Dyeing reagent for dyeing helicobacter pylori as well as preparation method and dyeing method thereof
A staining reagent was prepared by combining eosin, methylene blue, aniline blue, and methanol with glycerol as a stabilizer, which solved the problem of poor staining effect of Helicobacter pylori in the existing technology and achieved significant color difference contrast and rapid and accurate diagnosis.
Patent Information
- Application Number
- CN202510889670.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-11-21
AI Technical Summary
Existing Helicobacter pylori staining reagents cannot significantly improve the color difference between Helicobacter pylori and other tissues, resulting in low diagnostic efficiency and easy misdiagnosis or missed diagnosis.
A staining reagent was prepared by using a combination of eosin, methylene blue, aniline blue, and methanol in a ratio of (0.3-0.4):1, with glycerol added as a stabilizer. The reagent was prepared through specific solution mixing and treatment methods. Combined with gradient alcohol dehydration and xylene clearing treatment, the staining effect was significantly improved.
It significantly improves the color contrast between Helicobacter pylori and other tissues, shortens staining time, improves diagnostic accuracy and efficiency, and is non-toxic to humans with good stability.
Smart Images
Figure CN120992298A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to a staining reagent for staining Helicobacter pylori, its preparation method, and the staining method thereof. Background Technology
[0002] Helicobacter pylori (Hp) is a Gram-negative bacterium that primarily lives in the human stomach and duodenum. Numerous studies have shown that Hp is closely related to the development and progression of various gastric diseases, including gastritis, peptic ulcers, and gastric cancer. Accurate detection of Hp is crucial for the diagnosis, treatment, and prevention of gastric diseases. Currently, commercially available reagents for diagnosing Hp have limitations in staining effectiveness; the stained Hp is not clearly distinguishable from other tissues, causing significant difficulties for pathologists and easily leading to misdiagnosis or missed diagnosis. Based on this situation, we have initiated the development of novel Hp diagnostic reagents, aiming to improve the color difference between Hp and other tissues, providing a more reliable tool for pathological diagnosis.
[0003] Helicobacter pylori infection is widespread globally, affecting approximately half of the world's population. In my country, the infection rate is also high, exceeding 60% in some areas. Long-term Helicobacter pylori infection can lead to a range of gastric diseases, such as chronic active gastritis, duodenal ulcers, and gastric ulcers, and is classified as a Group 1 carcinogen for gastric cancer. Early and accurate detection and timely treatment of Helicobacter pylori infection can effectively reduce the risk of related gastric diseases and improve patients' health.
[0004] Currently, commonly used clinical diagnostic methods for Helicobacter pylori include the urea breath test, gastroscopy, and histological staining. Histological staining involves staining gastric mucosal tissue sections and observing the morphology of Helicobacter pylori under a microscope to determine infection status. However, existing diagnostic reagents fail to create a clear contrast between Helicobacter pylori and surrounding tissues during the staining process. This is because Helicobacter pylori bacteria are small, and their morphology is similar to some cell structures in gastric mucosa, making the staining difference between the two insignificant with traditional staining agents. In actual diagnosis, pathologists need to spend considerable time and effort carefully distinguishing them, resulting in low diagnostic efficiency and increasing the possibility of misdiagnosis and missed diagnosis. Therefore, there is an urgent clinical need to develop a diagnostic reagent that can significantly improve the color difference between Helicobacter pylori and other tissues. Summary of the Invention
[0005] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide a staining reagent for staining Helicobacter pylori, as well as its preparation method and staining method, thereby solving the technical problem of existing diagnostic reagents that significantly improve the color difference between Helicobacter pylori and other tissues.
[0006] To achieve the above-mentioned technical objectives, the present invention provides a staining reagent for staining Helicobacter pylori, characterized in that it comprises eosin, methylene blue, aniline blue and methanol; the mass ratio of eosin to methylene blue is (0.3-0.4):1.
[0007] In any embodiment, glycerol is also included.
[0008] In any embodiment, the material ratio of eosin to methanol is (0.3-0.4) g:(100-110) mL, and the amount of glycerol used is 2%-5% of the volume of methanol.
[0009] Furthermore, the present invention also provides a method for preparing the above-mentioned staining reagent for Helicobacter pylori staining, comprising the following steps:
[0010] Eosin, methylene blue and some methanol are ground and mixed, and then the remaining methanol is added and mixed to obtain solution A;
[0011] Aniline blue and first water are mixed, then heated to dissolve at 60-65°C, and then glacial acetic acid solution is added to obtain solution B;
[0012] Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and second water are mixed and dissolved to obtain solution C;
[0013] The staining reagent is obtained by mixing the A solution, the B solution, and the C solution.
[0014] In any embodiment, the ratio of aniline blue to the first water is (1-1.5) g: 100 mL.
[0015] In any embodiment, the addition of glycerol after mixing with the remaining methanol further comprises the addition of the mixture to obtain solution A.
[0016] In any embodiment, the mass ratio of sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride is (0.7-0.9):(2-3):10; the mass ratio of sodium chloride and the second water is (1-1.5):100.
[0017] In any embodiment, the volume ratio of solution A, solution B, and solution C is (4-5):(1-2):(3-5).
[0018] In any embodiment, the volume ratio of solution A, solution B, and solution C is 5:2:3.
[0019] In addition, the present invention also proposes a staining method for Helicobacter pylori, comprising the following steps: adding the above-mentioned staining reagent or the staining reagent prepared by the above-mentioned preparation method to the prepared Helicobacter pylori slide, then rinsing with running water until the staining solution on the slide no longer decolorizes, then performing gradient alcohol dehydration and xylene clearing on the slide, and then drying and mounting the slide.
[0020] Compared with existing technologies, the beneficial effects of this invention include: the staining reagent for Helicobacter pylori staining proposed in this invention comprises eosin, methylene blue, aniline blue, and methanol; the mass ratio of eosin to methylene blue is (0.3-0.4):1; it can rapidly and specifically stain Helicobacter pylori, making it exhibit a clear color difference from surrounding tissues under a microscope, facilitating rapid and accurate identification by pathologists. The color contrast between stained Helicobacter pylori and other tissues is significantly improved, and the staining stability is good, achieving consistent staining results under different laboratory environments and operating conditions. Furthermore, it has no significant toxicity to human tissues, and staining can be completed in just 1-1.5 minutes. The staining reagent is more specific than existing reagents such as methylene blue. Attached Figure Description
[0021] Figure 1 These are photographs of the staining reagents from Examples 1 and 2 of this invention after being left for 48 hours.
[0022] Figure 2 These are staining results of Helicobacter pylori using the staining reagents of Example 1 and Comparative Example 1 of this invention.
[0023] Figure 3 These are staining results of Helicobacter pylori using the staining reagents of Example 1 and Comparative Example 1 of this invention.
[0024] Figure 4 These are staining effect images of Helicobacter pylori using the staining reagents of Examples 1 and 2 of this invention.
[0025] Figure 5 These are staining results of Helicobacter pylori using the staining reagents of Example 1 and Comparative Example 2 of this invention. Detailed Implementation
[0026] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values of 1 and 2 are listed, and if maximum range values of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.
[0027] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.
[0028] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).
[0029] This specific embodiment provides a staining reagent for staining Helicobacter pylori, characterized in that it comprises eosin, methylene blue, aniline blue and methanol; the mass ratio of eosin to methylene blue is (0.3-0.4):1.
[0030] In some embodiments, glycerol is also included; the ratio of eosin to methanol is (0.3-0.4) g:(100-110) mL, and the amount of glycerol used is 2%-5% of the volume of methanol. The addition of glycerol prolongs the shelf life and stability of the staining reagent and also improves the staining effect.
[0031] This specific embodiment also proposes a method for preparing the staining reagent for Helicobacter pylori staining as described above, including the following steps:
[0032] Eosin, methylene blue and some methanol are ground and mixed, and then the remaining methanol is added and mixed to obtain solution A;
[0033] Aniline blue and first water are mixed and then heated to dissolve at 60-65°C. Then, glacial acetic acid solution is added to obtain solution B. The material ratio of aniline blue to first water is (1-1.5) g: 100 mL.
[0034] Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and second water are mixed and dissolved to obtain solution C; the mass ratio of sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride is (0.7-0.9):(2-3):10; the mass ratio of sodium chloride and second water is (1-1.5):100.
[0035] In some embodiments, the volume ratio of solution A, solution B, and solution C is (4-5):(1-2):(3-5).
[0036] In some embodiments, the volume ratio of solution A, solution B, and solution C is 5:2:3.
[0037] In addition, this specific embodiment also proposes a staining method for Helicobacter pylori, including the following steps: adding the above-mentioned staining reagent or the staining reagent prepared by the above preparation method to the prepared Helicobacter pylori slide, then rinsing with running water until the staining solution on the slide no longer decolorizes, then performing gradient alcohol dehydration and xylene clearing on the slide, and then drying and mounting the slide.
[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0039] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.
[0040] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.
[0041] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.
[0042] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.
[0043] Example 1
[0044] This embodiment proposes a staining reagent for staining Helicobacter pylori, comprising eosin, methylene blue, aniline blue, and methanol; wherein the mass ratio of eosin to methylene blue is 0.4:1.
[0045] This embodiment also proposes a method for preparing the staining reagent for Helicobacter pylori staining as described above, including the following steps:
[0046] Solution A: Take 0.4g of eosin and 1g of methylene blue, put them in a mortar, and gently crush the dye into a finer powder with a grinding stick. Add 20ml of methanol, grind and dissolve. After the powder has dissolved, add 80ml of methanol, let stand for a while, pour the upper liquid into a brown empty bottle, seal and store away from light.
[0047] Solution B: Take 1.5g of aniline blue, add it to 100ml of distilled water (i.e., first water), place it in a 60℃ water bath, heat and stir to dissolve. After complete dissolution, allow it to cool naturally, add 1ml of 1% glacial acetic acid solution, and store it sealed and protected from light at 4℃.
[0048] Solution C: Take 0.7g of sodium dihydrogen phosphate (NaH2PO4), 2g of disodium hydrogen phosphate (Na2HPO4), and 10g of sodium chloride (NaCl) and add them to 1000ml of distilled water (i.e., second water). Stir to dissolve and then seal for later use.
[0049] The staining reagent is prepared by mixing solutions A, B, and C in a volume ratio of 5:2:3.
[0050] We found that the staining reagent prepared in Example 1 was volatile. Based on this, we further studied the formulation and unexpectedly found that adding glycerol could improve the stability of the staining reagent. Therefore, we proposed an improved scheme of adding glycerol.
[0051] Example 2
[0052] The staining reagent used for Helicobacter pylori staining in this embodiment differs from that in Example 1 in that 5 mL of glycerol is added to solution A. Specifically, solution A is prepared by: taking 0.4 g of eosin and 1 g of methylene blue, placing them in a mortar, and gently crushing the dyes into a finer powder using a grinding stick. 20 mL of methanol is added, and the mixture is ground and dissolved. After the powder has completely dissolved, 80 mL of methanol is added, and the mixture is allowed to stand for a moment. The supernatant is then poured into an empty brown bottle, 5 mL of glycerol is added, and the mixture is shaken well and sealed to protect from light. All other raw materials, dosages, and procedures are the same as in Example 1.
[0053] The staining reagent openings of Examples 1 and 2 were left to stand for 48 hours, and the results were as follows: Figure 1 As shown. Combined with Figure 1 As can be seen, the amount of staining reagent in Example 1 decreased significantly over time, while the amount of staining reagent in Example 2 decreased less. This indicates that the stability of the staining reagent in Example 2 is superior to that in Example 1. Extending the open storage time to 8 days, we found that the liquid portion of the staining reagent in Example 1 completely evaporated, while the staining reagent in Example 2 still contained a considerable amount of liquid.
[0054] Example 3
[0055] This embodiment proposes a staining reagent for staining Helicobacter pylori, comprising eosin, methylene blue, aniline blue, and methanol; wherein the mass ratio of eosin to methylene blue is 0.3:1.
[0056] This embodiment also proposes a method for preparing the staining reagent for Helicobacter pylori staining as described above, including the following steps:
[0057] Solution A: Take 0.3g of eosin and 1g of methylene blue, put them in a mortar, and gently crush the dye into a finer powder with a grinding stick. Add 20ml of methanol, grind and dissolve. After the powder has dissolved, add 80ml of methanol, let stand for a while, pour the upper liquid into a brown empty bottle, seal and store away from light.
[0058] Solution B: Take 1.5g of aniline blue, add it to 100ml of distilled water (i.e., first water), place it in a 65℃ water bath, heat and stir to dissolve. After complete dissolution, allow it to cool naturally, add 1ml of 1% glacial acetic acid solution, and store it sealed and protected from light at 4℃.
[0059] Solution C: Take 0.9g of sodium dihydrogen phosphate (NaH2PO4), 3g of disodium hydrogen phosphate (Na2HPO4), and 10g of sodium chloride (NaCl) and add them to 1000ml of distilled water (i.e., second water). Stir to dissolve and then seal for later use.
[0060] The staining reagent is prepared by mixing solutions A, B, and C in a volume ratio of 4:1:5.
[0061] Comparative Example 1
[0062] The staining reagent used in this comparative example was commercially available methylene blue.
[0063] Comparative Example 2
[0064] The difference between this comparative example and Example 1 is that solution B was not added, as detailed below:
[0065] This comparative example presents a staining reagent for staining Helicobacter pylori, comprising eosin, methylene blue, and methanol; wherein the mass ratio of eosin to methylene blue is 0.4:1.
[0066] This comparative example also proposes a method for preparing the staining reagent for Helicobacter pylori staining described above, comprising the following steps:
[0067] Solution A: Take 0.4g of eosin and 1g of methylene blue, put them in a mortar, and gently crush the dye into a finer powder with a grinding stick. Add 20ml of methanol, grind and dissolve. After the powder is completely dissolved, add 80ml of methanol, let stand for a while, pour the upper liquid into a brown empty bottle, add 5ml of glycerol, shake well, seal and store away from light.
[0068] Solution C: Take 0.7g of sodium dihydrogen phosphate (NaH2PO4), 2g of disodium hydrogen phosphate (Na2HPO4), and 10g of sodium chloride (NaCl) and add them to 1000ml of distilled water. Stir to dissolve and then seal for later use.
[0069] The staining reagent is prepared by mixing solutions A and C at a volume ratio of 7:3. It should be noted that in this mixture, the amount of solution A used in this comparative example is the sum of the amounts of solutions A and B used in Example 1.
[0070] Application Example 1
[0071] This application example presents a staining method for Helicobacter pylori, including the following steps:
[0072] Step 1: Prepare staining reagents.
[0073] Step 2: After fixing, dehydrating and embedding the obtained gastric mucosal tissue sample containing Helicobacter pylori, dewax it with xylene, dehydrate it with graded alcohol, wash it with distilled water, and spin dry the surface moisture.
[0074] Step 3: Add the prepared staining reagent to the slide.
[0075] Step 4: After staining for 1-4 minutes, rinse with running water until the staining solution no longer discolors the slide.
[0076] Step 5: The sections are then dehydrated with a gradient of alcohols and cleared with xylene, dried at 65°C, and then mounted with neutral resin.
[0077] Step 6: Observe the tissue under a professional optical microscope.
[0078] The dyes of Example 1 and Comparative Example 1 were applied for 3.5 minutes following the steps in Application Example 1, and the results are as follows: Figure 2 and 3 As shown.
[0079] like Figure 2 As shown in the figure, HE: is a general structural morphology diagram of the stomach tissue.
[0080] IHC: Immunohistochemistry (IHC) is currently the gold standard for diagnosing Helicobacter pylori (H. pylori) infection. This method uses specific antibodies to label H. pylori in tissues, ensuring that all target bacteria are clearly detected, thereby improving diagnostic accuracy.
[0081] Comparing the staining results of Helicobacter pylori before and after optimization, it was found that before optimization (using commercial methylene blue in Comparative Example 1), when there were many bacteria, they tended to clump together and their morphology was unclear. After optimization (using the staining reagent in Example 1), the morphology of each bacterial cell was clearly visible.
[0082] Moreover, the combination Figure 3 Before optimization, substances resembling bacilli were found, but after optimization, they were not found at the same location, and the results after optimization were consistent with the results of immunohistochemistry for the gold index.
[0083] The prepared staining reagents from Examples 1 and 2 were sealed and left at room temperature for 24 hours, and then stained for 1.5 minutes according to the steps in Example 1. The results are as follows. Figure 4 As shown. Combined with Figure 4 It was found that Example 1 without glycerol affected the staining; under the microscope, the blue staining ability was weakened, affecting the staining of the bacteria. In Example 2 with glycerol, the bacteria were clearer under the microscope. This shows that the staining reagent in Example 2 has better stability, and the addition of glycerol improved the consistency and clarity of the staining.
[0084] The staining reagents prepared in Comparative Example 2 and Example 1 were used to stain for 1 minute according to the steps in Application Example 1. The results are as follows: Figure 5 As shown. Combined with Figure 5 The staining reagent in Example 1 clearly shows the bacterial cells, while the staining reagent in Comparative Example 2 does not. This demonstrates that the technical solution of Example 1 proposed in this application only requires a short staining time of 1 minute after adding aniline blue, whereas general staining reagents require 3-4 minutes. For example, the commercial methylene blue in Comparative Example 1 requires 3.5 minutes of staining, significantly shortening the staining time.
[0085] The staining reagent of Example 3 has a staining effect comparable to that of Example 2.
[0086] To further validate the clinical application value of the novel diagnostic reagent, we conducted clinical trials in multiple hospitals. Gastric mucosal tissue samples were collected from 50 patients suspected of Helicobacter pylori infection. The samples were stained and tested using both the novel diagnostic reagent described in Example 2 and commercially available diagnostic reagents, and blinded diagnoses were performed by experienced pathologists. Clinical trial results showed:
[0087] Improved diagnostic accuracy: The diagnostic accuracy using the new diagnostic reagents reached 90%, compared to 50% for existing reagents. The new reagents can more accurately detect Helicobacter pylori infection, reducing misdiagnosis and missed diagnosis.
[0088] Improved diagnostic efficiency: Due to the significant color difference after staining with the new diagnostic reagents, the average diagnostic time for pathologists has been reduced from 20 minutes with existing reagents to 5 minutes, greatly improving diagnostic efficiency and helping to speed up the patient's diagnosis and treatment process.
[0089] Other beneficial effects:
[0090] 1) Significantly improved color contrast: Experimental verification shows that after staining with the diagnostic reagents of Example 2 or 3 of this novel invention, the color contrast between Helicobacter pylori and other tissues is more than 3 times higher than that of reagents currently on the market. Under a microscope, Helicobacter pylori appears as a bright blue, forming a strong contrast with the color of the surrounding tissue, allowing pathologists to clearly identify the presence and distribution of Helicobacter pylori.
[0091] 2) Excellent staining stability: This reagent maintains consistent staining results under varying laboratory conditions (temperature, humidity, light, etc.) and when used by different operators following standard operating procedures. Long-term stability testing showed no significant decrease in staining performance within 12 months under specified storage conditions. 3) High safety: A series of safety assessment experiments, including cytotoxicity and animal studies, demonstrate that this novel diagnostic reagent has no significant toxicity or adverse reactions to human tissues and cells, and poses no additional health risks to patients.
[0092] 4) Compared with the traditional methylene blue staining method, the improved formula significantly shortens the staining time and the staining effect is closer to the gold standard.
[0093] 5) Compared to the simple eosin methylene blue method, the improved method with the addition of aniline blue can significantly shorten the staining time under the same experimental conditions.
[0094] 6) This study optimized the formula based on the simple eosin-methylene blue staining method by adding glycerol as a stabilizer. Experiments showed that the addition of glycerol significantly reduced reagent volatilization, enhanced the stability of the system, and thus improved the consistency and clarity of staining.
[0095] This invention develops a novel diagnostic reagent for Helicobacter pylori, effectively solving the problem of unsatisfactory staining results found in existing reagents. Through optimized staining formulation and techniques, this reagent significantly improves the color contrast between Helicobacter pylori and other tissues, exhibiting good staining stability and high safety. Clinical trial results show that the novel diagnostic reagent outperforms existing reagents in both diagnostic accuracy and efficiency, demonstrating broad application prospects and market potential. We will continue to increase R&D investment, continuously optimize product performance, and actively promote the industrialization and market expansion of the product, contributing to the diagnosis and prevention of Helicobacter pylori infection.
[0096] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A staining reagent for staining Helicobacter pylori, characterized in that, It includes eosin, methylene blue, aniline blue and methanol; the mass ratio of eosin to methylene blue is (0.3-0.4):
1.
2. The staining reagent for Helicobacter pylori staining according to claim 1, characterized in that, It also includes glycerin.
3. The staining reagent for Helicobacter pylori staining according to claim 2, characterized in that, The ratio of eosin to methanol is (0.3-0.4) g:(100-110) mL, and the amount of glycerol used is 2%-5% of the volume of methanol.
4. A method for preparing a staining reagent for Helicobacter pylori staining according to any one of claims 1-3, characterized in that, Includes the following steps: Eosin, methylene blue and some methanol are ground and mixed, and then the remaining methanol is added and mixed to obtain solution A; Aniline blue and first water are mixed, then heated to dissolve at 60-65°C, and then glacial acetic acid solution is added to obtain solution B; Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and second water are mixed and dissolved to obtain solution C; The staining reagent is obtained by mixing the A solution, the B solution, and the C solution.
5. The method for preparing the staining reagent for Helicobacter pylori staining according to claim 4, characterized in that, The ratio of aniline blue to the first water is (1-1.5) g: 100 mL.
6. The method for preparing the staining reagent for Helicobacter pylori staining according to claim 4, characterized in that, After adding the remaining methanol and mixing, glycerol is also added to obtain solution A.
7. The method for preparing the staining reagent for Helicobacter pylori staining according to claim 4, wherein the mass ratio of sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride is (0.7-0.9):(2-3):10; and the mass ratio of sodium chloride and the second water is (1-1.5):
100.
8. The method for preparing the staining reagent for Helicobacter pylori staining according to claim 4, characterized in that, The volume ratio of solution A, solution B, and solution C is (4-5):(1-2):(3-5).
9. The method for preparing the staining reagent for Helicobacter pylori staining according to claim 8, characterized in that, The volume ratio of solution A, solution B, and solution C is 5:2:
3.
10. A staining method for Helicobacter pylori, characterized in that, Includes the following steps: Add the staining reagent according to any one of claims 1-3 or the staining reagent prepared by the preparation method according to any one of claims 4-9 to the obtained Helicobacter pylori section, then rinse with running water until the staining solution on the section no longer decolorizes, then perform gradient alcohol dehydration and xylene clearing on the section, and then dry and mount the section.