A vaginal secretion staining solution and a preparation method and a staining method thereof
By using basic fuchsin and subunit blue staining agents combined with stabilizers and surfactants, the problem of misidentification of leukocytes and trichomonas in vaginal secretion staining methods has been solved, enabling rapid and accurate detection of vaginal secretions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG TAILIN MEDICAL ENG CO LTD
- Filing Date
- 2023-05-04
- Publication Date
- 2026-07-07
Smart Images
Figure CN116625778B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell staining technology, and in particular to a vaginal secretion staining solution and its preparation and staining methods. Background Technology
[0002] Bacterial vaginosis is a clinical syndrome characterized by a decrease in the number of normal reproductive tract flora (hydrogen peroxide-producing lactobacilli) and an increase in the number of a group of anaerobic bacteria, resulting in a disrupted vaginal ecosystem. Clinically, bacterial vaginosis presents with increased vaginal discharge; in 10% of patients, the discharge is frothy and has an unpleasant odor. A few patients may experience mild vulvar itching and burning. There are no signs of inflammation of the vaginal mucosa. Routine vaginal discharge tests show a mixed infection rate of bacteria, fungi, trichomonas, and bacterial vaginosis in 54.42% of cases. Abnormalities in routine vaginal discharge tests are closely related to mixed bacterial vaginosis infections and are a risk factor for salpingitis, pelvic inflammatory disease, ectopic pregnancy, infertility, urinary tract infections, postoperative infections, and gynecological tumors, thus requiring serious attention.
[0003] Routine methods for detecting vaginal secretions include dry chemical enzymatic methods, wet slide microscopy, nucleic acid hybridization, and staining. Among these, wet slide microscopy is advantageous due to its low cost, ease of operation, and good specificity, making it suitable for most laboratories; however, it is susceptible to operator bias. Staining methods offer high accuracy but are complex and time-consuming, making them unsuitable as routine methods. Combining wet slide microscopy and staining methods can create a low-cost, simple, and highly accurate method for detecting vaginal secretions.
[0004] CN201510484688.X discloses a vaginal secretion staining solution and its preparation and staining methods. The stained images are clear and easy to identify, clearly distinguishing components such as surface squamous epithelial cells, intermediate epithelial cells, basal epithelial cells, clue cells, leukocytes, erythrocytes, Candida albicans, and Trichomonas vaginalis, making it suitable for clinical screening of large samples. However, this staining method stains all of the above components. Since vaginal secretions often contain a mixture of various microorganisms, leukocytes and Trichomonas vaginalis have similar morphologies after staining, making them difficult to distinguish and easily leading to misdiagnosis in clinical practice. Summary of the Invention
[0005] To address the problem of misidentification of leukocytes and trichomonas in existing staining methods, this invention provides a vaginal secretion staining solution. This vaginal secretion staining solution uses basic fuchsin and subunit blue as staining agents, which can stain epithelial cells and leukocytes, but cannot stain trichomonas and fungi. The color difference clearly distinguishes leukocytes and trichomonas, avoiding misidentification caused by the similarity between leukocytes and trichomonas.
[0006] The specific technical solution of this invention is as follows:
[0007] In a first aspect, the present invention provides a vaginal secretion staining solution, comprising a staining agent and a staining solvent, a stabilizer, and a buffer salt;
[0008] The staining agents are basic fuchsin and methylene blue.
[0009] Compared to other test samples, vaginal secretions have a more complex composition. Normal vaginal secretions contain a large number of lactobacilli, no other bacteria, and epithelial cells and leukocytes. In other words, samples without pathological changes are mainly composed of epithelial cells, leukocytes, and lactobacilli. Pathological vaginal secretions, however, contain epithelial cells, leukocytes, clue cells, fungi, trichomonas, and a greater variety of other bacteria, including mycoplasma and chlamydia. Pathological samples may contain trichomonas, leukocytes, fungi, epithelial cells, clue cells, and bacteria. Epithelial cells and leukocytes have clearly visible morphologies under a microscope. The staining agent in this application, when mixed, can stain epithelial cells and leukocytes, but cannot stain trichomonas and fungi. This color difference clearly distinguishes leukocytes and trichomonas, reducing misdiagnosis caused by the similarity between leukocytes and trichomonas.
[0010] Preferably, the vaginal secretion staining solution contains 10-15 g / L of the basic dye fuchsin and 1-1.5 g / L of the sub-methylene blue dye.
[0011] This invention uses basic fuchsin and methylene blue as staining agents. Basic fuchsin stains the cell nucleus deep purple and the cytoplasm purple; methylene blue, as a nuclear dye, stains the cell nucleus blue. Based on experimental and theoretical research, the invention team discovered that the optimal concentration of basic fuchsin in the vaginal secretion staining solution is between 10 and 15 g / L, and the optimal concentration of methylene blue is between 1 and 1.5 g / L. At this dye ratio, the staining result is orange-red, which is superior to using both dyes as nuclear staining agents, where the cell nucleus stains deeper than the cytoplasm. If the basic fuchsin concentration is too low, the effect of methylene blue increases, the staining degree of the cytoplasm decreases, only the clearly visible cell nucleus is seen, and the stained cells appear bluish. If the basic fuchsin concentration increases, the effect of methylene blue decreases, the staining degree of the cell nucleus and cytoplasm is not significantly different, and both are purple, making them difficult to distinguish.
[0012] Preferably, the stabilizer includes cell membrane protectants and protein activity protectants, serum albumin, and antioxidants.
[0013] Preferably, the vaginal secretion staining solution contains 7-9 g / L of cell membrane protectant, 1-2 g / L of protein activity protectant, 0.4-0.6 g / L of serum albumin, and 2-4 g / L of antioxidant.
[0014] Preferably, the cell membrane protectant is one or more of trehalose, sucrose, mannitol, and glycine.
[0015] More preferably, the cell membrane protectant is sucrose.
[0016] Preferably, the protein activity protectant is ammonium sulfate.
[0017] Ammonium sulfate is an inert substance that does not react with bioactive substances and can protect protein activity.
[0018] Preferably, the serum albumin is BSA.
[0019] Serum albumin plays a role in maintaining osmotic pressure and pH buffering.
[0020] Preferably, the antioxidant is citric acid.
[0021] Citric acid is a natural preservative that inhibits the growth of harmful microorganisms and the production of toxins, thus working in conjunction with other preservatives for preservation.
[0022] Preferably, the dye solvent is one or more of anhydrous ethanol, methanol, and ethylene glycol.
[0023] Preferably, the dye solvent content in the vaginal secretion staining solution is 90-110 ml / L.
[0024] Preferably, the concentration of the buffer salt is 2–500 mmol / L, and the pH of the buffer salt is 5.0–7.8.
[0025] Preferably, the buffer salt is one or more of MES, borate, phosphate, Tris-HCl, Bistris-HCl, HEPES, and PIPES.
[0026] More preferably, the buffer salt is a phosphate.
[0027] Furthermore, the phosphate is Na2HPO4·12H2O or NaH2PO4·2H2O.
[0028] NaH₂PO₄·2H₂O is highly soluble in water, and its aqueous solution is acidic, often used as an acid buffer. Na₂HPO₄·12H₂O is also readily soluble in water, and its aqueous solution is weakly alkaline. A 1% aqueous solution has a pH of 8.8–9.2, and a 3.5% aqueous solution has a pH of 9.0–9.4. It is insoluble in alcohol.
[0029] Preferably, the vaginal secretion staining solution also includes surfactants and / or preservatives.
[0030] Preferably, the surfactant is sodium dodecyl sulfate.
[0031] This invention uses sodium dodecyl sulfate as a surfactant. Sodium dodecyl sulfate can increase cell membrane permeability, bind to the hydrophobic portions of membrane proteins and cause them to separate from the membrane, greatly improving the staining effect of the dye. Sodium dodecyl sulfate not only enhances the performance of the dye but also acts as a solubilizer, increasing the solubility of various solutes.
[0032] Preferably, the surfactant content in the vaginal secretion staining solution is 0.01–0.02 g / L.
[0033] Preferably, the preservative is Proclin 300.
[0034] Preferably, the preservative content in the vaginal secretion staining solution is 1-3 ml / L.
[0035] Secondly, this application provides a method for preparing a vaginal secretion staining solution, comprising the following steps:
[0036] (S1) Dissolve basic fuchsin and methylene blue in a staining solvent to obtain a staining solution;
[0037] (S2) Add a stabilizer to the above staining solution and adjust the pH to neutral with a buffer salt to obtain a buffer solution.
[0038] (S3) The above buffer solution was left to stand in the dark to obtain a vaginal secretion staining solution.
[0039] Thirdly, this application provides a staining method for vaginal secretion staining solution, which uses wet slide staining method to stain vaginal secretions. The wet slide staining method is as follows: vaginal secretions eluted with elution solution are dropped onto a glass slide or sample tube, and then the above-mentioned vaginal secretion staining solution is mixed evenly with the vaginal secretions so that the staining solution stains the vaginal secretions.
[0040] The staining method for vaginal secretion staining solution provided by the present invention can shorten the staining time to 20 seconds.
[0041] Compared with the prior art, the beneficial effects of the present invention are:
[0042] (1) The vaginal secretion staining solution of the present invention overcomes the problem that existing staining methods are prone to misidentification of leukocytes and trichomonas. This application uses basic fuchsin and subunit blue as staining agents, which can stain epithelial cells and leukocytes, but cannot stain trichomonas and fungi. By the color difference, leukocytes and trichomonas can be clearly distinguished.
[0043] (2) It overcomes the shortcomings of traditional staining methods, which are complicated and time-consuming. The vaginal secretion staining solution provided by this invention is simple to operate in one step, with a short cycle and fast staining, and can be used as a routine detection method.
[0044] (3) The present invention also provides a method for preparing a vaginal secretion staining solution, which is simple and quick and greatly improves the efficiency of the staining method. Attached Figure Description
[0045] Figure 1 The staining results of Trichomonas vaginalis and leukocytes in Example 1 are shown.
[0046] Figure 2 The staining results of mold-leukocytes in Example 1 are shown.
[0047] Figure 3 The staining results of mold-hyphae-leukocyte in Example 1 are shown.
[0048] Figure 4 The staining results of white blood cells in Example 1 are shown.
[0049] Figure 5 The staining results of small round epithelial cells in Example 1 are shown.
[0050] Figure 6 The staining results are for epithelial cells from Example 1.
[0051] Figure 7 The staining results are for clue cells in Example 1. Detailed Implementation
[0052] The present invention will be further described below with reference to embodiments.
[0053] General Implementation Examples
[0054] A vaginal secretion staining solution includes a staining agent and a staining solvent, a stabilizer, and a buffer salt;
[0055] The staining agents are basic fuchsin and methylene blue.
[0056] Preferably, the vaginal secretion staining solution contains 10-15 g / L of the basic dye fuchsin and 1-1.5 g / L of the sub-methylene blue dye.
[0057] Preferably, the stabilizer includes cell membrane protectants and protein activity protectants, serum albumin, and antioxidants.
[0058] Preferably, the vaginal secretion staining solution contains 7-9 g / L of cell membrane protectant, 1-2 g / L of protein activity protectant, 0.4-0.6 g / L of serum albumin, and 2-4 g / L of antioxidant.
[0059] Preferably, the cell membrane protectant is one or more of trehalose, sucrose, mannitol, and glycine.
[0060] More preferably, the cell membrane protectant is sucrose.
[0061] Preferably, the protein activity protectant is ammonium sulfate.
[0062] Preferably, the serum albumin is BSA.
[0063] Preferably, the antioxidant is citric acid.
[0064] Preferably, the dye solvent is one or more of anhydrous ethanol, methanol, and ethylene glycol.
[0065] Preferably, the dye solvent content in the vaginal secretion staining solution is 90-110 ml / L.
[0066] Preferably, the concentration of the buffer salt is 2–500 mmol / L, and the pH of the buffer salt is 5.0–7.8.
[0067] Preferably, the buffer salt is one or more of MES, borate, phosphate, Tris-HCl, Bistris-HCl, HEPES, and PIPES.
[0068] More preferably, the buffer salt is a phosphate.
[0069] Furthermore, the phosphate is Na2HPO4·12H2O or NaH2PO4·2H2O.
[0070] Preferably, the vaginal secretion staining solution also includes surfactants and / or preservatives.
[0071] Preferably, the surfactant is sodium dodecyl sulfate.
[0072] Preferably, the surfactant content in the vaginal secretion staining solution is 0.01–0.02 g / L.
[0073] Preferably, the preservative is Proclin 300.
[0074] Preferably, the preservative content in the vaginal secretion staining solution is 1-3 ml / L.
[0075] A method for preparing a vaginal secretion staining solution includes the following steps:
[0076] (S1) Dissolve basic fuchsin and methylene blue in a staining solvent to obtain a staining solution;
[0077] (S2) Add a stabilizer to the above staining solution and adjust the pH to neutral with a buffer salt to obtain a buffer solution.
[0078] (S3) The above buffer solution was left to stand in the dark to obtain a vaginal secretion staining solution.
[0079] A staining method for vaginal secretions using a wet mount staining method is provided. The wet mount staining method involves adding vaginal secretions eluted with elution solution onto a glass slide or sample tube, then mixing the vaginal secretion with the aforementioned vaginal secretion staining solution to stain the vaginal secretions.
[0080] Example 1
[0081] (1) Preparation of vaginal secretion staining solution:
[0082] Weigh 1.0 g of basic fuchsin and 0.1 g of methylene blue into a mortar and pestle using an electronic balance. Grind thoroughly and transfer the mixture to a beaker. Add 10 mL of anhydrous ethanol and stir until the dyes are fully dissolved. Then add 0.8 g of sucrose, 0.15 g of ammonium sulfate, 0.05 g of BSA, and 0.3 g of citric acid, stirring until the stabilizer is fully dissolved. Adjust the pH of the mixture to 7.4 using 0.537 g of Na₂HPO₄·12H₂O and 0.078 g of NaH₂PO₄·2H₂O. Add 90 mL of deionized water, 0.001 g of sodium dodecyl sulfate, and 0.2 mL of Proclin 300 and stir until homogeneous to prepare a solution. Let the mixture stand in the dark for 24 hours, then filter to obtain a vaginal secretion staining solution.
[0083] (2) The staining method for vaginal secretions is as follows:
[0084] Wash vaginal secretions from a disposable cotton swab into a soft tubing using 2ml of physiological saline. Add 2-3 drops of this staining solution, squeeze the tubing for one minute to ensure thorough staining, then transfer one drop of the sample solution to a glass slide for microscopic observation of the vaginal secretions. The staining results for Trichomonas vaginalis and leukocytes are shown below. Figure 1 As shown, the staining results of fungi and leukocytes are as follows: Figure 2 As shown, the staining results of mold-hyphae-leukocytes are as follows: Figure 3 As shown, the staining results of white blood cells are as follows: Figure 4 As shown, the staining results of the small round epithelial cells are as follows: Figure 5 As shown, the staining results of the epithelial cells are as follows: Figure 6 As shown, the staining results of the clue cells are as follows: Figure 7 As shown.
[0085] When the vaginal secretion staining solution prepared in Example 1 was used for staining, it was observed that trichomonas and fungi could not be stained, while leukocytes and epithelial cells stained normally.
[0086] Example 2
[0087] (1) Preparation of vaginal secretion staining solution:
[0088] Weigh 1.5g of basic fuchsin and 0.15g of methylene blue into a mortar and pestle using an electronic balance. Grind them thoroughly and place the mixture into a beaker. Add 11mL of methanol and stir until the dyes are fully dissolved. Then add 0.5g of trehalose, 0.4g of glycine, 0.2g of ammonium sulfate, 0.06g of BSA, and 0.4g of citric acid in sequence, stirring until the stabilizers are fully dissolved. Adjust the pH of the mixture with 0.537g of Na₂HPO₄·12H₂O and 0.078g of NaH₂PO₄·2H₂O. Add 90mL of deionized water, 0.001g of sodium dodecyl sulfate, and 0.1mL of Proclin 300 and stir until homogeneous to prepare a solution. Let the mixture stand in the dark for 24 hours, then filter to obtain a vaginal secretion staining solution.
[0089] (2) The staining method for vaginal secretions is as follows:
[0090] Use 2ml of physiological saline to wash vaginal secretions from a disposable cotton swab into a soft tubing. Add 2-3 drops of this staining solution, squeeze the tubing for one minute to allow the vaginal secretions to be fully stained, then take a drop of the sample solution onto a glass slide and observe the vaginal secretions under a microscope. The results showed that when using the vaginal secretion staining solution prepared in Example 2, trichomonas and fungi could not be stained, while leukocytes and epithelial cells stained normally.
[0091] Example 3
[0092] (1) Preparation of vaginal secretion staining solution:
[0093] Weigh 1.0 g of basic fuchsin and 0.1 g of methylene blue into a mortar and pestle using an electronic balance. Grind them thoroughly and place the mixture into a beaker. Add 9 mL of ethylene glycol and stir until the dyes are fully dissolved. Then add 0.7 g of mannitol, 0.1 g of ammonium sulfate, 0.04 g of BSA, and 0.2 g of citric acid sequentially, stirring until the stabilizers are fully dissolved. Adjust the pH of the mixture with 0.537 g of Na₂HPO₄·12H₂O and 0.078 g of NaH₂PO₄·2H₂O. Add 91 mL of deionized water, 0.002 g of sodium dodecyl sulfate, and 0.3 mL of Proclin 300 and stir until homogeneous to prepare a solution. Let the mixture stand in the dark for 24 hours, then filter to obtain a vaginal secretion staining solution.
[0094] (2) The staining method for vaginal secretions is as follows:
[0095] Use 2ml of physiological saline to wash vaginal secretions from a disposable cotton swab into a soft tubing. Add 2-3 drops of this staining solution, squeeze the tubing for one minute to allow the vaginal secretions to be fully stained, then take a drop of the sample solution onto a glass slide and observe the vaginal secretions under a microscope. The results showed that when using the vaginal secretion staining solution prepared in Example 3, trichomonas and fungi could not be stained, while leukocytes and epithelial cells stained normally.
[0096] Example 4
[0097] (1) Preparation of vaginal secretion staining solution:
[0098] Weigh 2.0 g of basic fuchsin and 0.15 g of methylene blue into a mortar and pestle using an electronic balance. Grind thoroughly and transfer the mixture to a beaker. Add 11 mL of methanol and stir until the dyes are fully dissolved. Then add 0.5 g of trehalose, 0.4 g of glycine, 0.2 g of ammonium sulfate, 0.06 g of BSA, and 0.4 g of citric acid, stirring until the stabilizers are fully dissolved. Adjust the pH of the mixture with 0.537 g of Na₂HPO₄·12H₂O and 0.078 g of NaH₂PO₄·2H₂O. Add 90 mL of deionized water, 0.001 g of sodium dodecyl sulfate, and 0.1 mL of Proclin 300 and stir until homogeneous to prepare a solution. Let the mixture stand in the dark for 24 hours, then filter to obtain a vaginal secretion staining solution.
[0099] (2) The staining method for vaginal secretions is as follows:
[0100] Wash the vaginal secretions from a disposable cotton swab into a soft tube using 2ml of physiological saline. Add 2-3 drops of this staining solution, squeeze the soft tube for one minute to fully stain the vaginal secretions, then take a drop of the sample solution onto a glass slide and observe the vaginal secretions under a microscope.
[0101] Compared with Example 2, it was found that when using the vaginal secretion staining solution prepared in Example 4, the effect of Methylene blue was reduced, and the staining degree of the cell nucleus and cytoplasm was not significantly different, both appearing purple and difficult to distinguish. Based on experimental and theoretical research, the invention team discovered that the optimal concentration of the basic dye fuchsin in the vaginal secretion staining solution is between 10 and 15 g / L, and the optimal concentration of Methylene blue is between 1 and 1.5 g / L. At this dye ratio, the staining result is orange-red, which is superior to using both dyes as nuclear staining agents, where the nuclear staining is deeper than the cytoplasm.
[0102] Example 5
[0103] (1) Preparation of vaginal secretion staining solution:
[0104] Weigh 0.5g of basic fuchsin and 0.1g of methylene blue into a mortar and pestle using an electronic balance. Grind them thoroughly and place the mixture into a beaker. Add 9mL of ethylene glycol and stir until the dyes are fully dissolved. Then add 0.7g of mannitol, 0.1g of ammonium sulfate, 0.04g of BSA, and 0.2g of citric acid sequentially, stirring until the stabilizers are fully dissolved. Adjust the pH of the mixture with 0.537g of Na₂HPO₄·12H₂O and 0.078g of NaH₂PO₄·2H₂O. Add 91mL of deionized water, 0.002g of sodium dodecyl sulfate, and 0.3mL of Proclin 300 and stir until homogeneous to prepare a solution. Let the mixture stand in the dark for 24 hours, then filter to obtain a vaginal secretion staining solution.
[0105] (2) The staining method for vaginal secretions is as follows:
[0106] Wash the vaginal secretions from a disposable cotton swab into a soft tube using 2ml of physiological saline. Add 2-3 drops of this staining solution, squeeze the soft tube for one minute to fully stain the vaginal secretions, then take a drop of the sample solution onto a glass slide and observe the vaginal secretions under a microscope.
[0107] Compared with Example 3, it was found that when using the vaginal secretion staining solution prepared in Example 5, the effect of Methylene blue increased, but the staining degree of cytoplasm decreased, and only clearly visible cell nuclei were observed, with the stained cells appearing bluish. Based on experimental and theoretical research, the invention team discovered that the optimal concentration of the basic dye fuchsin in the vaginal secretion staining solution is between 10 and 15 g / L, and the optimal concentration of Methylene blue is between 1 and 1.5 g / L. At this dye ratio, the staining result is orange-red, which is superior to using both dyes as nuclear staining agents, where the nuclear staining is deeper than the cytoplasm.
[0108] Example 6
[0109] (1) Preparation of vaginal secretion staining solution:
[0110] Weigh 1.0 g of basic fuchsin and 0.1 g of methylene blue into a mortar and pestle using an electronic balance. Grind thoroughly and transfer the mixture to a beaker. Add 10 mL of anhydrous ethanol and stir until the dyes are fully dissolved. Then add 0.8 g of sucrose, 0.15 g of ammonium sulfate, 0.05 g of BSA, and 0.3 g of citric acid, stirring until the stabilizers are fully dissolved. Adjust the pH of the mixture to 7.4 using 0.537 g of Na₂HPO₄·12H₂O and 0.078 g of NaH₂PO₄·2H₂O. Add 90 mL of deionized water, 0.001 g of sucrose ester, and 0.2 mL of Proclin 300 and stir until homogeneous to prepare a solution. Let the mixture stand in the dark for 24 hours, then filter to obtain a vaginal secretion staining solution.
[0111] (2) The staining method for vaginal secretions is as follows:
[0112] Wash the vaginal secretions from a disposable cotton swab into a soft tube using 2ml of physiological saline. Add 2-3 drops of this staining solution, squeeze the soft tube for one minute to fully stain the vaginal secretions, then take a drop of the sample solution onto a glass slide and observe the vaginal secretions under a microscope.
[0113] Example 6 uses sucrose ester as a surfactant. Compared with Example 1, staining with the vaginal secretion staining solution prepared in Example 6 showed that trichomonas and fungi could not be stained, while leukocytes and epithelial cells stained normally, but the staining effect was poor. Based on experimental and theoretical research, the invention team discovered that sodium dodecyl sulfate can not only improve the performance of dyes but also act as a solubilizer, increasing the solubility of various solutes. Using sodium dodecyl sulfate as a surfactant, it can increase cell membrane permeability, bind to the hydrophobic portions of membrane proteins, and separate them from the membrane, greatly improving the staining effect of the staining agent.
[0114] Comparative Example 1
[0115] (1) Preparation of vaginal secretion staining solution:
[0116] Weigh 1g of methyl red and 0.1g of toluidine blue into a mortar and pestle using an electronic balance. Grind thoroughly and transfer the mixture to a beaker. Add 10mL of anhydrous ethanol and stir until the dyes are fully dissolved. Then add 0.8g of sucrose, 0.15g of ammonium sulfate, 0.05g of BSA, and 0.3g of citric acid, stirring until the stabilizers are fully dissolved. Adjust the pH of the mixture to 7.4 using 0.537g of Na₂HPO₄·12H₂O and 0.078g of NaH₂PO₄·2H₂O. Add 90mL of deionized water, 0.001g of sodium dodecyl sulfate, and 0.2mL of Proclin 300 and stir until homogeneous to prepare a solution. Let the mixture stand in the dark for 24 hours, then filter to obtain a vaginal secretion staining solution.
[0117] (2) The staining method for vaginal secretions is as follows:
[0118] Use 2ml of physiological saline to wash vaginal secretions from a disposable cotton swab into a soft tubing. Add 2-3 drops of this staining solution, squeeze the tubing for one minute to allow the vaginal secretions to be fully stained, then take a drop of the sample solution onto a glass slide and observe the vaginal secretions under a microscope. The results showed that using the vaginal secretion staining solution prepared in Comparative Example 1, trichomonas, fungi, leukocytes, and epithelial cells could all be stained normally.
[0119] Comparative Example 2
[0120] (1) Preparation of vaginal secretion staining solution:
[0121] Weigh 1g of basic fuchsin and 0.1g of toluidine blue into a mortar and pestle using an electronic balance. Grind them thoroughly and place the mixture into a beaker. Add 10mL of anhydrous ethanol and stir until the dyes are fully dissolved. Then add 0.8g of sucrose, 0.15g of ammonium sulfate, 0.05g of BSA, and 0.3g of citric acid, stirring until the stabilizers are fully dissolved. Adjust the pH of the mixture to 7.4 using 0.537g of Na₂HPO₄·12H₂O and 0.078g of NaH₂PO₄·2H₂O. Add 90mL of deionized water, 0.001g of sodium dodecyl sulfate, and 0.2mL of Proclin 300 and stir until homogeneous to prepare a solution. Let the mixture stand in the dark for 24 hours, then filter to obtain a vaginal secretion staining solution.
[0122] (2) The staining method for vaginal secretions is as follows:
[0123] Use 2ml of physiological saline to wash vaginal secretions from a disposable cotton swab into a soft tubing. Add 2-3 drops of this staining solution, squeeze the tubing for one minute to allow the vaginal secretions to be fully stained, then take a drop of the sample solution onto a glass slide and observe the vaginal secretions under a microscope. The results showed that using the vaginal secretion staining solution prepared in Comparative Example 2, trichomonas, fungi, leukocytes, and epithelial cells could all be stained normally.
[0124] Comparative Example 3
[0125] (1) Preparation of vaginal secretion staining solution:
[0126] Weigh 1g of methyl red and 0.1g of methylene blue into a mortar and pestle using an electronic balance. Grind thoroughly and transfer the mixture to a beaker. Add 10mL of anhydrous ethanol and stir until the dyes are fully dissolved. Then add 0.8g of sucrose, 0.15g of ammonium sulfate, 0.05g of BSA, and 0.3g of citric acid, stirring until the stabilizer is fully dissolved. Adjust the pH of the mixture to 7.4 using 0.537g of Na₂HPO₄·12H₂O and 0.078g of NaH₂PO₄·2H₂O. Add 90mL of deionized water, 0.001g of sodium dodecyl sulfate, and 0.2mL of Proclin 300 and stir until homogeneous to prepare a solution. Let the mixture stand in the dark for 24 hours, then filter to obtain a vaginal secretion staining solution.
[0127] (2) The staining method for vaginal secretions is as follows:
[0128] Use 2ml of physiological saline to wash vaginal secretions from a disposable cotton swab into a soft tubing. Add 2-3 drops of this staining solution, squeeze the tubing for one minute to allow the vaginal secretions to be fully stained, then take a drop of the sample solution onto a glass slide and observe the vaginal secretions under a microscope. The results showed that using the vaginal secretion staining solution prepared in Comparative Example 3, trichomonas, fungi, leukocytes, and epithelial cells could all be stained normally.
[0129] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.
[0130] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.
Claims
1. A method for staining vaginal secretions to distinguish between leukocytes and trichomonas, characterized in that, Vaginal secretions were stained using a wet mount staining method. The wet mount staining method involves adding vaginal secretions eluted with elution buffer onto a glass slide or sample tube, then mixing the vaginal secretion staining solution with the secretions to allow the staining solution to stain the secretions. The vaginal secretion staining solution includes a staining agent, a staining solvent, a stabilizer, and a buffer salt. The staining agent is basic fuchsin and subunit blue. The stabilizer includes a cell membrane protectant, a protein activity protectant, serum albumin, and an antioxidant.
2. The staining method according to claim 1, characterized in that, The vaginal secretion staining solution contains 10-15 g / L of basic fuchsin and 1-1.5 g / L of methylene blue.
3. The staining method according to claim 1, characterized in that, The vaginal secretion staining solution contains 7-9 g / L of cell membrane protectant, 1-2 g / L of protein activity protectant, 0.4-0.6 g / L of serum albumin, and 2-4 g / L of antioxidant.
4. The staining method according to claim 1 or 2, characterized in that, The staining solvent is one or more of anhydrous ethanol, methanol, and ethylene glycol.
5. The staining method according to claim 1 or 2, characterized in that, The staining solvent in the vaginal secretion staining solution has a concentration of 90-110 ml / L.
6. The staining method according to claim 1 or 2, characterized in that, The buffer salt is one or more of MES, borate, phosphate, Tris-HCl, Bistris-HCl, HEPES, and PIPES.
7. The staining method according to claim 6, characterized in that, The buffer salt is a phosphate.
8. The staining method according to claim 7, characterized in that, The phosphate is Na2HPO4·12H2O or NaH2PO4·2H2O.
9. The staining method according to claim 1, characterized in that, The method for preparing the vaginal secretion staining solution includes the following steps: (S1) Dissolve basic fuchsin and methylene blue in a staining solvent to obtain a staining solution; (S2) Add a stabilizer to the above staining solution and adjust the pH to neutral with a buffer salt to obtain a buffer solution. (S3) The above buffer solution was left to stand in the dark to obtain a vaginal secretion staining solution.