Vagina micro-ecology detection dyeing method and kit

Abstract

The invention discloses a staining method for vagina micro-ecology detection. The staining method mainly comprises the following steps: 1) taking female genital tract secretions, and diluting the secretions with a diluent to form a suspension; (2) coating a glass slide with the suspension liquid; 3) drying and fixing the suspension on the glass slide; 4) immersing the glass slide into eosin dye liquor for dyeing, then taking out the glass slide, and washing the glass slide with running water; 5) immersing the glass slide into methylene blue dye liquor for dyeing, then taking out the glass slide, and washing the glass slide with running water; (6) carrying out gram staining on the glass slide; and (7) observing by using a microscope. According to the staining method, under the condition that the advantages of a gram staining method in the prior art are reserved, not only can the trophozoic form and flagellum form of trichomonas vaginalis be clearly displayed, but also leukocytes and trichomonas vaginalis can be accurately distinguished, the accuracy of trichomonas vaginalis detection can be improved, and the method is suitable for popularization and application. The comprehensive and accurate detection on the vagina micro-ecology is facilitated.

Description

Technical Field

[0001] This invention relates to a staining method and kit for vaginal microecology detection, belonging to the field of clinical laboratory technology for vaginal microecology detection. Background Technology

[0002] The detection of vaginal microecology in gynecological clinics (see: Collaborative Group for Infectious Diseases of the Obstetrics and Gynecology Branch of the Chinese Medical Association. Expert consensus on the clinical application of vaginal microecological evaluation [J]. Chinese Journal of Obstetrics and Gynecology, 2016, 51(10):721-723.DOI:10.3760 / cma.j.issn.0529-567x.2016.10.001) mainly adopts the Gram staining method to distinguish bacteria in vaginal secretions into Gram-positive bacteria and Gram-negative bacteria, and to report other formed elements in secretions, including: leukocytes, epithelial cells and Trichomonas vaginalis.

[0003] Gram staining (see: Zhang Fengxue, Yu Ailian, Song Guangle. Improvement of Gram staining method [J]. Journal of Taishan Medical College, 2002, 23(2):182-182.DOI:10.3969 / j.issn.1004-7115.2002.02.043) is a commonly used method for identifying bacteria. The staining principle is based on the differences in bacterial cell wall components. Gram-positive bacteria have thick cell walls, and the peptidoglycan network molecules form a permeability barrier. When destained with ethanol, the peptidoglycan dehydrates and the pore barrier shrinks, thus retaining the crystal violet-iodine complex on the cell wall, which appears purple. In contrast, Gram-negative bacteria have thin peptidoglycan layers and loose cross-linking. Ethanol decolorization cannot cause their structure to shrink. Their lipid content is high, and ethanol dissolves the lipids, widening the gaps. The crystal violet-iodine complex dissolves out of the cell wall, so when stained with counterstaining solutions (such as safranin), they appear red. The clinical significance of Gram staining lies in: (1) it can classify bacteria into two major categories: Gram-positive bacteria and Gram-negative bacteria; (2) Gram staining is the basis for clinical selection of treatment drugs: some antibiotics are sensitive to Gram-positive bacteria, while some antibiotics are sensitive to Gram-negative bacteria; (3) it is related to the occurrence and development of diseases: Gram-positive bacteria produce exotoxins, and Gram-negative bacteria produce endotoxins. The above are the reasons why Gram staining is recommended for reporting vaginal microecological testing.

[0004] Gram staining is based on the differences in bacterial cell wall composition. However, Trichomonas vaginalis, leukocytes, and epithelial cells lack cell walls. When a patient is infected with Trichomonas vaginalis, causing a large number of leukocytes, Gram staining makes it difficult to distinguish the morphology of Trichomonas vaginalis and leukocytes. Therefore, trichomoniasis is easily missed in vaginal microecological testing reports. Furthermore, gonococcal infection can also cause an inflammatory response, resulting in a large number of leukocytes staining pink, which mixes with the pink-stained gonococci, making them difficult to identify and also leading to missed detection. Therefore, vaginal microecological testing technically suffers from two major challenges: underreporting of gonorrhea and trichomoniasis.

[0005] Among gynecological infections, trichomoniasis and gonorrhea account for a significant proportion. If not detected and treated early, they may become chronic, increasing the difficulty of treatment and potentially causing various complications. Vaginal microecological testing, as one of the pioneering diagnostic methods for gynecological infections, typically provides results within one hour of sample collection. Accurate reporting of gonorrhea and trichomoniasis in vaginal microecological testing avoids missed diagnoses of these patients, eliminating the need for further nucleic acid testing. Results are available the following day, greatly benefiting patients and improving the quality of medical care.

[0006] The applicant's earlier patent application (publication number CN116202839A) used a Gram staining method with a dried suspension for microscopic examination of Trichomonas vaginalis. This method avoids the damage to Trichomonas vaginalis caused by traditional smear techniques, keeping the trophozoites of Trichomonas vaginalis intact. After Gram staining, the trophozoite morphology is easily identifiable. However, when Trichomonas vaginalis is mixed with a large number of white blood cells, the white blood cells are stained pink after Gram staining, and the Trichomonas vaginalis, which is also stained pink, become clumps together, making it difficult to distinguish Trichomonas vaginalis from the white blood cells. Inflammation is a common symptom in gynecological infections, and vaginal secretions often contain a large number of white blood cells. When white blood cells clump together and are adsorbed by Trichomonas vaginalis, the delicate flagella are not easily observed, still leading to missed detection of Trichomonas vaginalis. As a result, the cause of the patient's inflammatory reaction remains unclear in clinical practice, leading to inaccurate medication. Therefore, accurately displaying the morphological characteristics of Trichomonas vaginalis against a Gram staining background and clearly distinguishing it from other formed elements, especially leukocytes, under a microscope is a pressing technical problem that needs to be solved. The reporting of gonorrhea in vaginal microecological testing follows the same principle as that of trichomoniasis. Summary of the Invention

[0007] Gram staining solutions can stain bacterial cell wall structures, but their staining effect on proteins and nuclei is poor. Vaginal microecology requires reporting the microbiota structure, inevitably necessitating the use of Gram staining solutions. However, vaginal secretions differ from the staining and identification of pure bacterial cultures in a microbiology laboratory; vaginal secretions contain more and more complex formed elements, such as leukocytes, epithelial cells, various cell debris, protein mucus filaments, and Trichomonas vaginalis. Therefore, developing a novel combined staining solution that can both preserve bacterial Gram staining results and specifically stain Trichomonas vaginalis flagella and leukocyte nuclei is crucial. Improving the contrast and comparison between the microscopic morphology of Trichomonas vaginalis and Neisseria gonorrhoeae and other formed elements in secretions, and enhancing the identification of various formed elements, is a problem that needs to be addressed in clinical vaginal microecology testing techniques. To overcome the problems existing in the prior art, this invention provides a novel vaginal microecological detection staining method and kit. While retaining the advantages of existing Gram staining, it can improve the contrast and comparison between the microscopic morphology of Trichomonas vaginalis or Neisseria gonorrhoeae and other formed elements in secretions, thereby improving the accuracy and convenience of pathogen reporting. The specific technical solution is as follows.

[0008] A vaginal microecological detection staining method mainly includes the following steps: 1) Collect female reproductive tract secretions and dilute them with a diluent to form a suspension; 2) Coat the suspension onto a glass slide; 3) Dry the glass slides; 4) Immerse the slide in eosin staining solution, then remove the slide and rinse it with running water; 5) Immerse the slide in methylene blue staining solution, then remove the slide and rinse it with running water; 6) Gram stain the slides; 7) Observe using a microscope.

[0009] Furthermore, the diluent in step 1) is distilled water, deionized water, or physiological saline.

[0010] Furthermore, in step 2), 20-50 μL of suspension is coated onto a glass slide using a pipette or dropper.

[0011] Furthermore, the drying process in step 3) is either heater drying or natural air drying; preferably, drying is carried out at a temperature range of 40℃-60℃. Drying helps to shorten the inspection time and improve efficiency, and does not affect the staining of bacteria, trichomonas, and leukocytes.

[0012] Furthermore, in step 4), the glass slide is immersed in eosin staining solution for 20s-30s.

[0013] Furthermore, in step 5), the glass slide is immersed in methylene blue staining solution for 20s-30s.

[0014] Furthermore, the Gram staining method in step 6) is a very common staining method, and its specific operation includes the following steps: S1: Crystal violet staining (referred to as primary staining step); S2: Rinse with distilled water; S3: Mordant treatment: Covering the surface with iodine solution for staining (referred to as mordant step); S4: Wash with water and use absorbent paper to remove moisture; S5: Add a few drops of ethanol and / or acetone, and gently shake to decolorize, then wash with water and absorb the moisture (referred to as the decolorization step). S6: After staining with safranin solution (referred to as the counterstaining step), rinse with distilled water and dry.

[0015] Based on the same inventive concept, this invention also relates to a staining kit, including eosin Y stain, methylene blue stain, and Gram stain. The eosin Y stain, methylene blue stain, and Gram stain are all commercially available.

[0016] Eosin staining solution can also be prepared at home: In a clean container, add 0.5 g of eosin to 100 ml of 95% alcohol or methanol and stir until dissolved; prepare 900 ml of distilled water, slowly add the distilled water and continue stirring until the mixture is completely dissolved.

[0017] Methylene blue staining solutions can also be prepared at home. To prepare an aqueous solution, in a clean container, add 1.0 g of methylene blue powder to 80 ml of distilled water and stir until dissolved. Prepare 900 ml of distilled water and slowly add it, continuing to stir until the mixture is completely dissolved. To prepare Löffler's methylene blue, in a clean container, dissolve 1.0 g of methylene blue powder in 30 mL of 95% ethanol, then mix with 100 mL of 0.01% KOH solution (or 1% borax) and let stand for 24 hours before filtering. Both aqueous methylene blue solutions and Löffler's methylene blue have similar staining effects.

[0018] The specific concentrations and formulations of eosin and methylene blue staining solutions can be fine-tuned according to experimental needs. It is recommended to refer to relevant standard experimental manuals (such as "Bancroft Staining Techniques").

[0019] The main principles and technical effects of the staining method of the present invention are as follows.

[0020] This invention stains the flagella, a unique structure of Trichomonas vaginalis, and also stains the nuclei of leukocytes. The staining method of this invention is based on Gram staining detection of vaginal secretions, therefore, this method ensures that it will not cause any deviation in the Gram staining results of bacteria; for example, it will not result in Gram-positive bacteria staining as Gram-negative or vice versa.

[0021] The main principle of the staining method of this invention is as follows.

[0022] I. Slide Preparation Method: This invention uses the "suspension drying method" to prepare slides, which avoids the damage to the morphology of Trichomonas vaginalis caused by the traditional smear method in vaginal microecological testing projects, and completely preserves the morphology of Trichomonas vaginalis trophozoites and the integrity of other cell morphologies.

[0023] II. The main basis for the coloration of flagella—a special structure that enables Trichomonas vaginalis trophozoites: 1) Gram staining is a commonly used method for bacterial identification. The principle of Gram staining is based on the differences in bacterial cell wall components. Gram-positive bacteria have thick cell walls, and their peptidoglycan network molecules form a permeability barrier. When destained with ethanol, the peptidoglycan dehydrates, and the pore barrier shrinks, thus retaining the crystal violet-iodine complex on the cell wall, resulting in a purple color. In contrast, Gram-negative bacteria have thin peptidoglycan layers with loose cross-linking. Ethanol destained staining cannot cause their structure to shrink. Their high lipid content causes ethanol to dissolve the lipids, widening the gaps and allowing the crystal violet-iodine complex to dissolve out of the cell wall. Therefore, when stained with counterstaining solutions (such as safranin), they appear red.

[0024] 2) Trichomonas vaginalis belongs to the protozoan kingdom and is a single-celled organism, while bacteria are prokaryotes.

[0025] 3) Trichomonas vaginalis and bacteria have significant structural differences. Trichomonas vaginalis has only one cell membrane and no cell wall, while most bacteria have cell walls.

[0026] 4) The flagella at the anterior end of Trichomonas vaginalis are mainly composed of filamentous structures made of protein, and the main component of these flagella is composed of fine filamentous protein.

[0027] Therefore, Gram staining solution has no specificity for staining the morphology of Trichomonas vaginalis. To stain the flagella, a key morphological feature of the Trichomonas vaginalis trophozoite, a staining solution capable of staining intracellular proteins is needed. Repeated experiments revealed that Eosin Y Stain showed good staining results for the flagella of Trichomonas vaginalis before using Gram staining solution.

[0028] III. The main basis for the staining of white blood cell nuclei: Methylene blue is a basic dye with a positive charge, while nucleic acids (especially DNA and RNA) in the cell nucleus carry a negative charge. This attraction between the positive and negative charges allows methylene blue to bind to the cell nucleus. This electrostatic interaction is the main working mechanism of methylene blue staining. Additionally, the cell nucleus contains a large number of nucleic acids and acidic proteins, providing numerous negatively charged binding sites. Proteins in the cytoplasm may carry different charges, so during staining, methylene blue tends to bind to the cell nucleus, resulting in a deep staining of the nucleus and a lighter staining of the cytoplasm. Thirdly, methylene blue has oxidizing properties. Living cells may reduce methylene blue to a colorless form, while dead cells, due to the cessation of metabolic activity, cannot reduce the dye and are thus stained. This is related to cell viability testing. However, in vaginal microecological testing using the drying method to fix cells in a secretion suspension, the cells are fixed and inactive, allowing methylene blue to stably stain the cell nucleus. When the nuclei of leukocytes are stained deep blue by methylene blue, the pink-stained gonococci are easily distinguished from the blue leukocytes, greatly reducing the probability of missed detection of gonococci.

[0029] Extensive experimental procedures have confirmed that using a protein-specific dye (eosin) to stain flagella and a nuclear dye (methylene blue) to stain leukocyte nuclei, combined with a bacterial dye (Gram stain), can achieve "one stain, three manifestations" of bacteria, flagellar proteins, and leukocyte nuclei. Combining eosin, methylene blue, and Gram stains together can create a novel microecological detection kit: eosin + methylene blue + Gram. This kit achieves specific staining of Trichomonas flagella and leukocyte nuclei without altering bacterial Gram staining results. This improves the contrast and identification of Trichomonas vaginalis and Neisseria gonorrhoeae under the microscope with other formed elements in secretions, resulting in a more comprehensive and accurate report for clinical vaginal microecological testing. Attached Figure Description

[0030] Figure 1 Comparative Example 1 shows the field of view under a microscope; Figure 2 Comparative Example 2 shows the field of view under a microscope; Figure 3 This is a microscopic view of Example 1; Figure 4 This is another field-of-view image under the microscope in Example 1. Detailed Implementation

[0031] The following are related explanations of the embodiments and comparative examples: 1. Both the comparative and example samples used the same real clinical sample (to ensure no sample differences). 2. The comparative examples and the embodiment examples all use the same tablet preparation method (suspension-dried tablets). 3. All dyeing steps were timed using a stopwatch (±2s).

[0032] 4. Using the staining results of Comparative Example 1 (Gram staining of dried suspension slides) as the standard, evaluate the staining effect of the combined staining solution.

[0033] Comparative Example 1 (Gram staining of dried suspension slides) The staining method for detecting Trichomonas vaginalis in Comparative Example 1 mainly includes the following steps: 1) Use cotton swabs to collect female reproductive tract secretions and dilute them directly with a diluent (distilled water, deionized water, or physiological saline) to form a suspension; 2) Use a pipette or dropper to coat 50 μl of the suspension onto a glass slide to form a thin liquid surface; 3) Dry the glass slide at a temperature of 60℃; 4) Gram stain the dried slide: S1: Soak the crystal violet (initial staining solution) in the staining tank for 10 seconds, then soak it in the water washing tank (with inlet) and gently shake it for 20 seconds. Remove the slide and shake it dry. S2: Immerse the slide in the iodine solution (mordant) for 10 seconds, then immerse it in the water rinse tank (with inlet) and gently shake for 20 seconds. Remove the slide and spin dry. S3: Enter the covered 95% alcohol (decolorizing solution) tank, gently shake for 5 seconds to decolorize, soak in the water washing solution tank (with water inlet) and gently shake for 20 seconds, then remove the glass slide and spin dry. S4: Soak the slide in the staining tank of safranin solution (counterstaining solution) for 5 seconds, then soak it in the rinsing tank (with water inlet) and gently shake it for 20 seconds. Remove the slide and dry it with an electric fan.

[0034] 5) Observe using a microscope (oil immersion), the observed field of view is as follows: Figure 1 As shown.

[0035] Figure 1 The image clearly shows the morphology of Trichomonas vaginalis (within the yellow box). Gram-positive cocci, Gram-negative bacilli (bacteria, including pink Gram-negative bacilli and dark purple Gram-positive cocci, are also visible in the field of view, as well as leukocytes (within the black box). The box colors in Comparative Example 2 and Example 1 refer to the same objects as the box colors in Comparative Example 1.

[0036] Comparative Example 2 (Eosin stain + Gram stain) The staining method for detecting Trichomonas vaginalis in Comparative Example 2 mainly includes the following steps: 1) Use cotton swabs to collect female reproductive tract secretions and dilute them directly with a diluent (distilled water, deionized water, or physiological saline) to form a suspension; 2) Use a pipette or dropper to coat 50 μl of the suspension onto a glass slide to form a thin liquid surface; 3) Dry the glass slide at a temperature of 60℃; 4) Immerse the fixed suspension slide in eosin staining solution for 25 seconds, then remove the slide and rinse it with water. 5) Gram stain the slide: S1: Soak the crystal violet (initial staining solution) in the staining tank for 10 seconds, then soak it in the water washing tank (with inlet) and gently shake it for 20 seconds. Remove the slide and shake it dry. S2: Immerse the slide in the iodine solution (mordant) for 10 seconds, then immerse it in the water rinse tank (with inlet) and gently shake for 20 seconds. Remove the slide and spin dry. S3: Enter the covered 95% alcohol (decolorizing solution) tank, gently shake for 5 seconds to decolorize, soak in the water washing solution tank (with water inlet) and gently shake for 20 seconds, then remove the glass slide and spin dry. S4: Soak the slide in the staining tank of safranin solution (counterstaining solution) for 5 seconds, then soak it in the rinsing tank (with water inlet) and gently shake it for 20 seconds. Remove the slide and dry it with an electric fan.

[0037] 6) Observe using a microscope (oil immersion), the observed field of view is as follows: Figure 2 As shown.

[0038] In step 1), the diluent is physiological saline.

[0039] The kit used in the detection staining method of Comparative Example 2 includes eosin stain and Gram stain, wherein the Gram stain includes primary stain, mordant, destaining solution and counterstain; the kit includes 5 containers (bottles) to contain eosin stain, primary stain, mordant, destaining solution and counterstain respectively.

[0040] Figure 2 The staining method in Comparative Example 2 clearly and completely presents the morphology of Trichomonas vaginalis, especially the four anterior flagella, which are clearly visible. Compared with the staining results of Comparative Example 1, the staining method in Comparative Example 2 provides clearer and more complete observation of the flagella of Trichomonas vaginalis, with higher identification rate, thus improving the accuracy of Trichomonas vaginalis detection and greatly reducing the requirements for the operator's clinical slide reading experience. Figure 2 Gram-positive cocci, Gram-negative bacilli, and leukocytes are visible in the sample. Figure 1 The difference in staining between bacteria was compared.

[0041] Example 1 (Eosin stain + Methylene blue stain + Gram stain) The staining method for detecting Trichomonas vaginalis in Example 1 mainly includes the following steps: 1) Use cotton swabs to collect female reproductive tract secretions and dilute them directly with a diluent (distilled water, deionized water, or physiological saline) to form a suspension; 2) Use a pipette or dropper to coat 50 μl of the suspension onto a glass slide to form a thin liquid surface; 3) Dry the glass slides at a temperature of 60℃ (experiments show that the staining effect is the same when the drying temperature is 40℃). 4) Immerse the fixed suspension slide in eosin staining solution for 25 seconds, then remove the slide and rinse it with water. 5) Immerse the slide in methylene blue staining solution for 25 seconds, then remove the slide and rinse it with water. 6) Gram stain the slide: S1: Soak the crystal violet (initial staining solution) in the staining tank for 10 seconds, then soak it in the water washing tank (with inlet) and gently shake it for 20 seconds. Remove the slide and shake it dry. S2: Immerse the slide in the iodine solution (mordant) for 10 seconds, then immerse it in the water rinse tank (with inlet) and gently shake for 20 seconds. Remove the slide and spin dry. S3: Enter the covered 95% alcohol (decolorizing solution) tank, gently shake for 5 seconds to decolorize, soak in the water washing solution tank (with water inlet) and gently shake for 20 seconds, then remove the glass slide and spin dry. S4: Soak the slide in the staining tank of safranin solution (counterstaining solution) for 5 seconds, then soak it in the rinsing tank (with water inlet) and gently shake it for 20 seconds. Remove the slide and dry it with an electric fan.

[0042] 7) Observe using a microscope (oil immersion), the observed field of view is as follows: Figure 3 , Figure 4 As shown.

[0043] In step 1), the diluent is physiological saline.

[0044] The kit used in the detection staining method of Example 1 includes eosin stain, methylene blue stain and Gram stain, wherein the Gram stain includes primary stain, mordant, destaining solution and counterstain; the kit includes 6 containers (bottles) to contain eosin stain, methylene blue stain, primary stain, mordant, destaining solution and counterstain respectively.

[0045] Figure 3 and Figure 2 The similarities are: they can clearly and completely present the morphology of Trichomonas vaginalis, especially the four anterior flagella are very clearly visible, and Gram-positive cocci, Gram-negative bacilli, and white blood cells can also be seen; Figure 3 and Figure 2The difference lies in the fact that the nuclei of leukocytes are stained dark blue with methylene blue, making it easier to distinguish leukocytes from Trichomonas vaginalis in Example 1 compared to Comparative Example 2. Additionally, from... Figure 4 As can be seen, after adopting the staining scheme of Example 1, it is possible to better distinguish between leukocytes (within the black box) and gonococci (within the red box), thereby improving the identification of gonococci and reducing the probability of missed detection of gonococci.

[0046] By comparison Figure 1 , Figure 2 and Figure 3 It is clearly evident that after the staining regimen of Comparative Example 1, Trichomonas vaginalis remains morphologically intact, making it difficult to identify when mixed with a large number of leukocytes. After the staining regimen of Comparative Example 2, the flagella of Trichomonas vaginalis are clearly stained, highlighting its characteristics and making Trichomonas vaginalis easily identifiable. However, other formed elements (including leukocytes) are stained Gram-negative (red), and it is still difficult to distinguish them when Trichomonas vaginalis, Neisseria gonorrhoeae, and leukocytes are mixed together. After the staining regimen of Example 1, the flagella of Trichomonas vaginalis are clearly visible, and the nuclear staining of leukocytes is prominent. Even when Trichomonas vaginalis, Neisseria gonorrhoeae, and leukocytes are mixed together, the three can be easily distinguished, thereby improving the identification of various formed elements and making clinical vaginal microecological testing more accurate and efficient.

Claims

1. A staining method for detecting vaginal microecology, characterized in that, The main steps include: 1) Collect female reproductive tract secretions and dilute them with a diluent to form a suspension; 2) Coat the suspension onto a glass slide; 3) Dry the glass slides; 4) Immerse the slide in eosin staining solution, then remove the slide and rinse it with running water; 5) Immerse the slide in methylene blue staining solution, then remove the slide and rinse it with running water; 6) Gram stain the slides; 7) Observe using a microscope.

2. The vaginal microecological detection staining method according to claim 1, characterized in that, The diluent in step 1) is distilled water, deionized water, or physiological saline.

3. The vaginal microecological detection staining method according to claim 1, characterized in that, In step 2), 20-50 μL of suspension is coated onto a glass slide using a pipette or dropper.

4. The vaginal microecological detection staining method according to claim 1, characterized in that, The drying process in step 3) is either drying with a heater or air drying.

5. The vaginal microecological detection staining method according to claim 4, characterized in that, Dry within a temperature range of 40℃-60℃.

6. The vaginal microecological detection staining method according to claim 1, characterized in that, In step 4), the glass slide is immersed in eosin staining solution for 20s-30s.

7. The vaginal microecological detection staining method according to claim 1, characterized in that, In step 5), the glass slide is immersed in methylene blue staining solution for 20-30 seconds.

8. The vaginal microecological detection staining method according to claim 1, characterized in that, Step 6) of the Gram staining method includes the following steps: S1: Crystal violet staining; S2: Rinse with distilled water; S3: Iodine solution is added to cover and stain the surface; S4: Wash with water and use absorbent paper to remove moisture; S5: Add a few drops of ethanol and / or acetone, and gently shake to decolorize, then wash with water and blot away the moisture; S6: After staining with safranin solution, rinse with distilled water and dry.

9. A staining kit comprising eosin stain, methylene blue stain, and Gram stain.

10. The staining kit according to claim 9, characterized in that, The Gram staining solution includes primary staining solution, mordant solution, decolorizing solution, and counterstaining solution.

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