (also called tubercle bacillus). Tuberculosis usually infects and destroys the lung and lymphatic system, but other organs such as the brain, central nervous system, circulatory system, urinary system, bones, joints, and even the skin can be infected. Tuberculosis mortality is high, and early, rapid and effective etiological diagnosis is extremely important for treating tuberculosis immediately and reducing the mortality and sequelae.
At present, under the framework of the current national tuberculosis prevention and treatment plan, the control of the basic tuberculosis is mainly to discover and control the infectious sources, and the tuberculosis patients with positive acid-fast bacilli of the sputum smear are the most main infectious sources in the society, so the microscopic examination result of the acid-fast bacilli of the sputum smear is an important index for diagnosing the infectious tuberculosis. The bacteriological experimental technique of sputum smear microscopy is very in line with the cost-benefit principle for developing countries with low income and serious tuberculosis epidemic. How to discuss the preparation method of the sputum smear staining solution more suitable for basic laboratory personnel can enable the basic laboratory personnel to master the more scientific and simple preparation method of the staining solution.
Disclosure of Invention
The invention aims to provide an acid-fast staining solution for mycobacterium tuberculosis, which is easy to stain, and the stained specimen has bright color, is convenient to observe, and can improve the detection rate of the mycobacterium tuberculosis.
The invention also aims to provide a preparation method of the mycobacterium tuberculosis acid-fast staining solution, which is simple and convenient to operate, and can reduce unnecessary workload, reduce laboratory pollution and improve working efficiency.
The invention also aims to provide a staining method of the mycobacterium tuberculosis acid-fast staining solution, which improves the staining effect of the stained specimen, deepens the color depth of the specimen, is more convenient for inspectors to identify the acid-fast bacillus, improves the working efficiency, can reduce the staining cost of the specimen and has better economic benefit.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides an acid-fast staining solution for mycobacterium tuberculosis, which comprises the following components:
the working solution, taking 500mL as an example, comprises the following components: 2-8g of basic fuchsin, 40-60mL of 95% ethanol, 20-30mL of liquid carbolic acid and the balance of distilled water;
the destaining solution, taking 500mL as an example, comprises the following components: 20-50mL of concentrated hydrochloric acid with the mass fraction of 36-38% and 450-480mL of 95% ethanol;
the compound dyeing liquid, taking 500mL as an example, comprises the following components: 0.5-1g of methylene blue, 40-60mL of 95% ethanol and the balance of distilled water.
The invention provides a preparation method of mycobacterium tuberculosis acid-fast staining solution, which comprises the following steps:
adding 95% ethanol into basic fuchsin, shaking for 5-15min to obtain fuchsin solution, heating and liquefying carbolic acid in an acid water bath, then adding liquid carbolic acid into the fuchsin solution, adding distilled water to a constant volume of 500mL, and mixing to obtain a working solution;
adding concentrated hydrochloric acid into 95% ethanol, and mixing to obtain decolorized solution;
adding methylene blue into 95% ethanol, shaking for 5-15min, and adding distilled water to a constant volume of 500mL to obtain a complex dye solution.
The invention provides a staining method of mycobacterium tuberculosis acid-fast staining solution, which comprises the following steps:
s1, heating and fixing a specimen smear, dripping working solution on the surface of the specimen smear, heating for 5-10min, standing for 5-15min, and washing with water;
s2, dripping a decoloring solution from the outer edge of the sample smear washed by the water in the S1, decoloring and washing;
and S3, dripping a re-staining solution into the specimen smear washed by the water in the S2, and carrying out dyeing, washing, drying and microscopic examination.
The embodiment of the invention at least has the following beneficial effects:
according to the invention, the final concentration of the counter-staining solution can reach 0.16% in the above proportion, the counter-staining solution has the characteristic of short staining time, the appearance color of the specimen is bright after counter-staining, the background under a microscope is bright, and the acid-resistant bacillus can be identified by inspectors more conveniently, so that the detection rate of the acid-resistant bacillus is improved. The staining solution is easy to stain, and the stained specimen has bright color, is convenient to observe, and can improve the detection rate of the mycobacterium tuberculosis.
Compared with the classical old dyeing method (the Zille method), the preparation method can reduce the preparation steps of the dyeing solution, avoid repeatedly grinding the basic reddish dye in the preparation process, prevent the environment pollution caused by reagent overflow in the mixing process and improve the preparation speed of the working solution. The preparation method is simple and convenient to operate, unnecessary workload can be reduced, laboratory pollution is reduced, and working efficiency is improved.
According to the invention, through the steps of dyeing-decoloring-redyeing, the coloring effect of the specimen after dyeing can be improved, the color depth of the specimen is deepened, the acid-fast bacillus can be more conveniently identified by an inspector, the working efficiency is improved, the specimen dyeing cost can be reduced, and the method has better economic benefit.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.
An acid-fast staining solution for mycobacterium tuberculosis, comprising:
the working solution, taking 500mL as an example, comprises the following components: 2-8g of basic fuchsin, 40-60mL of 95% ethanol, 20-30mL of liquid carbolic acid and the balance of distilled water;
the destaining solution, taking 500mL as an example, comprises the following components: 20-50mL of concentrated hydrochloric acid with the mass fraction of 36-38% and 450-480mL of 95% ethanol;
the compound dyeing liquid, taking 500mL as an example, comprises the following components: 0.5-1g of methylene blue, 40-60mL of 95% ethanol and the balance of distilled water.
Basic fuchsin, an organic compound of formula C 19 H 18 ClN 3 It is mainly used as biological coloring agent, and is red-purple when dissolved in cold and hot water, and is very easy to dissolve in alcohol.
Carbolic acid, also known as phenol, is an organic compound formed by directly linking a hydroxyl group (-OH) with an aromatic nucleus (a benzene ring or a fused benzene ring).
Methylene blue is an aromatic heterocyclic compound, and an aqueous solution of methylene blue is blue in an oxidizing environment.
The final concentration of the counterstain solution can reach 0.16% in the proportion, the counterstain solution has the characteristic of short staining time, the appearance of the specimen is bright after counterstain, the background under a microscope is bright, and the inspector can conveniently identify the acid-resistant bacillus, so that the detection rate of the acid-resistant bacillus is improved. The staining solution is easy to stain, and the stained specimen has bright color, is convenient to observe, and can improve the detection rate of the mycobacterium tuberculosis.
Compared with a kit (produced by Wuhan Beisuo company), the staining solution of the invention has the advantages that the sputum membrane has slightly light appearance color after staining by the kit, the background under a microscope is dark, the kit is expensive, and the cost of each sputum specimen is about 5 yuan. The sputum slice dyed by the dyeing liquid is easy to observe, the cost is low, each sputum specimen is about 1 yuan, the working cost can be reduced, and the economic efficiency is improved.
A preparation method of mycobacterium tuberculosis acid-fast staining solution comprises the following steps:
adding 95% ethanol into basic fuchsin, shaking for 5-15min to obtain fuchsin solution, heating carbolic acid in water bath at 40-50 ℃ for liquefaction, then adding liquid carbolic acid into the fuchsin solution, adding distilled water to a constant volume of 500mL, and mixing to obtain working solution;
adding concentrated hydrochloric acid into 95% ethanol at a rate of 0.1-0.8mL/min, and mixing to obtain decolorized solution;
adding methylene blue into 95% ethanol, shaking for 5-15min, and adding distilled water to a constant volume of 500mL to obtain a counterstain solution.
When preparing the carbolic acid reddening working solution by a classical old dyeing method (a Nie method), three steps are needed, namely firstly preparing an alkaline reddening storage solution, then preparing a carbolic acid aqueous solution, and finally mixing the alkaline reddening storage solution and the carbolic acid aqueous solution according to a proportion. The steps are complicated, the reagent is easy to overflow, the environment is polluted, and the alkaline reddish dye needs to be repeatedly milled in the preparation process, so that the labor intensity is increased. The invention only needs to weigh two dyes of alkaline re-reddening and liquid carbolic acid (phenol) and pour the dyes into the same reagent bottle for mixing, can prepare a large amount of carbolic acid re-reddening working solution at one time, is beneficial to obviously improving the working efficiency, and is suitable for large-scale screening of sputum specimens of suspected tuberculosis patients in laboratories of rural and town health hospitals.
The preparation of methylene blue working solution by the classical old dyeing method (the Cel-Nie method) needs two steps, namely, a methylene blue storage solution is prepared firstly, then the storage solution is diluted by 5 times of distilled water, and the final concentration is 0.06%. When a large-scale sputum specimen is screened in a laboratory of a rural and township health institute, a large amount of mucus sputum and saliva sputum are used, a sputum film is thin and even drops after smear dyeing, when the old methylene blue working solution is used for counterdyeing, the dyeing time is long, staining is not easy, the appearance color of a dyed sputum piece is light, the background under a microscope is dark, and acid-fast bacillus is not easy to distinguish. The methylene blue working solution prepared by the method has high concentration and short coloring time, the appearance color of the counterstained sputum slice is bright, the background under a microscope is bright, and acid-fast bacilli are easier to identify for inexperienced inspectors, so that the detection rate is improved.
Compared with the classical old dyeing method (the common Zille-Nicol method), the preparation method of the invention can reduce the preparation steps of the dyeing liquid, avoid repeatedly grinding the basic reddish dye in the preparation process, prevent the environment pollution caused by reagent overflow in the mixing process and improve the preparation speed of the working liquid. The preparation method is simple and convenient to operate, unnecessary workload can be reduced, laboratory pollution is reduced, and working efficiency is improved.
A staining method of mycobacterium tuberculosis acid-fast staining solution comprises the following steps:
s1, heating and fixing a specimen smear, dripping working solution on the surface of the specimen smear, heating for 5-10min, standing for 5-15min, and washing with water;
s2, dripping a decoloring solution from the outer edge of the specimen smear washed by the water in the S1, decoloring for 1min, and washing with water;
and S3, dripping a re-staining solution into the specimen smear washed by the water in the S2, staining for 30S, washing by the water, drying and performing microscopic examination.
In step S1, the specimen is covered with the working solution when the working solution is dropped, and the specimen is always covered with the working solution during the staining, and the staining solution may be added continuously if necessary.
In step S3, the dried specimen is visually bright blue without red patches, and is a qualified slide.
Through the steps of dyeing, decoloring and counterdyeing, the staining effect of the stained specimen can be improved, the color depth of the specimen is deepened, the acid-fast bacillus can be more conveniently identified by inspectors, the working efficiency is improved, the specimen staining cost can be reduced, and the method has better economic benefit.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
A preparation method of mycobacterium tuberculosis acid-fast staining solution comprises the following steps:
the working solution, taking 500mL as an example, comprises the following components: 4g of basic fuchsin, 50mL of 95% ethanol, 22.5mL of liquid carbolic acid and the balance of distilled water;
the preparation method of the working solution comprises the following steps: adding 95% ethanol into basic fuchsin, shaking for 10min to obtain fuchsin solution, heating and liquefying carbolic acid in a water bath at 45 ℃, then adding liquid carbolic acid into the fuchsin solution, adding distilled water to a constant volume of 500mL, and mixing to obtain a working solution;
the destaining solution, taking 500mL as an example, comprises the following components: 35mL of concentrated hydrochloric acid with the mass fraction of 36% and 465mL of 95% ethanol;
the preparation method of the destaining solution comprises the following steps: adding concentrated hydrochloric acid into 95% ethanol at a rate of 0.2mL/min, and mixing to obtain decolorized solution;
the compound dyeing liquid, taking 500mL as an example, comprises the following components: 0.8g of methylene blue, 50mL of 95% ethanol and the balance of distilled water;
the preparation method of the dyeing liquor comprises the following steps: adding methylene blue into 95% ethanol, shaking for 10min, and adding distilled water to a constant volume of 500mL to obtain a double-dyeing liquid.
Example 2
A preparation method of mycobacterium tuberculosis acid-fast staining solution comprises the following steps:
the working solution, taking 500mL as an example, comprises the following components: 2g of basic fuchsin, 40mL of 95% ethanol, 20mL of liquid carbolic acid and the balance of distilled water;
the preparation method of the working solution comprises the following steps: adding 95% ethanol into basic fuchsin, shaking for 5min to obtain fuchsin solution, heating and liquefying carbolic acid in a water bath at 40 ℃, then adding liquid carbolic acid into the fuchsin solution, adding distilled water to a constant volume of 500mL, and mixing to obtain a working solution;
the destaining solution, taking 500mL as an example, comprises the following components: 20mL of concentrated hydrochloric acid with the mass fraction of 36% and 480mL of 95% ethanol;
the preparation method of the destaining solution comprises the following steps: adding concentrated hydrochloric acid into 95% ethanol at a rate of 0.1mL/min, and mixing to obtain decolorized solution;
the compound dyeing liquid, taking 500mL as an example, comprises the following components: 0.5g of methylene blue, 40mL of 95% ethanol and the balance of distilled water;
the preparation method of the dyeing liquor comprises the following steps: adding methylene blue into 95% ethanol, shaking for 5min, and adding distilled water to a constant volume of 500mL to obtain a double-dyeing liquid.
Example 3
A preparation method of mycobacterium tuberculosis acid-fast staining solution comprises the following steps:
the working solution, taking 500mL as an example, comprises the following components: 8g of basic fuchsin, 60mL of 95% ethanol, 30mL of liquid carbolic acid and the balance of distilled water;
the preparation method of the working solution comprises the following steps: adding 95% ethanol into basic fuchsin, shaking for 15min to obtain fuchsin solution, heating and liquefying carbolic acid in a water bath at 50 ℃, then adding liquid carbolic acid into the fuchsin solution, adding distilled water to a constant volume of 500mL, and mixing to obtain a working solution;
the destaining solution, taking 500mL as an example, comprises the following components: 50mL of concentrated hydrochloric acid with the mass fraction of 38% and 450mL of 95% ethanol;
the preparation method of the destaining solution comprises the following steps: adding concentrated hydrochloric acid into 95% ethanol at a rate of 0.8mL/min, and mixing to obtain decolorized solution;
the compound dyeing liquid, taking 500mL as an example, comprises the following components: 1g of methylene blue, 60mL of 95% ethanol and the balance of distilled water;
the preparation method of the dyeing liquor comprises the following steps: adding methylene blue into 95% ethanol, shaking for 15min, and adding distilled water to a constant volume of 500mL to obtain a double-dyeing liquid.
Example 4
A staining method of mycobacterium tuberculosis acid-fast staining solution comprises the following steps:
s1, heating and fixing a specimen smear, dripping working solution to the surface of the specimen smear, heating for 5min, standing for 5min, and washing with water;
s2, dripping a decoloring solution from the outer edge of the specimen smear washed by the water in the S1, decoloring for 1min, and washing with water;
and S3, dripping a re-staining solution into the specimen smear washed by the water in the S2, staining for 30S, washing by the water, drying and performing microscopic examination.
Test results
The method comprises the steps of randomly selecting 100 parts of qualified sputum specimens of patients with tuberculosis prevention and treatment outpatient clinic daily visits for 3-10 months in 2015 by a Guangfeng region disease prevention and control center, coating 3 sputum specimens on each specimen, dyeing by using a dyeing solution prepared by an old method (Zille-Niber dyeing), dyeing by using a kit reagent (produced by Wuhan Betso Co., ltd.) for 1 piece, and dyeing by using the dyeing solution prepared in the embodiment 1 of the invention. All 3 smears were uniformly stained by the staining method of the present invention.
The preparation method of the sputum smear comprises the following steps:
1. using a new scratch-free glass slide with a frosted surface at one end, degreasing by 95% ethanol, drying, and cleaning for later use;
2. the experimental serial number and the sample serial number are marked on the frosted surface by using a 2B pencil;
3. ensure that the serial number of the slide is the same as that on the sputum box
4. In the biological safety cabinet, the container for bearing the sputum sample is carefully opened to prevent aerosol or overflow of the sample;
5. carefully observing the specimen, using the broken bamboo stick stubble end, picking out cheese sample, pus sample or about 0.05mL of part of the cheese sample and the pus sample in the sputum specimen, and slightly and annularly and uniformly smearing the cheese sample, the pus sample or the part of the pus sample on the front surface of the glass slide to form an oval sputum membrane of 10mm multiplied by 20 mm;
6. standing the sputum membrane upwards in a biological safety cabinet for natural drying, and dyeing.
Grading standard of microscopic examination result:
1. acid fast bacilli negative with ehl-niemann stain: continuously observing 300 different visual fields, and not finding acid-fast bacillus;
2. number of positive acid-fast bacilli of Ziehelia nileriensis staining: 1-8 strips/300 field of view;
3. ehl-niemann-staining positive for acid fast bacilli (1 +): 3-9 strips per 100 fields, and continuously observing 300 fields;
4. ehl-niemann-staining positive for acid fast bacilli (2 +): 1-9 strips/10 fields, and continuously observing 100 fields;
5. ehl-niemann-staining positive for acid fast bacilli (3 +): 1-9 strips/1 field;
6. ehl-niemann-staining positive for acid fast bacilli (4 +): not less than 10 strips/1 visual field.
At least 300 fields were observed when "1+" was reported, at least 100 fields were observed when "2+" was reported, and at least 50 fields were observed when "3+", and "4 +".
TABLE 1 analysis of the stained sputum smear
As can be seen from Table 1, the sputum smear of example 1 was able to observe more sputum cells in the same field of view after staining, indicating that the sputum cells were better observed after staining with the staining solution of example 1. After staining, the size of the sputum smear of example 1 was comparable to that of the sputum smear of the kit group, indicating that the sputum smear of example 1 and the kit had less effect on the size after staining. The thickness of the sputum smear of the example 1, the reagent kit group and the old staining solution group is equivalent, which shows that the thickness of the sputum smear is slightly influenced after the three groups are stained. The apparent staining number of example 1 was much greater than the kit and old staining solution groups, indicating that the staining solution formulated in example 1 was more prone to staining when stained.
TABLE 2 grading of sputum smear staining results
| Group of | 1-8 strips/example | 1 +/example | 2 +/example | 3 +/example | 4 +/example |
| Reagent kit group | 3 | 9 | 4 | 4 | 1 |
| Old dyeing liquid group | 3 | 9 | 4 | 4 | 1 |
| Example 1 | 3 | 9 | 4 | 4 | 1 |
As is clear from Table 2, 21 positive specimens and 79 negative specimens were examined in 3 groups of 100 specimens, and the results were consistent. And 3 groups of positive tablets are classified according to positive grades, 1-8 strips: 3 examples, 1+:9 cases, 2+:4 cases, 3+:4 examples, 4+: in 1 case, the dyeing results of the three groups are consistent.
In addition, through microscope observation, the sputum membrane of one group of smears dyed by the staining solution prepared in the embodiment of the invention is more in accordance with the standardized requirements than the appearance color of the other 2 groups of smears, the background under the microscope is brighter and brighter than the other 2 groups of smears, while the sputum membrane of the sputum sheet dyed by the reagent box group and the old staining solution group is lighter in color, and the background under the microscope is dark.
In conclusion, the mycobacterium tuberculosis acid-fast staining solution provided by the embodiment of the invention has the characteristics that the final concentration of the re-staining solution can reach 0.16% in the above proportion, the staining time is short, the appearance color of the specimen after re-staining is bright, the background under a microscope is bright, and the inspector can conveniently identify the acid-fast bacillus, so that the detection rate of the acid-fast bacillus is improved. The staining solution is easy to stain, and the stained specimen has bright color, is convenient to observe, and can improve the detection rate of the mycobacterium tuberculosis.
Compared with the classical old staining method (the Ziehelian method), the preparation method of the mycobacterium tuberculosis acid-fast staining solution provided by the embodiment of the invention can reduce the preparation steps of the staining solution, avoid repeated grinding of basic red-recovery dye in the preparation process, prevent the environment pollution caused by reagent overflow in the mixing process, and improve the preparation speed of the working solution. The preparation method is simple and convenient to operate, unnecessary workload can be reduced, laboratory pollution is reduced, and working efficiency is improved.
According to the staining method of the mycobacterium tuberculosis acid-fast staining solution provided by the embodiment of the invention, through the steps of staining, decoloring and redyeing, the staining effect of the stained specimen can be improved, the color depth of the specimen is deepened, the acid-fast bacillus can be more conveniently identified by inspectors, the working efficiency is improved, the staining cost of the specimen can be reduced, and the method has better economic benefits.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (7)
1. An acid-fast staining solution for mycobacterium tuberculosis, which is characterized by comprising:
the working solution, taking 500mL as an example, comprises the following components: 2-8g of basic fuchsin, 40-60mL of 95% ethanol, 20-30mL of liquid carbolic acid and the balance of distilled water;
the destaining solution, taking 500mL as an example, comprises the following components: 20-50mL of concentrated hydrochloric acid with the mass fraction of 36-38% and 450-480mL of 95% ethanol;
the compound dyeing liquid, taking 500mL as an example, comprises the following components: 0.5-1g of methylene blue, 40-60mL of 95% ethanol and the balance of distilled water.
2. A method for preparing an acid-fast staining solution for Mycobacterium tuberculosis according to claim 1, comprising the steps of:
working fluid: adding 95% ethanol into basic fuchsin, shaking for 5-15min to obtain fuchsin solution, heating and liquefying carbolic acid in an acid water bath, then adding liquid carbolic acid into the fuchsin solution, adding distilled water to a constant volume of 500mL, and mixing;
decoloring liquid: adding concentrated hydrochloric acid into 95% ethanol, and mixing;
dyeing liquor again: adding methylene blue into 95% ethanol, shaking for 5-15min, and adding distilled water to a constant volume of 500mL.
3. The method for preparing an acid-fast staining solution for Mycobacterium tuberculosis as claimed in claim 2, wherein the heating temperature is 40-50 ℃ when the carbolic acid is heated and liquefied in the acid bath.
4. The method for preparing an acid-fast staining solution for Mycobacterium tuberculosis as claimed in claim 3, wherein the addition rate of concentrated hydrochloric acid to 95% ethanol is 0.1-0.8mL/min.
5. A staining method of mycobacterium tuberculosis acid-fast staining solution as set forth in any one of claims 2 to 4, comprising the steps of:
s1, heating and fixing a specimen smear, dripping working solution on the surface of the specimen smear, heating for 5-10min, standing for 5-15min, and washing with water;
s2, dripping a decoloring solution from the outer edge of the sample smear washed by the water in the S1, decoloring and washing;
and S3, dripping a re-staining solution into the specimen smear washed by the water in the S2, and carrying out dyeing, washing, drying and microscopic examination.
6. The method for staining Mycobacterium tuberculosis with an acid-fast staining solution according to claim 5, wherein, in step S2, the staining is performed for 1min.
7. The method for staining Mycobacterium tuberculosis acid-fast staining solution according to claim 5, wherein in step S3, the staining is performed for 30S.
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