A vaginal microecological detection staining method and kit

Abstract

The application discloses a vaginal micro-ecological detection staining method, mainly comprising the following steps: 1) taking female genital tract secretion and diluting the same by a diluent to form a suspension; 2) coating the suspension on a glass slide; 3) drying and fixing the suspension on the glass slide; 4) immersing the glass slide in a eosin dye solution for staining, and then taking out the glass slide and washing the same by flowing water; 5) immersing the glass slide in a methylene blue dye solution for staining, and then taking out the glass slide and washing the same by flowing water; 6) performing gram staining on the glass slide; and 7) observing by using a microscope. The staining method can not only clearly show the trophozoite shape and flagellum shape of Trichomonas vaginalis, but also accurately distinguish leukocytes and trichomonads, is favorable to improving the accuracy of trichomonas vaginalis detection, and is favorable to comprehensively and accurately detecting the vaginal micro-ecology.

Description

TECHNICAL FIELD

[0001] The present application relates to a vaginal microecological detection staining method and kit, belonging to the technical field of clinical examination of vaginal microecological detection. BACKGROUND

[0002] The detection of gynecological clinical vaginal microecology (see: Chinese Medical Association Obstetrics and Gynecology Branch Infectious Disease Group. 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 uses Gram staining method to distinguish bacteria in vaginal secretions into Gram-positive bacteria and Gram-negative bacteria, and reports other formed components in the secretions, including: leukocytes, epithelial cells and Trichomonas vaginalis.

[0003] Gram staining (see: Zhang Fengxue, Yu Ailian, Song Guangle. Improvement of Gram Stain [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 identification method for bacteria, and the staining principle is based on the difference in bacterial cell wall components. Gram-positive bacteria have thick cell walls, and peptidoglycan network molecules form a permeability barrier. When ethanol is decolorized, peptidoglycan dehydrates and the pore barrier shrinks, so the crystal violet-iodine complex is retained on the cell wall, showing purple color. Gram-negative bacteria have thin peptidoglycan layers, and the cross-linking is loose. Ethanol decolorization cannot make its structure shrink, and its lipid content is high. Ethanol will dissolve the lipid, the gap will be enlarged, and the crystal violet-iodine complex will be dissolved out of the cell wall, so when stained with a counterstain (such as saffron), it will show red. The clinical significance of Gram staining is: (1) bacteria can be divided into two categories: Gram-positive bacteria and Gram-negative bacteria; (2) Gram staining is the basis for choosing treatment drugs: some antibiotics are sensitive to Gram-positive bacteria, while some antibiotics are sensitive to Gram-negative bacteria; (3) related to the occurrence and development of diseases: Gram-positive bacteria produce exotoxins, and Gram-negative bacteria produce endotoxins. The above is the basis for recommending the use of Gram staining for reporting vaginal microecological detection.

[0004] The principle of Gram Stain is based on the difference of bacterial cell wall components, but Trichomonas vaginalis, leukocytes, and epithelial cells do not have cell walls. When a patient is infected with Trichomonas vaginalis and causes a large number of leukocytes, it is not easy to distinguish the morphology of Trichomonas vaginalis and leukocytes using Gram staining, so it is easy to miss reporting Trichomonas vaginalis disease in the vaginal microecological detection report. In addition, the infection of gonococci can also cause an inflammatory response, and a large number of leukocytes are stained pink and mixed with gonococci also stained pink, which is not easy to identify and is also easy to miss detection. Therefore, there are two technical problems of missing reporting gonorrhea and trichomoniasis in vaginal microecological detection.

[0005] In gynecological infections, trichomoniasis and gonorrhea account for a not small proportion. If they cannot be found early and treated in time, they may become chronic, increasing the difficulty of treatment, and may also cause various complications. Vaginal microecological detection is one of the pioneer detection projects for the diagnosis and treatment of gynecological infectious diseases. The results can be obtained within one hour after the doctor takes a sample. If gonorrhea and trichomoniasis can be accurately reported in vaginal microecological detection, it avoids the missed diagnosis of this part of patients, and there is no need for further nucleic acid detection, waiting for the results the next day, which greatly benefits the patients and improves the quality of medical care.

[0006] The applicant's prior patent application (publication number CN116202839A) uses a suspension liquid drying sheet Gram stain method to examine Trichomonas vaginalis, which avoids the damage to Trichomonas vaginalis caused by traditional smear operation, so that the trophozoites of Trichomonas vaginalis remain intact, and the morphology of the trophozoites of Trichomonas vaginalis is easy to identify after Gram staining. However, when the Trichomonas vaginalis is mixed with a large number of leukocytes, the leukocytes are stained pink after Gram staining, and the Trichomonas vaginalis also stained pink is mixed together, which makes it difficult to distinguish the Trichomonas vaginalis from the leukocytes. Inflammatory response is a common symptom in gynecological infectious diseases, and there are often a large number of leukocytes in vaginal secretions. When leukocytes gather into a group and are adsorbed together by Trichomonas vaginalis, the fine flagella are not easy to be observed, which still causes the missed detection of Trichomonas vaginalis, so that the cause of the inflammatory response of the patient is not clear, causing inaccurate medication. Therefore, under the background of Gram staining, how to more accurately display the morphological characteristics of Trichomonas vaginalis and distinguish it from other formed elements, especially leukocytes, under a microscope, is a technical problem that needs to be solved. The report of gonorrhea in vaginal microecological detection is the same as that of trichomoniasis. SUMMARY

[0007] Gram staining solution can be stained according to the structural characteristics of bacterial cell wall, but the staining effect on protein and nucleus is poor. The vaginal microecology needs to report the flora structure, and it is inevitable to use the Gram staining solution, but the vaginal secretion is different from the staining identification of bacterial pure culture in the microbial room, and the vaginal secretion has more complex formed elements, such as white blood cells, epithelial cells, various cell fragments, protein mucus silk, trichomonas vaginalis and the like. Therefore, a new type of combined staining solution can be developed, which can not change the Gram staining result of bacteria, and can perform specific staining on the flagella of trichomonas vaginalis and the nucleus of white blood cells. Improve the contrast and contrast of trichomonas vaginalis and gonococcus under the microscope and other formed elements in the secretion, and improve the recognition of various formed elements, which is a problem that needs to be overcome in the clinical vaginal microecology detection technology. In order to overcome the problems existing in the prior art, the present application provides a new vaginal microecology detection staining method and kit, which can improve the contrast and contrast of trichomonas vaginalis or gonococcus under the microscope and other formed elements in the secretion, and improve the accuracy and convenience of pathogenic bacteria report. The specific technical scheme is as follows.

[0008] A vaginal microecology detection staining method mainly comprises the following steps:

[0009] 1) Take the female genital tract secretion and dilute it to form a suspension liquid by using a diluent;

[0010] 2) Coating the suspension liquid on the slide;

[0011] 3) Dry the slide;

[0012] 4) Stain the slide by immersing it in eosin Y stain, and then take out the slide and rinse it with running water;

[0013] 5) Stain the slide by immersing it in methylene blue stain, and then take out the slide and rinse it with running water;

[0014] 6) Gram staining of the slide;

[0015] 7) Observation by microscope.

[0016] Further, the diluent in step 1) is distilled water, deionized water or physiological saline.

[0017] Further, in step 2), 20-50 μL of the suspension liquid is coated on the slide by using a pipette or a dropper.

[0018] Further, the drying treatment in step 3) is a heater drying or natural air drying; preferably, drying at a temperature range of 40-60°C. The drying is advantageous to shorten the inspection time, improve the efficiency, and does not affect the staining of bacteria, trichomonas, and leukocytes.

[0019] Further, in step 4), the slide is immersed in the eosin dye solution for 20-30s.

[0020] Further, in step 5), the slide is immersed in the methylene blue dye solution for 20-30s.

[0021] Further, the Gram staining method in step 6) is a very common staining method, and the specific operation includes the following steps:

[0022] S1: crystal violet staining (referred to as initial staining step);

[0023] S2: distilled water washing;

[0024] S3: iodine solution covering surface staining (referred to as mordant step);

[0025] S4: water washing, and water absorption paper is used to absorb water;

[0026] S5: adding a few drops of ethanol and or acetone, and gently shaking for decolorization, then water washing, and water absorption (referred to as decolorization step);

[0027] S6: after staining with safranin dye solution (referred to as re-staining step), distilled water washing, and drying.

[0028] Based on the same inventive concept, the present application also relates to a staining kit, which comprises eosin dye solution, methylene blue dye solution, and Gram dye solution. The eosin dye solution (Eosin Y Stain), methylene blue dye solution (Methylene blue Stain), and Gram dye solution can be purchased through commercial channels.

[0029] The eosin dye solution can also be prepared by itself: in a clean container, 0.5g of eosin is added to 100ml of 95% alcohol or methanol, and stirred until dissolved; 900ml of distilled water is prepared, and the distilled water is slowly added and continuously stirred until the mixture is completely dissolved.

[0030] Methylene blue staining solution can also be prepared by yourself. If preparing an aqueous solution, in a clean container, 1.0 g of methylene blue powder is added to 80 ml of distilled water, and stirred until dissolved; 900 ml of distilled water is prepared, and the distilled water is slowly added, and continue to stir until the mixture is completely dissolved. If preparing Loffler methylene blue, in a clean container, 1.0 g of methylene blue powder is dissolved in 30 mL of 95% ethanol, and then mixed with 100 mL of 0.01% KOH solution (or 1% borax) and stand for 24 hours, and then filtered for use. The aqueous solution of methylene blue and Loffler methylene blue have similar staining effects.

[0031] The specific concentration and formula of eosin staining solution and methylene blue staining solution can be adjusted according to experimental needs, and it is recommended to refer to relevant standard experimental manuals (such as “Bancroft Staining Technology”).

[0032] The main principle and technical effect of the staining method of the present application are as follows.

[0033] The present application can color the special structure of Trichomonas vaginalis, i.e. flagellum, and also stain the nucleus of leukocytes. The staining method of the present application is based on the detection of vaginal secretion micro-ecological Gram staining, so the staining method of the present application can ensure that there is no deviation in the Gram staining results of bacteria, such as Gram-positive bacteria being stained as Gram-negative and Gram-negative bacteria being stained as Gram-positive.

[0034] The main principle of the staining method of the present application is as follows.

[0035] I. Preparation method: the present application adopts “suspension liquid drying method” to prepare the slice, which avoids the damage to the morphology of Trichomonas vaginalis in the traditional smearing method of vaginal micro-ecological detection, and completely preserves the morphology of Trichomonas vaginalis trophozoite and the integrity of other various cell morphologies.

[0036] II. The main basis for coloring the special structure of Trichomonas vaginalis trophozoite, i.e. flagellum, is as follows.

[0037] 1) Gram staining is a commonly used method for bacterial identification, and the principle of Gram staining is based on the difference in the composition of bacterial cell walls. The cell wall of Gram-positive bacteria is thick, and the peptidoglycan network molecules form a permeability barrier. When ethanol is decolorized, the peptidoglycan dehydrates and the pore barrier shrinks, so the crystal violet-iodine complex is retained on the cell wall, showing purple color. The peptidoglycan layer of Gram-negative bacteria is thin, and the cross-linking is loose. Ethanol cannot make its structure contract, and its lipid content is high. Ethanol will dissolve the lipid, the gap will be enlarged, and the crystal violet-iodine complex will be dissolved out of the cell wall, so when stained with a counterstaining solution (such as saffron), it will show red color.

[0038] 2)Trichomonas vaginalis belongs to the kingdom of protozoa, is a single-celled organism, while bacteria are prokaryotes.

[0039] 3)Trichomonas vaginalis and bacteria have significant differences in structure, Trichomonas vaginalis has only one cell membrane, no cell wall, while most bacteria have cell walls.

[0040] 4)The flagella at the front of Trichomonas vaginalis is mainly a filamentous structure composed of protein, and the main component of these flagella is composed of filaments.

[0041] So the Gram stain solution has no specificity for the morphological staining of Trichomonas vaginalis. And to stain the morphological characteristics of Trichomonas vaginalis body-whip, we need to find a dye that can stain the intracellular protein. After repeated experiments, it was found that Eosin Y Stain has good staining effect on the flagella of Trichomonas vaginalis before using Gram stain.

[0042] Three, the main basis for staining the nucleus of white blood cells:

[0043] Methylene blue is a basic dye with a positive charge, while the nucleic acids in the nucleus (especially DNA and RNA) have a negative charge, so the positive and negative charges attract each other, so methylene blue can bind to the nucleus. This electrostatic interaction is the main working mechanism of methylene blue stain. In addition, the nucleus contains a large amount of nucleic acids and acidic proteins, which provide a large number of negative charge binding sites. The proteins in the cytoplasm may have different charges, so during staining, methylene blue tends to bind to the nucleus, resulting in deep staining of the nucleus and light staining of the cytoplasm. Third, methylene blue has oxidizing properties, and cells in a living state may reduce methylene blue to a colorless form, while dead cells cannot reduce the dye because their metabolic activity has stopped, so dead cells will be stained, which is related to cell activity detection, but in the case of the vaginal microecological detection of the dried suspension solution of the secretions, the cells have been fixed and lost activity, so methylene blue can stably stain the nucleus. When the nucleus of white blood cells is stained dark blue by methylene blue, gonococci stained pink are easily distinguished from blue white blood cells, greatly reducing the false negative probability of gonococci.

[0044] 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

[0045] Figure 1 This is a microscopic view of the field of view in Comparative Example 1.

[0046] Figure 2 Comparative Example 2 shows the field of view under a microscope;

[0047] Figure 3 This is a microscopic view of Example 1;

[0048] Figure 4 This is another field-of-view image under the microscope in Example 1. Detailed Implementation

[0049] The following are related explanations of the embodiments and comparative examples:

[0050] 1. Both the comparative and example samples used the same real clinical sample (to ensure no sample differences).

[0051] 2. The comparative examples and the embodiment examples all use the same tablet preparation method (suspension-dried tablets).

[0052] 3. All dyeing steps were timed using a stopwatch (±2s).

[0053] 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.

[0054] Comparative Example 1 (Gram staining of dried suspension slides)

[0055] The staining method for detecting Trichomonas vaginalis in Comparative Example 1 mainly includes the following steps:

[0056] 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;

[0057] 2) Use a pipette or dropper to apply 50 μl of the suspension to the slide to form a thin layer of liquid;

[0058] 3) Dry the slide, and bake it to a temperature of 60°C;

[0059] 4) Perform Gram staining on the dried slide:

[0060] S1: Soak the slide in a crystal violet (primary stain) dye vat for 10 seconds, soak in a water wash vat (with inlet) and gently shake for 20 seconds, and then remove the slide and spin dry;

[0061] S2: Soak the slide in an iodine solution (mordant) dye vat for 10 seconds, soak in a water wash vat (with inlet) and gently shake for 20 seconds, and then remove the slide and spin dry;

[0062] S3: Soak the slide in a 95% alcohol (decolorizing solution) vat with a lid, gently shake for 5 seconds to decolorize, soak in a water wash vat (with inlet) and gently shake for 20 seconds, and then remove the slide and spin dry;

[0063] S4: Soak the slide in a safranin solution (counterstain) dye vat for 5 seconds, soak in a water wash vat (with inlet) and gently shake for 20 seconds, and then remove the slide and spin dry.

[0064] 5) Use a microscope (oil immersion lens) to observe, and the observed field of view is as shown in Figure 1 .

[0065] Figure 1 The morphology of Trichomonas vaginalis (in the yellow box) can be more completely presented, and in the field of view, Gram-positive cocci, Gram-negative bacilli (green box for bacteria, including pink Gram-negative bacilli and deep purple Gram-positive cocci), and leukocytes (black box) can also be seen. The colors of the boxes in Comparative Example 2 and Example 1 refer to the same objects as the colors of the boxes in Comparative Example 1.

[0066] Comparative Example 2 (eosin stain + Gram stain)

[0067] The Trichomonas vaginalis detection staining method of Comparative Example 2 mainly includes the following steps:

[0068] 1) Use a cotton swab to take female genital tract secretions and directly dilute them with a diluent (distilled water, deionized water, or physiological saline) to form a suspension;

[0069] 2) Use a pipette or dropper to apply 50 μl of the suspension to the slide to form a thin layer of liquid;

[0070] 3) Dry the slide, and bake it to a temperature of 60°C;

[0071] 4) Immersed the fixed suspension slide into the eosin dye solution for 25s, then removed the slide and washed it with clean water;

[0072] 5) Gram staining of the slide:

[0073] S1: Crystal violet (primary dye) dye solution tank immersion for 10s, clean water solution tank (with inlet) immersion and gently shaking for 20s, remove the slide and dry it;

[0074] S2: Iodine solution (mordant) dye solution tank immersion for 10s, clean water solution tank (with inlet) immersion and gently shaking for 20s, remove the slide and dry it;

[0075] S3: Enter the 95% alcohol (decoloring solution) tank with cover, gently shake for 5s for decolorization, clean water solution tank (with inlet) immersion and gently shaking for 20s, remove the slide and dry it;

[0076] S4: Safranin solution (counterstain) dye solution tank immersion for 5s, clean water solution tank (with inlet) immersion and gently shaking for 20s, remove the slide and dry it with an electric fan.

[0077] 6) Observed under a microscope (oil immersion lens), the observed field is as shown in Figure 2 .

[0078] The diluent in step 1) is physiological saline.

[0079] The kit used in the detection and staining method of Comparative Example 2 includes eosin dye solution and gram dye solution, wherein the gram dye solution includes primary dye, mordant, decolorizing solution and counterstain; the kit includes 5 containers (bottles) for containing eosin dye solution, primary dye, mordant, decolorizing solution and counterstain, respectively.

[0080] Figure 2 The morphology of Trichomonas vaginalis can be clearly and completely presented, especially the four front flagella are very clear and visible, compared with the staining result of Comparative Example 1, the observation of flagella of Trichomonas vaginalis in the staining scheme of Comparative Example 2 is more clear, complete and higher in recognition, which improves the accuracy of detection of Trichomonas vaginalis and greatly reduces the requirement for clinical reading experience of the operator. Figure 2 Gram-positive cocci, gram-negative bacilli and white blood cells can be seen, and there is no staining difference between bacteria compared with Figure 1 .

[0081] Example 1 (eosin dye solution + methylene blue dye solution + gram dye solution)

[0082] The Trichomonas vaginalis detection and staining method of Example 1 mainly includes the following steps:

[0083] 1) Using a cotton swab to take female genital tract secretions and directly dilute with a diluent (distilled water, deionized water or physiological saline) to form a suspension;

[0084] 2) Using a pipette or dropper to apply 50 μl of the suspension to a glass slide to form a thin liquid surface;

[0085] 3) Dry the glass slide, and dry at a temperature of 60°C (tests show that the staining effect is the same when the drying temperature is 40°C);

[0086] 4) Immerse the fixed suspension glass slide in the eosin dye solution for 25 s, then take out the glass slide and rinse it with clean water;

[0087] 5) Immerse the glass slide in the methylene blue dye solution for 25 s, then take out the glass slide and rinse it with clean water;

[0088] 6) Perform Gram staining on the glass slide:

[0089] S1: Crystal violet (primary dye) dye cylinder immersion for 10 s, clean water washing cylinder (with water inlet) immersion and gentle shaking for 20 s, take out the glass slide and dry;

[0090] S2: Iodine solution (mordant) dye cylinder immersion for 10 s, clean water washing cylinder (with water inlet) immersion and gentle shaking for 20 s, take out the glass slide and dry;

[0091] S3: Enter the 95% alcohol (decoloring solution) cylinder with a cover, gently shake for 5 s for decolorization, clean water washing cylinder (with water inlet) immersion and gentle shaking for 20 s, take out the glass slide and dry;

[0092] S4: Safranin solution (counterstain) dye cylinder immersion for 5 s, clean water washing cylinder (with water inlet) immersion and gentle shaking for 20 s, take out the glass slide and dry with an electric air dryer.

[0093] 7) Observe under a microscope (oil immersion lens), and the observed field is as shown in Figure 3 、 Figure 4 .

[0094] Among them, the diluent in step 1) is physiological saline.

[0095] The kit used in the detection and staining method of Example 1 includes eosin dye, methylene blue dye and Gram dye, wherein the Gram dye includes primary dye, mordant, decolorizing solution and counterstain; the kit includes 6 containers (bottles) for containing eosin dye, methylene blue dye, primary dye, mordant, decolorizing solution and counterstain.

[0096] Figure 3 and Figure 2The same is that the morphology of Trichomonas vaginalis can be clearly and completely presented, especially the four front flagella are very clear, and Gram-positive cocci, Gram-negative bacilli and leukocytes can also be seen; Figure 3 With Figure 2 The difference is that the nucleus of leukocytes is dyed dark blue by methylene blue, so the scheme of example 1 is more easily distinguishable than the scheme of comparative example 2 between leukocytes and Trichomonas vaginalis. In addition, it can be seen from Figure 4 that after using the staining scheme of example 1, leukocytes (in the black frame) and gonococci (in the red frame) can be better distinguished, thereby improving the recognition of gonococci and reducing the missed report probability of gonococci.

[0097] By comparing Figure 1 , Figure 2 and Figure 3 , it can be clearly seen that after the staining scheme of comparative example 1, the morphology of Trichomonas vaginalis is complete, and it is not easy to identify when mixed with a large number of leukocytes. After the staining scheme of comparative example 2, the flagella of Trichomonas vaginalis are obviously dyed, and the characteristics are prominent, and Trichomonas vaginalis is easy to identify, but other components (including leukocytes) are dyed Gram-negative (red), and when Trichomonas vaginalis, gonococci and leukocytes are mixed together, it is still difficult to distinguish. After the staining scheme of example 1, the flagella of Trichomonas vaginalis are very obvious, and the nucleus of leukocytes is dyed prominently, so even when Trichomonas vaginalis, gonococci and leukocytes are mixed together, the three can be easily distinguished, thereby improving the recognition of various components, making the clinical vaginal microecological detection more accurate and efficient.

Claims

1. A method for detecting vaginal microflora staining, characterized by, The method mainly comprises the following steps: 1) taking female genital tract secretion and diluting the secretion with a diluent to form a suspension; 2) coating the suspension on a glass slide; 3) drying the glass slide; i.e. using the suspension drying method to prepare the sample; 4) immersing the glass slide in a safranine dye solution for dyeing, and then taking out the glass slide and washing it with running water; 5) immersing the glass slide in a methylene blue dye solution for dyeing, and then taking out the glass slide and washing it with running water; 6) performing gram staining on the glass slide; 7) observing under a microscope, wherein the Trichomonas vaginalis is dyed purple, the leukocytes are dyed dark blue, and the gonococci are dyed dark purple.

2. The method according to claim 1, wherein, The diluent in step 1) is distilled water, deionized water or normal saline.

3. The method according to claim 1, wherein the method is characterized by, In step 2), 20-50 μL of the suspension is coated on the glass slide using a pipette or a dropper.

4. The method according to claim 1, wherein the method is a method for detecting and staining vaginal microflora. The drying treatment in step 3) is heating drying or natural air drying.

5. The method according to claim 4, wherein the method is a method for detecting and staining vaginal microflora. The drying is performed at a temperature of 40-60 °C.

6. The method according to claim 1, wherein the method is a method for detecting and staining vaginal microflora. In step 4), the glass slide is immersed in the safranine dye solution for 20-30 s.

7. The method according to claim 1, wherein the method is a method for detecting and staining vaginal microflora. In step 5), the glass slide is immersed in the methylene blue dye solution for 20-30 s.

8. The method according to claim 1, wherein the method is a method for detecting and staining vaginal microflora. The gram staining method in step 6) comprises the following steps: S1: crystal violet staining; S2: distilled water washing; S3: covering the surface with iodine solution for dyeing; S4: water washing, and absorbing the water with a water-absorbing paper; S5: adding a few drops of ethanol and / or acetone, and gently shaking for decolorization, then water washing, and absorbing the water; S6: staining with safranine dye solution, then distilled water washing, and drying.

9. A dyeing kit comprising a safranine dye solution, a methylene blue dye solution and a gram dye solution.

10. The staining kit according to claim 9, characterized in that, The gram dye solution comprises a primary dye solution, a mordant dye solution, a decolorizing solution and a counterstain solution.

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