Dyeing method and kit for vagina micro-ecology detection

Abstract

The invention discloses a dyeing method for vagina micro-ecology detection. The dyeing method mainly comprises the following steps: 1) taking female genital tract secretions, and directly diluting the secretions with a diluent to form a suspension; (2) coating the suspension liquid on a glass slide by using a pipettor; 3) drying and fixing the suspension on the glass slide; 4) immersing the fixed suspension liquid glass slide into an eosin dye solution, and washing the glass slide with running water; (5) carrying out gram staining on the glass slide; and 6) observing by using a microscope. The invention also relates to a staining kit. According to the staining method, under the condition that the advantages of a gram staining method in the prior art are reserved, simple and rapid detection of trichomonad vaginalis can be achieved, the trophozoic form and flagellum form of trichomonad vaginalis can be clearly displayed, the accuracy of trichomonad 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 testing, belonging to the field of clinical testing technology for vaginal microecology testing. Background Technology

[0002] Currently, vaginal microecology (see: Infectious Diseases Collaborative Group 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).

[0003] The detection method (DOI:10.3760 / cma.j.issn.0529-567x.2016.10.001) mainly uses Gram staining to distinguish bacteria in vaginal secretions into Gram-positive and Gram-negative bacteria, and reports other formed elements, including: leukocytes, epithelial cells, and Trichomonas vaginalis.

[0004] 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 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 the peptidoglycan network molecules form a permeability barrier. When decolorized 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 significance of Gram staining lies in: (1) it can classify bacteria into two major categories: Gram-positive bacteria and Gram-negative bacteria; (2) it facilitates clinical drug selection: some antibiotics are sensitive to Gram-positive bacteria, while others are sensitive to Gram-negative bacteria; (3) it is related to pathogenicity: Gram-positive bacteria produce exotoxins, while Gram-negative bacteria produce endotoxins. This is why Gram staining is used for vaginal microecological testing instead of other staining methods.

[0005] Gram staining solution cannot stain the nucleus and flagella of Trichomonas vaginalis. When there are a large number of white blood cells in the secretions, Trichomonas vaginalis and white blood cells are not easily distinguishable morphologically after staining. Therefore, Trichomonas vaginalis is very easy to miss in vaginal microecological testing. Trichomonas vaginalis (TV) (see: HirtRP. Trichomonas vaginalis virulence factors: an integrative overview. Sex Transm Infect. 2013 Sep; 89(6):439-43. doi:10.1136 / sextrans-2013-051105. Epub 2013May21. PMID:23694938; PMCID:PMC3749517) belongs to the protozoan kingdom. It is a single-celled organism with a cell membrane but no obvious cell wall. According to the morphological description of Trichomonas vaginalis in the textbook "Clinical Laboratory Parasitology," the trophozoite (parasite body) of Trichomonas vaginalis is pear-shaped or oval, with four anterior flagella at the anterior end and a slender, transparent axonal column running longitudinally through the trophozoite and extending out of the body from the posterior end (see...). Figure 1 The flagella at the anterior end of Trichomonas vaginalis are mainly filamentous structures made of protein, used for cell movement and positioning. The main component of these flagella is filamentous protein.

[0006] Trichomonas vaginalis primarily parasitizes the vagina and urethra in women. Clinically, it can cause vaginitis, urethritis, pelvic inflammatory disease, etc., manifesting as vaginal erythema, itching, grayish-green or yellowish-green discharge, and difficulty urinating. The incidence of trichomoniasis varies greatly in different countries and regions, ranging from 0.3% to 20%.

[0007] Currently in domestic clinical practice, there is no independent test for Trichomonas vaginalis. It is only a sub-item in the report of vaginal microecology testing. There are no other testing methods implemented in clinical practice. Therefore, the morphological examination of Trichomonas vaginalis is very important. However, vaginal microecology testing based on Gram staining as the reporting standard cannot accurately report Trichomonas vaginalis morphologically. First, the traditional preparation of dried vaginal secretion slides (see: Hematology and Body Fluid Group of Chinese Society of Laboratory Medicine, Infectious Diseases Collaboration Group of Chinese Society of Obstetrics and Gynecology. Guidelines for the standardization of clinical testing and result reporting of vaginal secretions [J]. Chinese Medical Journal, 2023, 103(1):10-17.DOI:10.3760 / cma.j.cn112137-20220804-01687) cannot identify the trophozoites of Trichomonas vaginalis. In vaginal microecology testing, the most important reported bacterial species are the secretions scraped from the lateral wall of the female reproductive tract with a dry cotton swab and then smeared onto a glass slide. Cell breakage during this process does not affect the bacterial report in the microecology results. However, during the smear process of rubbing the cotton swab against the glass slide, most of the trophozoites of Trichomonas vaginalis are already damaged. Therefore, under the microscope, they appear as Gram-negative pink fragments, making it difficult to identify the trophozoites. Secondly, Trichomonas vaginalis and bacteria have significant structural differences. Gram staining primarily targets the cell wall of bacteria, while Trichomonas vaginalis has only one cell membrane and no obvious cell wall. Therefore, Gram staining is very ineffective for Trichomonas vaginalis.

[0008] Under traditional Gram staining of vaginal secretions, the morphological features of Trichomonas vaginalis are atypical. Figure 2 These are images of Trichomonas vaginalis from the book "Atlas of Female Vaginal Microecology" published by People's Medical Publishing House. Figure 2 The arrow points to Trichomonas vaginalis, whose trophozoite (parasite) morphology is not obvious and is difficult to identify. Figure 3 These are microscopic images of vaginal trichomoniasis from a hospital in Beijing, illustrating the current state of testing in hospitals of all sizes across China.

[0009] With the development of biotechnology and the improvement of medical standards and diagnostic efficiency, we need more convenient and efficient diagnostic methods to further improve the accuracy and efficiency of medical testing. If we can also reduce medical costs and shorten testing time, this would undoubtedly be a benefit to patients.

[0010] The inventors' prior patent application (publication number CN116202839A) utilizes a Gram staining method with a dried suspension for microscopic examination of Trichomonas vaginalis, avoiding the damage caused to Trichomonas vaginalis by traditional smear techniques. This results in a very complete trophozoite morphology of Trichomonas vaginalis after Gram staining, and the flagella can also be observed. Figure 4 As shown.

[0011] Although the inventor's prior patent application allows observation of the complete trophozoite and some flagella of Trichomonas vaginalis, the typical morphology of the flagella is still not clear enough. When a patient is infected with Trichomonas vaginalis and microscopic examination reveals a large number of white blood cells, morphological identification is difficult, easily leading to missed detection. If a method could be found to accurately display the morphological characteristics of Trichomonas vaginalis against a Gram staining background, clearly distinguishing it from other formed elements under the microscope, it would greatly improve the accuracy of clinical Trichomonas vaginalis detection, significantly reduce the clinical experience required for morphological identification, greatly improve the timeliness of clinical testing, and save medical costs, thus reducing patient treatment expenses. Therefore, how to further improve the convenience and accuracy of Trichomonas vaginalis detection based on Gram staining in vaginal secretion microecological testing is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0012] Gram staining cannot reveal the morphological characteristics of Trichomonas vaginalis, therefore a novel combined staining solution is needed that can stain the flagella of Trichomonas vaginalis (i.e., stain cellular proteins) without affecting the results of bacterial Gram staining. This would allow for a more comprehensive and accurate detection of the vaginal microecology using a single staining method. To overcome the problems existing in the prior art, this invention provides a staining method and kit for vaginal microecology detection. While retaining the advantages of existing Gram staining, it can more clearly and completely display the trophozoite morphology of Trichomonas vaginalis and more clearly demonstrate the flagellar characteristics of Trichomonas vaginalis. The specific technical solution is as follows.

[0013] A staining method for vaginal microecology detection mainly includes the following steps:

[0014] 1) Collect female reproductive tract secretions and dilute them directly with a diluent to form a suspension;

[0015] 2) Use a pipette or dropper to coat the suspension onto a glass slide;

[0016] 3) Dry and fix the suspension on the glass slide;

[0017] 4) Immerse the fixed suspension slide in eosin Y stain and rinse the slide with running water;

[0018] 5) Gram stain the slides;

[0019] 6) Observe using a microscope.

[0020] Furthermore, the diluent in step 1) is distilled water, deionized water, or physiological saline.

[0021] Furthermore, in step 2), 20-50 μL of suspension is coated onto a glass slide using a pipette or dropper.

[0022] Furthermore, the drying and fixing process in step 3) is either heater drying or natural air drying; preferably, drying is performed within a temperature range of 40℃-60℃. Drying helps to shorten the inspection time and improve efficiency without affecting bacterial staining.

[0023] Furthermore, in step 4), the fixed suspension slide is immersed in eosin staining solution for 20s-30s.

[0024] Furthermore, the Gram staining method in step 5) is a very common staining method, and its specific operation includes the following steps:

[0025] S1: Crystal violet staining (referred to as the primary staining step);

[0026] S2: Rinse with distilled water;

[0027] S3: Mordanting step: Covering the surface with iodine solution for staining (referred to as mordanting step);

[0028] S4: Wash with water and use absorbent paper to remove moisture;

[0029] 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);

[0030] S6: After staining with safranin solution (referred to as the counterstaining step), rinse with distilled water and dry.

[0031] Based on the same inventive concept, this invention also relates to a staining kit, including eosin Y stain and Gram stain. Both eosin Y stain and Gram stain are commercially available. The eosin stain can also be prepared by adding 0.5 g of eosin to 100 ml of 95% ethanol or methanol in a clean container and stirring until dissolved; then, slowly add 900 ml of distilled water while continuing to stir until the mixture is completely dissolved.

[0032] The main principles and technical effects of the staining method of the present invention are as follows.

[0033] This invention enables the flagella, a distinctive structure of Trichomonas vaginalis, to stain. The staining method of this invention is based on Gram staining for vaginal secretion microecological detection; therefore, this staining method ensures that it does not cause any deviation in the Gram staining results of bacteria, such as preventing Gram-positive bacteria from being stained as Gram-negative and vice versa.

[0034] The main principle of the staining method of this invention is as follows.

[0035] 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.

[0036] II. The main basis for the coloration of flagella—a special structure that enables Trichomonas vaginalis trophozoites:

[0037] 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 composition. 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.

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

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

[0040] 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.

[0041] Therefore, Gram staining is not specific for staining the morphology of Trichomonas vaginalis. If we want to stain the flagella, a morphological feature of the Trichomonas vaginalis trophozoite, we need to find a staining solution that can stain cytoplasm and proteins. Repeated experiments have shown that Eosin Y Stain is effective in staining the flagella of Trichomonas vaginalis before Gram staining.

[0042] Third, the newly added staining agent must not cause any deviation in the Gram staining results of bacteria, such as Gram-positive bacteria being stained as Gram-negative, or Gram-negative bacteria being stained as Gram-positive.

[0043] Based on the above theoretical explanation, and adhering to the principle of staining cytoplasm and proteins without altering Gram staining results, the inventors selected a suitable eosin staining solution. Its main component is eosin Y, an acidic dye with an affinity for basic components in cells and tissues, such as proteins in the cytoplasm and DNA in the cell nucleus. Under a microscope, stained cells and tissues typically appear red or pink.

[0044] Eosin staining solution is generally unsuitable for staining bacteria, mainly due to the following reasons: 1. Insufficient dye affinity: The dyes in eosin staining solution, such as eosin B, are primarily acidic dyes with low affinity for bacterial cell walls and structures. Bacterial cell walls are typically composed of polysaccharides, proteins, and lipids, which have poor affinity for eosin dyes, resulting in poor staining effects. 2. Unclear staining effect: Because bacterial cells are small and have simple structures, eosin staining solution often cannot provide sufficient contrast, making it difficult to clearly observe bacterial morphology.

[0045] Therefore, while eosin staining solution is effective for staining cells, it is not suitable for staining bacteria. Eosin staining solution perfectly achieves the goal of staining Trichomonas vaginalis without affecting the Gram staining results of bacteria.

[0046] Eosin Y stain can be prepared in the laboratory or purchased commercially. By combining eosin stain with Gram stain, a kit can be made: eosin stain + Gram. This kit can stain the flagella of Trichomonas vaginalis without affecting the Gram staining results of bacteria, thereby improving the comprehensive and accurate detection of the existing vaginal microecology. Attached Figure Description

[0047] Figure 1 It is a picture of Trichomonas vaginalis from the textbook "Clinical Laboratory Parasitology";

[0048] Figure 2 The image is of Trichomonas vaginalis from the book "Atlas of Female Vaginal Microecology" published by People's Medical Publishing House.

[0049] Figure 3 This is a microscopic view of vaginal trichomoniasis from a hospital in Beijing.

[0050] Figure 4 It is a microscopic view of Trichomonas vaginalis in the inventor's prior patent application.

[0051] Figure 5 This is a microscopic view of Trichomonas vaginalis in Comparative Example 1.

[0052] Figure 6 This is a microscopic view of Trichomonas vaginalis in Example 1. Detailed Implementation

[0053] The following are related explanations of the embodiments and comparative examples:

[0054] 1. Both the comparative and example samples used the same real clinical sample (to ensure no sample differences);

[0055] 2. The comparative examples and the embodiment examples all use the same tablet preparation method (suspension-dried tablets).

[0056] 3. All dyeing steps were timed using a stopwatch (±2s).

[0057] 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.

[0058] Comparative Example 1 (Gram staining of dried slides after suspension)

[0059] The method for detecting Trichomonas vaginalis in Comparative Example 1 mainly includes the following steps:

[0060] 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;

[0061] 2) Use a pipette to coat 50 μl of suspension onto a glass slide. Use the pipette tip to draw up the suspension and squeeze the liquid onto the glass slide at an angle, then guide the liquid along the tip to form a thin liquid surface.

[0062] 3) Dry the suspension on the glass slide at a temperature of 60°C;

[0063] 4) Gram stain the dried suspension slides:

[0064] S1: Soak the slide in the crystal violet (initial staining solution) for 10 seconds, then soak it in the water washing solution (with inlet) and gently shake it for 20 seconds. Remove the slide and spin it dry.

[0065] S2: Immerse the slide in the iodine solution (mordant) staining tank for 10 seconds, then immerse it in the water washing tank (with inlet) and gently shake it for 20 seconds. Remove the slide and spin it dry.

[0066] 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.

[0067] S4: Soak the slide in the staining tank of safranin solution (counterstain) for 5 seconds, then soak it in the rinsing tank (with inlet) of clean water and gently shake it for 20 seconds. Remove the slide and dry it with an electric fan.

[0068] 5) Observe using a microscope (oil immersion), the observed field of view is as follows: Figure 5 As shown.

[0069] Figure 5 The morphology of Trichomonas vaginalis can be presented relatively completely, and some flagella can be identified. Gram-positive cocci, Gram-negative bacilli, and leukocytes can be seen in the field of view.

[0070] Example 1 (Eosin stain + Gram stain)

[0071] The method for detecting Trichomonas vaginalis in Example 1 mainly includes the following steps:

[0072] 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;

[0073] 2) Use a pipette to coat 50 μl of suspension onto a glass slide. Use the pipette tip to draw up the suspension and squeeze the liquid onto the glass slide at an angle, then guide the liquid along the tip to form a thin liquid surface.

[0074] 3) Dry and fix the suspension on the glass slide at a temperature of 60℃;

[0075] 4) Immerse the fixed suspension slide in the additional staining solution for 25 seconds, and then rinse the slide with water; the additional staining solution is eosin staining solution.

[0076] 5) Stain the slide using the Gram staining method:

[0077] S1: Soak the slide in the crystal violet (initial staining solution) for 10 seconds, then soak it in the water washing solution (with inlet) and gently shake it for 20 seconds. Remove the slide and spin it dry.

[0078] S2: Immerse the slide in the iodine solution (mordant) staining tank for 10 seconds, then immerse it in the water washing tank (with inlet) and gently shake it for 20 seconds. Remove the slide and spin it dry.

[0079] 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.

[0080] S4: Soak the slide in the staining tank of safranin solution (counterstain) for 5 seconds, then soak it in the rinsing tank (with inlet) of clean water and gently shake it for 20 seconds. Remove the slide and dry it with an electric fan.

[0081] 6) Observe using a microscope (oil immersion), the observed field of view is as follows: Figure 6 As shown.

[0082] In step 1), the diluent is physiological saline.

[0083] The kit used in the detection method of Example 1 includes eosin staining solution and Gram staining solution, wherein the Gram staining solution includes primary staining solution, mordant solution, destaining solution and counterstaining solution; the kit includes 5 containers (bottles) for containing eosin staining solution, primary staining solution, mordant solution, destaining solution and counterstaining solution respectively.

[0084] Figure 6The staining method in Example 1 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 observation of Trichomonas vaginalis flagella in Example 1 is clearer, more complete, and has higher identification accuracy, thus improving the accuracy of Trichomonas vaginalis detection and greatly reducing the requirements for clinical reading experience of operators. Figure 6 Gram-positive cocci, Gram-negative bacilli, and leukocytes are visible in the sample. Figure 5 The difference in staining between bacteria was compared.

[0085] Where there is no conflict, the embodiments and features described herein can be combined with each other. This invention is not limited to the specific embodiments described above; the specific embodiments described above are merely illustrative and not limiting. Those skilled in the art, under the guidance of this invention, can make many other modifications without departing from the spirit and scope of the claims, and all of these modifications fall within the scope of protection of this invention.

Claims

1. A staining method for vaginal microecological detection, characterized in that, The main steps include: 1) Collect female reproductive tract secretions and dilute them directly with a diluent to form a suspension; 2) Use a pipette or dropper to coat the suspension onto a glass slide; 3) Dry and fix the suspension on the glass slide; 4) Immerse the fixed suspension slide in the additional staining solution and rinse the slide with running water; the additional staining solution is a staining solution that can stain cytoplasm and proteins without changing the Gram staining results. 5) Gram stain the slides; 6) Observe using a microscope.

2. An improved Gram staining method for detecting Trichomonas vaginalis, characterized in that, The main steps include: 1) Collect female reproductive tract secretions and dilute them directly with a diluent to form a suspension; 2) Use a pipette or dropper to coat the suspension onto a glass slide; 3) Dry and fix the suspension on the glass slide; 4) Immerse the fixed suspension slide in eosin staining solution and rinse the slide with running water. 5) Gram stain the slides; 6) Observe using a microscope.

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

4. A staining method for vaginal microecological detection according to claim 1 or 2, characterized in that, The drying and fixing process in step 3) is either drying with a heater or air drying.

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

6. A staining method for vaginal microecological detection according to claim 1 or 2, characterized in that, Apply 20-50 μL of the suspension onto a glass slide using a pipette or dropper.

7. An improved Gram staining method for detecting Trichomonas vaginalis according to claim 1 or 2, a staining method for vaginal microecological detection, characterized in that... In step 4), the fixed suspension slide is immersed in eosin staining solution for 20s-30s.

8. A staining kit comprising eosin staining solution and Gram staining solution.

9. The staining kit according to claim 8, characterized in that, The Gram staining solution includes primary staining solution, mordant solution, decolorizing solution, and counterstaining solution.

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