Vaginal secretion staining fluid, preparation method and method for staining by applying vaginal secretion staining fluid

Abstract

The invention provides a vaginal secretion staining solution, a preparation method and a method for staining by applying the vaginal secretion staining solution, and belongs to the technical field of cell staining. The alkaline toluidine blue basic dye is used as a coloring agent, the phosphate buffer solution is used as a solvent, the sodium hydroxide is used as a regulator, the components are simple, the preparation is convenient, the dyeing method is rapid, epithelial cells, leukocytes and bacilli can be dyed, trichomonad and mould cannot be dyed, the leukocytes and the trichomonad can be obviously distinguished through color difference, and the method is suitable for large-scale popularization and application. In addition, the colored bacilli can intuitively assist in cleanliness discrimination and clue cell identification, so that personnel misjudgment caused by similar forms of detection targets can be avoided, and the problems that the current dye liquor has many components, the preparation method is tedious, the dyeing method is complex, the detection time is short and the like are solved. And the problem of lack of a sample tube dropwise adding dye liquor proportioning scheme is solved.

Description

Technical Field

[0001] The invention relates to the technical field of cell staining, and in particular to a vaginal secretion staining liquid, a preparation method and a staining method using the same. Background Art

[0002] Vaginal secretion examination is the most common gynecological examination item and the simplest and most commonly used examination method for diagnosing female external genital tract infection. Through the examination of the appearance, cleanliness, epithelial cells, white blood cells, various bacteria, fungi and parasites of female vaginal secretions, a systematic evaluation of the microecology in the vagina is made to understand the health status of women.

[0003] The conventional inspection methods for vaginal secretions include direct smear method and wet mount method. Among them, the direct smear method is rarely used in clinical practice due to the defects of uneven smear and local excessive thickness that affect microscopic observation; the saline wet mount method is the inspection method currently commonly used in clinical practice. The advantages of the saline wet mount method are uniform smear and easier microscopic observation compared with the direct smear method. The disadvantages are that the inspector needs to have rich clinical experience, and the diagnosis is more subjective and the missed detection rate is high; the staining method is a further development of the saline wet mount method, which belongs to the wet mount method and is more conducive to improving the detection rate of the cause of disease. It is one of the means currently widely used. However, the existing staining method uses a dyeing solution with many components, a cumbersome preparation method, and a complex staining method. In the existing vaginal secretion staining solution, there are more than a dozen components and as few as five or six, and the process of dyeing solution preparation is also cumbersome. The staining process is also mostly stained on a slide, and the staining process is complicated. Among them, CN 105067412 A discloses a vaginal secretion staining solution and a preparation method and a staining method thereof. Although a relatively simple method of dripping the staining solution into a sample tube is described, no suitable staining ratio scheme is given.

[0004] In recent years, the development of lens-free imaging microscopy has further promoted the diversification of vaginal secretion sample detection methods. However, due to the technical limitations of its resolution, if the bacilli that are diagnostically significant in the clinical diagnosis of vaginal secretions are not stained, the recognition rate will decrease. Summary of the invention

[0005] In view of this, the present invention provides a vaginal secretion dyeing liquid. The dyeing liquid of the present invention has simple ingredients, is easy to prepare, and has a quick dyeing method. It can dye epithelial cells, leukocytes and bacilli, but cannot dye trichomonas and molds. Through color difference, leukocytes and trichomonas can be clearly distinguished, as well as broken leukocytes and spores.

[0006] The vaginal secretion dyeing liquid of the present invention comprises a dye, a dyeing solvent and a regulator, wherein the dye is a toluidine blue basic dye, the dyeing solvent is a buffered salt solution, and the regulator is an alkaline solution.

[0007] Furthermore, the dye is toluidine blue O, the dyeing solvent is phosphate buffer, and the regulator is sodium hydroxide solution; the concentration of the sodium hydroxide solution is 140-145 g / L.

[0008] Furthermore, the contents of the dye, dyeing solvent and regulator are 0.6-1.0 g / L, 990-1000 ml / L and 0.7-2 ml / L respectively. The concentrations of the three components of the dye solution are all in the optimal content range. If the concentration is lower than the range, the visible components will be lightly stained, and the contrast will be poor. If the concentration is higher than the range, the visible components will be darkly stained, and the background and the effective components will be integrated. The target objects will be colored, and there will be no specificity of the dye solution, which is not conducive to identification.

[0009] The vaginal secretion staining solution according to the present invention has simple ingredients, is easy to prepare, and has a quick staining method. It can stain epithelial cells, leukocytes and bacilli, but cannot stain trichomonas and molds. Through the color difference, leukocytes and trichomonas, as well as broken leukocytes and spores can be clearly distinguished. In addition, the stained bacilli can intuitively assist in cleanliness judgment and clue cell identification, and can avoid personnel misjudgment due to the similar morphology of the detection targets. The current dye solution has many ingredients, a cumbersome preparation method, a complicated staining method, and a lack of a sample tube dripping dye solution ratio scheme.

[0010] The preparation method of the vaginal secretion dyeing liquid of the present invention comprises the following steps:

[0011] (1) mixing a dye and a dye solvent in a certain proportion and stirring them to fully dissolve the dye to obtain a dye buffer;

[0012] (2) adding a regulator to the staining buffer described in step (1), stirring and oscillating to obtain a mixed solution;

[0013] (3) Filtering the mixed solution of step (2) to obtain the vaginal secretion dye solution. The pore size of the filter membrane used for filtration is selected to be 0.22um or 0.45um. A pore size of 0.22um can basically effectively filter out all unnecessary particulate impurities in the dye solution, and can also filter out bacteria to achieve a sterilization effect; a filter membrane with a pore size of 0.45um or a relatively close pore size can allow some small molecules of the dye solution to enter, while still effectively filtering out most large particulate impurities and bacteria.

[0014] The present invention also provides an application of the vaginal secretion staining solution, wherein the vaginal secretion staining solution is used for wet mount staining, and the staining method comprises the following steps:

[0015] Vaginal secretions were collected by swab, and physiological saline was added for elution to obtain physiological saline eluate of vaginal secretions, and then the vaginal secretion staining solution was added dropwise, mixed evenly, and the staining condition was observed after 10 to 20 seconds.

[0016] Preferably, the volume ratio of the physiological saline eluent of vaginal secretions to the vaginal secretion staining solution is 2:1-2.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The vaginal secretion staining solution of the present invention has simple ingredients, is easy to prepare, and is easy to stain. The staining effect of the calibrated object is good and the stability is strong. It not only reduces the preparation cost of the staining solution, but also improves the staining efficiency, especially improves the imaging effect of bacilli under lens-free microscopic imaging technology.

[0019] (2) The vaginal secretion staining solution of the present invention overcomes the component problems of the traditional staining solution, and is composed of three components and is easy to obtain.

[0020] (3) The preparation method of the present invention overcomes the problem of cumbersome operation of the traditional dyeing solution preparation method, which is mixing, filtering and stirring at room temperature without other requirements such as time, temperature, sequence, etc.

[0021] (4) The vaginal secretion staining solution of the present invention can be used to quickly stain bacilli in the wet mount method, thus overcoming the defect that bacilli are not easy to stain in the wet mount method on the market. It can effectively assist in the determination of the cleanliness of leucorrhea and provide background information for dysbiosis, and can effectively reduce the missed detection rate, especially in clinical diagnosis using lensless microscopic imaging technology.

[0022] (5) The vaginal secretion staining solution of the present invention can effectively distinguish leukocytes from trichomonas, broken leukocytes from spores, and the staining of clue cells is prominent with high contrast, which can effectively and quickly improve the detection rate of vaginitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the staining result of Trichomonas-leukocytes in Example 1.

[0024] Figure 2 This is the staining result of mold-leukocytes in Example 2.

[0025] Figure 3 This is the staining result of clue cells in Example 3.

[0026] Figure 4 This is the staining result of the bacillus in Example 4.

[0027] Figure 5 This is the staining result of epithelial cells and bacilli in Comparative Example 1.

[0028] Figure 6 This is the staining result of Trichomonas-leukocytes in Comparative Example 2.

[0029] Figure 7 This is the staining result of clue cells in comparative example 3.

[0030] Figure 8 This is the staining result of bacillus in comparative example 4. DETAILED DESCRIPTION

[0031] The embodiments of the present invention provide a vaginal secretion dyeing liquid, a preparation method and an application thereof. In the specific embodiments of the present invention, the methods used are conventional methods in the art unless otherwise specified.

[0032] The present invention will be further described below in conjunction with the embodiments.

[0033] Example 1

[0034] A vaginal secretion dyeing solution, composed of the following raw materials:

[0035] Toluidine blue O 0.007g, phosphate buffer 10mL, sodium hydroxide solution with a mass concentration of 140.024g / L 12ul.

[0036] The preparation method of the vaginal secretion dyeing solution comprises the following steps:

[0037] (1) mixing toluidine blue O and phosphate buffer in proportion and stirring to fully dissolve the dye to obtain a dye buffer;

[0038] (2) adding sodium hydroxide solution to the staining buffer described in step (1), stirring and shaking to mix, to obtain a mixed solution;

[0039] (3) Filter the mixed solution of step (2) using a filter membrane with a pore size of 0.45 μm to obtain the vaginal secretion staining solution.

[0040] The vaginal secretion staining solution is used for wet mount staining, and the steps are as follows:

[0041] Vaginal secretions were collected by swab, 2 mL of saline was added to elute, and then 1 mL of the vaginal secretion staining solution was added dropwise, mixed evenly, and the staining was observed for 10 to 20 seconds. Samples were taken for microscopic examination, and the staining results of Trichomonas-leukocytes were obtained as follows Figure 1 As shown, from Figure 1 It can be seen that Trichomonas is not stained, while leukocytes are stained, and Trichomonas and leukocytes are clearly separated.

[0042] Example 2

[0043] A vaginal secretion dyeing solution, composed of the following raw materials:

[0044] Toluidine blue O 0.009 g, phosphate buffer 11 mL, sodium hydroxide solution with a mass concentration of 140.024 g / L 10 ul.

[0045] The preparation method of the vaginal secretion dyeing solution comprises the following steps:

[0046] (1) mixing toluidine blue O and phosphate buffer in proportion and stirring to fully dissolve the dye to obtain a dye buffer;

[0047] (2) adding sodium hydroxide solution to the staining buffer described in step (1), stirring and shaking to mix, to obtain a mixed solution;

[0048] (3) Filter the mixed solution of step (2) using a filter membrane with a pore size of 0.22 μm to obtain the vaginal secretion staining solution.

[0049] The vaginal secretion staining solution is used for wet mount staining, and the steps are as follows:

[0050] Vaginal secretions were collected by swab, 2 mL of physiological saline was added to elute, and then 2 mL of the vaginal secretion staining solution was added dropwise, mixed evenly, and the staining was observed for 10 to 20 seconds. Samples were taken for microscopic examination, and the mold-leukocyte staining results were obtained as follows Figure 2 As shown, from Figure 2 It can be seen that the mold spores are not stained, the white blood cells are stained, and the contrast is high.

[0051] Example 3

[0052] A vaginal secretion dyeing solution, composed of the following raw materials:

[0053] Toluidine blue O 0.012g, phosphate buffer 12mL, sodium hydroxide solution with a mass concentration of 140.024g / L 10ul.

[0054] The preparation method of the vaginal secretion dyeing solution comprises the following steps:

[0055] (1) mixing toluidine blue O and phosphate buffer in proportion and stirring to fully dissolve the dye to obtain a dye buffer;

[0056] (2) adding sodium hydroxide solution to the staining buffer described in step (1), stirring and shaking to mix, to obtain a mixed solution;

[0057] (3) Filter the mixed solution of step (2) using a filter membrane with a pore size of 0.22 μm to obtain the vaginal secretion staining solution.

[0058] The vaginal secretion staining solution is used for wet mount staining, and the steps are as follows:

[0059] Vaginal secretions were collected by swab, 2 mL of saline was added to elute, and then 1 mL of the vaginal secretion staining solution was added dropwise, mixed evenly, and the staining was observed for 10 to 20 seconds. Samples were taken for microscopic examination, and the staining results of clue cells were obtained as follows Figure 3 As shown, from Figure 3 It can be seen that the clue cells with attached bacteria have a high contrast with the normal epithelium.

[0060] Example 4

[0061] A vaginal secretion dyeing solution, composed of the following raw materials:

[0062] Toluidine blue O 0.01g, phosphate buffer 10mL, sodium hydroxide solution with a mass concentration of 140.024g / L 12ul.

[0063] The preparation method of the vaginal secretion dyeing solution comprises the following steps:

[0064] (1) mixing toluidine blue O and phosphate buffer in proportion and stirring to fully dissolve the dye to obtain a dye buffer;

[0065] (2) adding sodium hydroxide solution to the staining buffer described in step (1), stirring and shaking to mix, to obtain a mixed solution;

[0066] (3) Filter the mixed solution of step (2) using a filter membrane with a pore size of 0.22 μm to obtain the vaginal secretion staining solution.

[0067] The vaginal secretion staining solution is used for wet mount staining, and the steps are as follows:

[0068] Vaginal secretions were collected by swab, 2 mL of saline was added to elute, and then 2 mL of the vaginal secretion staining solution was added dropwise, mixed evenly, and the staining was observed for 10 to 20 seconds. Samples were taken for microscopic examination, and the staining results of the bacilli were obtained as follows: Figure 4 As shown, from Figure 4 It can be seen that all bacilli are colored and have clear morphology.

[0069] Comparative Example 1

[0070] A vaginal secretion dyeing solution, composed of the following raw materials:

[0071] Toluidine blue O 0.002g, phosphate buffer 10mL, sodium hydroxide solution with a mass concentration of 140.024g / L 12ul.

[0072] The preparation method of the vaginal secretion dyeing solution comprises the following steps:

[0073] (1) mixing toluidine blue O and phosphate buffer in proportion and stirring to fully dissolve the dye to obtain a dye buffer;

[0074] (2) adding sodium hydroxide solution to the staining buffer described in step (1), stirring and shaking to mix, to obtain a mixed solution;

[0075] (3) Filter the mixed solution of step (2) using a filter membrane with a pore size of 0.22 μm to obtain the vaginal secretion staining solution.

[0076] The vaginal secretion staining solution is used for wet mount staining, and the steps are as follows:

[0077] Vaginal secretions were collected by swab, 2 mL of saline was added to elute, and then 2 mL of the vaginal secretion staining solution was added dropwise, mixed evenly, and the staining was observed for 10 to 20 seconds. Samples were taken for microscopic examination, and the staining results of epithelial cells and bacilli were obtained as follows: Figure 5 As shown, from Figure 5 It can be seen that all epithelial cells are lightly stained, and most of the bacilli are not stained, making counting and observation difficult.

[0078] Comparative Example 2

[0079] A vaginal secretion dyeing solution, composed of the following raw materials:

[0080] Toluidine blue O 0.02g, phosphate buffer 10mL, sodium hydroxide solution with a mass concentration of 140.024g / L 12ul.

[0081] The preparation method of the vaginal secretion dyeing solution comprises the following steps:

[0082] (1) mixing toluidine blue O and phosphate buffer in proportion and stirring to fully dissolve the dye to obtain a dye buffer;

[0083] (2) adding sodium hydroxide solution to the staining buffer described in step (1), stirring and shaking to mix, to obtain a mixed solution;

[0084] (3) Filter the mixed solution of step (2) using a filter membrane with a pore size of 0.22 μm to obtain the vaginal secretion staining solution.

[0085] The vaginal secretion staining solution is used for wet mount staining, and the steps are as follows:

[0086] Vaginal secretions were collected by swab, 2 mL of physiological saline was added to elute, and then 2 mL of the vaginal secretion staining solution was added dropwise, mixed evenly, and the staining was observed for 10 to 20 seconds. Samples were taken for microscopic examination, and the staining results of Trichomonas-leukocytes were obtained as follows Figure 6 As shown, from Figure 6 It can be seen that both Trichomonas and white blood cells are stained and difficult to distinguish.

[0087] Comparative Example 3

[0088] (1) A product commonly purchased on the market was selected as a vaginal secretion staining solution, and the staining solution was a methylene blue solution purchased from Zhuhai Beso Biotechnology Co., Ltd., batch number C221002.

[0089] (2) The vaginal secretion staining wet mount method is as follows:

[0090] Vaginal secretions were collected by swab, 2 mL of saline was added to elute, and then 1 mL of body fluid staining solution was added, mixed evenly, and the staining was observed for 10 to 20 seconds. Samples were taken for microscopic examination, and the staining results of clue cells were obtained as follows Figure 7 As shown, from Figure 7 It can be seen that the contrast between clue cells and normal epithelial cells is not obvious.

[0091] Comparative Example 4

[0092] (1) A product commonly purchased on the market was selected as a vaginal secretion staining solution, and the staining solution was a methylene blue solution purchased from Zhuhai Beso Biotechnology Co., Ltd., batch number C221002.

[0093] (2) Vaginal secretion staining wet mount method, the steps are as follows:

[0094] Swab the vaginal secretions, add 2mL of saline to elute, then add 1mL of body fluid staining solution, mix evenly, and observe the staining for 10 to 20 seconds. Take a sample for microscopic examination, and get the bacillus staining results as follows Figure 8 As shown, from Figure 8 It can be seen that the bacilli are not colored, identification is unclear, and it is difficult to count.

[0095] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A vaginal secretion dyeing solution, comprising a dye, a dyeing solvent and a conditioning agent, characterized in that: The dye is a toluidine blue basic dye, the dyeing solvent is a buffered salt solution, and the regulator is an alkaline solution.

2. The vaginal secretion dyeing solution according to claim 1, characterized in that: The dye is toluidine blue O, the dye solvent is phosphate buffer, and the regulator is sodium hydroxide solution; the concentration of the sodium hydroxide solution is 140-145 g / L.

3. The vaginal secretion dyeing solution according to claim 2, characterized in that: The contents of the dye, dyeing solvent and regulator are 0.6-1.0 g / L, 990-1000 ml / L and 0.7-2 ml / L respectively.

4. The method for preparing a vaginal secretion dyeing solution according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) mixing a dye and a dye solvent in a certain proportion and stirring them to fully dissolve the dye to obtain a dye buffer; (2) adding a regulator to the staining buffer described in step (1), stirring and oscillating to obtain a mixed solution; (3) Filtering the mixed solution of step (2) to obtain the vaginal secretion staining solution.

5. The use of the vaginal secretion dyeing solution according to any one of claims 1 to 3, characterized in that: The vaginal secretion dyeing liquid is used for dyeing, and the dyeing method comprises the following steps: Vaginal secretions are collected by swab, and physiological saline is added for elution to obtain physiological saline eluate of vaginal secretions, and then the vaginal secretion staining solution is added dropwise, mixed evenly, and the staining condition is observed after 10 to 20 seconds; the volume ratio of the physiological saline eluate of vaginal secretions to the vaginal secretion staining solution is 2:1 to 2.

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