CN113375999B - Acid-fast dyeing method and quality monitoring method - Google Patents

Acid-fast dyeing method and quality monitoring method Download PDF

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CN113375999B
CN113375999B CN202110575523.9A CN202110575523A CN113375999B CN 113375999 B CN113375999 B CN 113375999B CN 202110575523 A CN202110575523 A CN 202110575523A CN 113375999 B CN113375999 B CN 113375999B
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formaldehyde
acid
dyeing
incubation
dewaxing
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CN113375999A (en
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钟学军
黎锋
孙成刚
赖辉龙
张�杰
覃韦双
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Guangzhou Kingmed Diagnostics Central Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G01N21/783—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour for analysing gases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving

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Abstract

The invention relates to an acid-fast staining method and a quality monitoring method, and belongs to the technical field of tissue staining. The method comprises a formaldehyde detection step in the dyeing incubation process, and when the formaldehyde concentration in the detection incubation environment is smaller than a threshold value, the dyeing process is judged to be qualified, otherwise, the dyeing process is judged to be unqualified. The method can monitor the quality of the anti-acid dyeing process, avoid the problem of incapability of decoloring and improve the detection accuracy.

Description

Acid-fast dyeing method and quality monitoring method
Technical Field
The invention relates to the technical field of tissue staining, in particular to an acid-resistant staining method and a quality monitoring method.
Background
Acid fast staining is a method commonly used for revealing mycobacterium such as tubercle bacillus, leprosy bacillus and the like. The cell wall of mycobacteria contains a large amount of lipids, and when combined with a phenol basic fuchsin dye, resists the decolorization of acidic decolorizers (hydrochloric acid, sulfuric acid, etc.), and is also known as acid fast bacilli.
In acid fast staining, the step of decolorizing and differentiating with acidic decolorizing agent is important, and if decolorizing is not clean, the whole tissue section background is red, which brings great interference to the observation of mycobacteria under the lens. Sometimes, a red dyeing of an acid-fast dyeing background occurs in the laboratory process, and the phenomenon that the background cannot be removed cleanly after the background is decolored by 20% sulfuric acid. If the decolorization is not clean, the detection of the tubercle bacillus can be influenced, and the detection result can be influenced.
Disclosure of Invention
Accordingly, it is desirable to provide an acid-fast dyeing quality monitoring method for quality monitoring of an acid dyeing process to avoid the problem of incapability of decoloring.
A method for monitoring acid-resistant dyeing quality includes formaldehyde detection step in the dyeing incubation process, judging that the dyeing process is qualified when the formaldehyde concentration in the detection incubation environment is smaller than a threshold value, otherwise judging that the dyeing process is unqualified.
The inventor found in experiments that a red staining of an acid-fast staining background sometimes occurs in the laboratory process, and the phenomenon that the red staining cannot be removed by 20% sulfuric acid for decolorization is also caused. Through a systematic review and analysis of the problem, it was confirmed that the cause of the problem was the interference of formaldehyde in the dyeing environment.
After the reason is found, the acid-resistant dyeing quality monitoring method is provided, and the problem that the dyeing cannot be decolorized after the subsequent dyeing is avoided by monitoring the method, so that the detection accuracy is improved.
In one embodiment, the threshold is 0.20.+ -. 0.10mg/m 3 。
In one embodiment, the formaldehyde detection step is: and placing the formaldehyde detection reagent in an incubation box, detecting the concentration of formaldehyde in the incubation box, taking the formaldehyde detection reagent out after the formaldehyde detection reagent is judged to be qualified, and placing the slice to be dyed in the incubation box for dyeing.
The invention also discloses an acid-fast staining method, which comprises the following steps:
dewaxing: taking a tissue embedding slice, and dewaxing;
removing formaldehyde: taking an incubation box, removing formaldehyde in the incubation box, detecting the formaldehyde concentration in the incubation box by the formaldehyde detection method, and continuously removing the formaldehyde in the incubation box when the detection result shows that the formaldehyde concentration is greater than or equal to a threshold value until the detection result shows that the formaldehyde concentration is less than the threshold value;
dyeing: and placing the slices to be dyed after dewaxing treatment in an incubation box, dripping phenol alkaline fuchsin liquid as a dye liquid, and carrying out acid-resistant dyeing.
In one embodiment, in the formaldehyde removal step, formaldehyde in the incubation cassette is removed by: soaking in 84 disinfectant, cleaning, washing with running water, and air drying.
In one embodiment, the formaldehyde removal step is performed by soaking in 84 disinfectant for 1-2 hours and rinsing with running water for 5-10 minutes.
In one embodiment, the dewaxing step is performed using a xylene-free dewaxing agent.
In one embodiment, the phenol basic fuchsin liquid is formulated by the following method: dissolving 5g of basic fuchsin in 100ml of ethanol solution with the volume percentage concentration of 95% to obtain basic fuchsin ethanol solution; mixing 10ml basic fuchsin ethanol solution with 90ml5 v% (volume percent) phenol water solution.
In one embodiment, in the staining step, the acid fast staining further comprises the steps of:
1) Taking out tissues from the dye liquor, and washing off superfluous dye liquor by running water;
2) Differentiating with sulfuric acid until the tissue color is reddish after the washing with flowing water;
3) Continuously flushing with running water;
4) Lining with hematoxylin dye liquor;
5) Flushing with running water;
6) The tissue is air-dried or baked, transparentized with xylene, and then fixed with neutral resin.
In one embodiment, in the step 2), the sulfuric acid concentration is 20+ -5 v%, and the Mayer hematoxylin dye solution is used for padding for 30+ -5 s.
Compared with the prior art, the invention has the following beneficial effects:
according to the acid-resistant dyeing quality monitoring method, the formaldehyde detection step is added in the dyeing incubation process, so that the problem that the subsequent dyeing cannot be decolorized is avoided, and the detection accuracy is improved.
According to the acid-resistant staining method, through the steps of formaldehyde removal and the like, the incubation process is ensured to be carried out in an environment with the formaldehyde content meeting the requirement, so that the problem that the subsequent staining cannot be decolorized is avoided, the obtained sections are easy to observe, the contrast is clear, and the detection accuracy is improved.
Drawings
FIG. 1 is a view under a microscope of the tissue incapable of being decolorized in example 1 (40X);
FIG. 2 is a view under a normal stained tissue microscope (40X) in example 1;
FIG. 3 is a view under a group C tissue microscope (40X) of example 1;
FIG. 4 is a view under a group D tissue microscope (40X) of example 1.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The reagents used in the following examples are commercially available unless otherwise specified; the methods used in the examples below, unless otherwise specified, were all conventional.
Example 1
The cause of the occurrence of discoloration in acid-fast staining was investigated.
1. Problems were found.
The inventors of the present invention conducted acid fast staining according to a conventional method, found that a red staining of the acid fast stained background was sometimes observed, and the phenomenon that the removal of the red stain was impossible even if the red stain was decolorized with 20% sulfuric acid was observed, the section was dark red, and the whole of the tissue section was purplish red when observed under a mirror as shown in FIG. 1, the background was not clear, the contrast was poor, and it was difficult to distinguish whether or not mycobacteria were present.
After re-slicing and staining, the slice was observed visually to be reddish, as shown in FIG. 2, and the tubercle bacillus was observed to be reddish, the number was large, the observation was easy, the background was bluish, and the contrast was clear.
2. And (5) primary screening of reasons.
To systematically find the root of the problem, we list the key links and elements in the experimental process for systematic review and analysis.
1) Personnel: review of the list of operators who have had this problem, find that almost all technicians have had this problem, and initially eliminate the human operator.
2) Reagent: the same reagent is reworked for one time, and the problem of incomplete background decolorization is eliminated. Factors of the reagent are primarily excluded.
3) The operation method comprises the following steps: technicians all operate strictly according to SOP of departments, and abnormal irregular operation is not found. In the dyeing process, the phenomenon of dry sheets does not occur.
4) Environment: no abnormalities were found in both the storage environment of the retrospective reagent and in the humidity environment of the laboratory room temperature. Is it suggested by the skilled artisan that what is caused by interference from other staining reagents during acid fast staining? Thus reviewing the last few experimental recordings of this problem, it was found that acid fast staining and sirius scarlet staining were performed simultaneously in the same covered incubator. The reagent that stains sirius scarlet is primarily suspected to interfere with acid fast staining.
3. The reasons are explored.
To confirm the above hypothesis, the following experiments were designed:
1) The method comprises the following steps: 6 tissue sections positive for the known tubercle bacillus were taken and divided into A, B groups of 3 sections each. The acid-fast staining is carried out on the group A alone, and the acid-fast staining is carried out on the group B and 10 slices with the picric acid-sirius scarlet dye solution dropwise added in the same incubation box.
2) Results: the A group slice can be successfully differentiated after dyeing, the red dyed background is decolorized, the macroscopic tissue is slightly light red after flushing, the background is clean after microscopic observation, and the tubercle bacillus can be clearly observed; the differentiation conditions of the B-group section are the same as those of the A-group section, but the red-stained background cannot be decolorized (even if the differentiation time is prolonged), the tissue is deeply red by naked eyes, the background of the tissue section is also deep purple when observed under a lens, and whether mycobacteria exist or not is difficult to distinguish.
Experimental results prove that the problem of unclean acid-fast staining and decolorization is caused by interference of picric acid-sirius scarlet dye liquor. The components of the picric acid-sirius scarlet dye liquor comprise saturated picric acid liquor and sirius scarlet dye. Because picric acid belongs to a controlled reagent and is relatively difficult to be purchased in the market at present, the sirius dye liquor is prepared by using the bouin's fixing liquor to replace the saturated picric acid aqueous solution, and the dyeing effect is satisfactory. The components of bouin's fixative include: picric acid, formaldehyde, glacial acetic acid. Therefore, the picric acid-sirius scarlet dye liquor used by us comprises the following components: picric acid, formaldehyde, glacial acetic acid, and sirius scarlet.
To confirm the interference caused by which specific reagent components in picric acid-sirius scarlet dye liquor, the following test was further designed: taking 5 incubation boxes with covers, wherein the number of the incubation boxes is C, D, E, F, G, 5 groups are altogether, 10 clean blank glass slides are laid in each incubation box, and the 10 glass slides in each group are respectively dripped with interfering reagents: 1ml each of 0.1% aqueous solution of sirius red was added to group C, 1ml each of 25% aqueous solution of formaldehyde was added to group D, 1ml each of 5% aqueous solution of glacial acetic acid was added to group E, 1ml each of saturated aqueous solution of picric acid was added to group F, and 1ml each of distilled water was added to group G.
Selecting one of known tubercle bacillus positive control wax blocks, continuously slicing 15 sheets, dewaxing to water, tiling 3 sheets in each of the groups C-G, dripping phenol basic fuchsin liquid, covering and dyeing for 30 minutes at room temperature, differentiating for 10 minutes with 20% sulfuric acid, and shallow staining cell nuclei with Mayer hematoxylin for 1 minute.
The experimental results are shown in the following table, the C, E, F, G group slice background is light blue, the tubercle bacillus is red and thin rod-shaped, and the comparison is clear; the background of the slice of the group D is dark purple, and tubercle bacillus is difficult to observe, and the contrast ratio is poor.
TABLE 1 test results
As shown in the above results, the 25% aqueous formaldehyde solution of group D interferes with the acid fast staining, resulting in red staining of the whole acid fast stained tissue section, which is difficult to decolorize and differentiate cleanly with 20% sulfuric acid, and tuberculosis is difficult to observe under the lens. C. The reagent of the E, F, G group does not contain formaldehyde component, and after acid-fast staining of the tissue slices, the tissue slices can be normally differentiated and decolored by using 20% sulfuric acid solution, and tubercle bacillus is easy to observe under a lens, so that the existence of formaldehyde is judged to interfere with the decoloring process of the acid-fast stained tissue slices.
Formaldehyde is a common reagent in the pathology department, is colorless aqueous solution or gas, has pungent odor, and is easy to volatilize. The inventors of the present invention confirmed through the above experiments that formaldehyde volatilized in a closed incubator to form vapor, and mixed into a phenol basic fuchsin dye solution, and the aldehyde group acted on basic fuchsin to form a mauve stable complex, so that decolorization and differentiation were difficult with sulfuric acid solution. To solve this problem, the acid-fast staining is performed in the same closed environment (e.g. in an incubation box) as the reagent containing formaldehyde, and the reagent and the vessel in each step of the staining process are prevented from being polluted by formaldehyde. The usual special dyeings containing formaldehyde are also reticular fibre dyeings, the Masson trichromatic method, etc., and particular attention is paid to avoiding cross-contamination by these dyeings during acid-fast dyeings. Therefore, according to the requirements of the quality control system of the pathology department, we prescribe in the working instruction for acid fast staining: acid-fast staining is performed by using a special incubation box, and can not be performed with other stained sections; after the dyeing is completed every day, technicians must clean and air-dry the incubation box, so as to ensure that the incubation box is clean and free of formaldehyde pollution before use. The problem of unclean differentiation and decolorization after acid-fast dyeing is thoroughly solved.
Example 2
And (5) researching formaldehyde content standard in acid-fast dyeing quality monitoring.
1. The method.
An environmental formaldehyde concentration monitoring link is arranged in the experimental process, and the influence of acid dyeing resistance under different environmental formaldehyde concentration conditions is compared.
2. As a result.
After finishing the experimental results, the following steps are found: when the formaldehyde concentration in the environment is less than 0.20mg/m 3 When the slice is dyed, the acidic decolorizer can be used smoothlyDecoloring and differentiating, washing the slices with water, visually observing the tissue to be slightly light red, observing the tissue under a microscope to see that the background is clean, and clearly observing the tubercle bacillus; when the formaldehyde concentration in the environment is more than 0.20mg/m 3 When the red background of the slice is not decolorized by an acidic decolorizer, the tissue is deeply red after the slice is washed by water, and the background of the tissue slice is also deep purple when being observed under a microscope, so that whether mycobacteria exist or not is difficult to distinguish. And the higher the formaldehyde concentration in the environment, the poorer the decolorization effect with an acidic decolorizer.
That is, in acid fast staining, the tolerable formaldehyde concentration threshold is 0.20.+ -. 0.10mg/m 3 。
Example 3
An acid-resistant staining quality monitoring method comprises formaldehyde detection steps in the staining incubation process, and specifically comprises the following steps:
1. the method comprises the following steps: the formaldehyde detection reagent is placed at one corner of an incubation box (transparent and covered, and the condition in the box is convenient to observe), an absorption box in the formaldehyde detection reagent box (brand: green source, model: Z-0434) is placed, and then all detection liquid in the formaldehyde detection reagent box is poured into the absorption box and uniformly mixed. The lid of the incubation box was closed and the reagents were allowed to react for 30 minutes. And pouring the color reagent in the formaldehyde detection kit into an absorption box, uniformly mixing, and standing for 10 minutes.
2. Observation results: and (3) colorimetric color comparison is carried out by using the absorption box and a colorimetric card in the formaldehyde detection kit, so as to obtain a formaldehyde concentration result in the incubation box.
3. And (3) monitoring and judging: when the formaldehyde concentration in the detection incubation environment is less than 0.20mg/m 3 If the dyeing process is judged to be qualified, the subsequent dyeing process can be continued, otherwise, the dyeing process is judged to be unqualified, and formaldehyde removal measures are continued to be taken for the incubation box.
Example 4
An acid-fast staining quality monitoring method comprises the following steps:
1. reagent preparation
(1) Basic fuchsin ethanol solution: basic fuchsin 5g+95% ethanol 100ml;
(2) Phenol basic fuchsin liquid: 90ml of 5% phenol water solution and 10ml of alkaline fuchsin ethanol solution;
2. dyeing step
(1) Tissue was fixed in 10% neutral buffered formalin fixation, routinely dehydrated embedded; the slice thickness was 4. Mu.m.
(2) Dewaxing with an environment-friendly dewaxing agent: ( Name of dewaxing agent: EZ PREP SOLUTION, manufacturer: roche diagnostic product (Shanghai) limited, product characteristics: the water-soluble reagent does not contain dimethylbenzene, is not easy to destroy lipid on the cell wall of the tubercle bacillus, can keep the acid-resistant property of the tubercle bacillus, and improves the positive detection rate; xylene damages lipids on the cell wall of tubercle bacillus, and has low positive detection rate. )
The dewaxing procedure was as follows:
1. a first cylinder: EZ PREP SOLUTION for 5-10 minutes
2. A second cylinder: EZ PREP SOLUTION for 5-10 minutes
3. And a third cylinder: EZ PREP SOLUTION for 5-10 minutes
4. Flushing with running water for 3-5 min
(3) The incubation box removes formaldehyde: preparing an incubation box (transparent, with a cover, convenient for observing the condition in the box), and soaking for 1-2 hours with 84 disinfectant; then brushing and cleaning by using a test tube brush, flushing for 5-10 minutes by using flowing water, and airing. So as to be beneficial to clean interference components such as formaldehyde and the like possibly existing in the incubation box.
(4) Detecting formaldehyde concentration: and (3) using the formaldehyde detection kit, placing an absorption box in the formaldehyde detection kit at one corner of the incubation kit, and pouring all detection liquid in the formaldehyde detection kit into the absorption box to be uniformly mixed. The lid of the incubation box was closed and the reagents were allowed to react for 30 minutes. And pouring the color reagent in the formaldehyde detection kit into an absorption box, uniformly mixing, and standing for 10 minutes.
Observation results: and (3) colorimetric color comparison is carried out by using the absorption box and a colorimetric card in the formaldehyde detection kit, so as to obtain a formaldehyde concentration result in the incubation box. The detection result is more than or equal to 0.20mg/m 3 When the incubation box is needed, formaldehyde removal measures are needed again (namely, the step 3 is repeated).
(5) Formaldehyde concentration of incubation box<0.20mg/m 3 When in use, the anti-cancer agent can be normally usedAcid staining. Sections to be stained were placed flat in an incubation box, and phenol basic fuchsin solution (preferably with sufficient coverage of tissue) was added dropwise and stained at room temperature for 30 minutes.
(6) Excess dye liquor (phenol basic fuchsin liquor) is washed off by running water.
(7) 20% sulfuric acid is differentiated for 3-10 minutes.
(8) The tissue is washed clean by running water and has reddish color.
(9) If the slices are still red after flushing with running water, the slices can be differentiated by using 20% sulfuric acid for 10 seconds to 1 minute and immediately flushed. (if still too red, repeating the above differentiation steps).
(10) Flow flushing for 5-10 min
(11) Mayer hematoxylin dye liquor counterstain for 30 seconds.
(12) Washing with running water for 5-10 min.
(13) The tissue is air-dried or baked, the dimethylbenzene is transparent, and the neutral resin is fixedly sealed.
3. Dyeing results
Acid fast bacteria (including leprosy bacillus and tubercle bacillus) are red, cell nuclei are blue, and typical pictures are shown in fig. 2 and 3.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (6)

1. An acid fast staining method, comprising the steps of:
dewaxing: taking a tissue embedding slice, and dewaxing;
removing formaldehyde: taking an incubation box, removing formaldehyde in the incubation box, detecting the formaldehyde concentration in the incubation box, placing a formaldehyde detection reagent in the incubation box, and continuously removing the formaldehyde in the incubation box when the detection result shows that the formaldehyde concentration is greater than or equal to a threshold value, until the detection result shows that the formaldehyde concentration is less than the threshold value, wherein the threshold value is 0.20g/m 3 Placing the slice to be dyed into the incubation box for dyeing;
dyeing: placing the slices to be dyed after dewaxing treatment in an incubation box, dripping phenol alkaline fuchsin liquid as dye liquor, and carrying out acid-resistant dyeing;
taking out tissues from the dye liquor, and washing off superfluous dye liquor by running water;
differentiating with sulfuric acid until the tissue color is reddish after the washing with flowing water;
continuously flushing with running water;
lining with hematoxylin dye liquor;
flushing with running water;
air-drying or baking the tissue, performing transparency treatment by using dimethylbenzene, and then performing solid sealing by using neutral resin;
the incubation process is ensured to be carried out in an environment with the formaldehyde content meeting the requirement, the phenomenon that the subsequent dyeing cannot be decolorized is avoided, and the obtained section can observe tubercle bacillus and is clear in comparison.
2. The acid-fast staining method according to claim 1, wherein in the step of removing formaldehyde, formaldehyde in the incubation cassette is removed by: soaking in 84 disinfectant, cleaning, washing with running water, and air drying.
3. The acid fast staining method according to claim 2, wherein in the formaldehyde removal step, 84 disinfectant is used for soaking for 1-2 hours, and running water is used for washing for 5-10 minutes.
4. The acid fast staining method according to claim 1, wherein dewaxing is performed with a dewaxing agent containing no xylene in the dewaxing step.
5. The acid fast staining method according to claim 1, wherein the phenol basic fuchsin solution is formulated by the following method: dissolving 5g of basic fuchsin in 100ml of ethanol solution with the volume percentage concentration of 95% to obtain basic fuchsin ethanol solution; mixing 10ml basic fuchsin ethanol solution with 90ml 5% v/v phenol water solution.
6. The method according to claim 1, wherein the differentiation is carried out with sulfuric acid at a concentration of 20% v/v, and the cells are counterstained with Mayer hematoxylin for 30.+ -. 5s.
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