WO2021036368A1

WO2021036368A1 - Epidermis fungus sampling and staining method

Info

Publication number: WO2021036368A1
Application number: PCT/CN2020/092135
Authority: WO
Inventors: 禹洋; 江颖纯; 黎璐; 马敬信; 林焱; 吴玥; 马洁
Original assignee: 昆氏(深圳)生物科技有限公司
Priority date: 2019-08-28
Filing date: 2020-05-25
Publication date: 2021-03-04
Also published as: CN110455604A; CN110455604B

Abstract

An epidermis fungus sampling and staining method. The epidermis fungus sampling and staining method comprises: providing a transparent detection card (100) having a detection area (11), a transparent viscous material layer (111) being attached to the detection area (11), and a surface of the transparent viscous material layer (111) being covered with a protection film (112); combing in the hair direction of a subject under test for multiple times by using a sampling comb subjected to disinfection treatment, so that dandruff is sticked to the sampling comb; uncovering the protection film (112) on the transparent detection card (100), and repeatedly attaching tooth tips where the dandruff is sticked of the sampling comb to the surface of the transparent viscous material layer (111) to enable sufficient dandruff to be sticked to the transparent viscous material layer (111); sequentially dropwise adding equal amounts of dyes A and B to the surface of the transparent viscous material layer (111), standing, and washing with deionized water; and transferring the transparent detection card (100) under a microscope for microscopy. The epidermis fungus sampling and staining method has the characteristics that sampling is convenient, samples are easy to transfer and fix and not easy to pollute, the staining time is short, and the like, and the success rate and efficiency of sampling and staining are improved.

Description

Epidermal fungus sampling and staining method

Technical field

The invention belongs to the technical field of fungal detection and processing, and in particular relates to a method for sampling and dyeing epidermal fungi.

Background technique

The detection of epidermal fungi plays an important guiding role in the diagnosis and prevention of skin and hair diseases. Among them, direct sampling and staining microscopy is one of the fastest judgment methods, and it can also provide key guidance information for further culture identification.

The traditional method of epidermal sampling is to scrape the dander from the test site with a scalpel or the end of a glass slide, collect it in an appropriate package, and then transfer it to the laboratory, where it is stained on the glass slide for microscopic examination. This method of sampling and staining has the following defects: (1) Sampling of the head skin is not suitable for use with razor blades or glass slides in areas with hair; (2) The scraped sample needs to be transferred with packaging, and multiple transfers may easily lose the sample. (3) The dander sample is difficult to fix after being transferred to the glass slide, and it is washed away by the dye and lotion during the dyeing process, causing the sample to be lost. The dyeing operation is difficult; (4) The sample is heavy. Swarf and fungus will be dyed at the same time. Even if a specific dye is used, it is difficult to distinguish dander and fungus in color, which makes it difficult to identify the fungus attached or hidden in the dander. In addition, the entire sampling and dyeing is very dependent on the experience and level of the operator.

Summary of the invention

technical problem

The solution to the problem

Technical solutions

Aiming at the problems of high sampling difficulty in sampling and dyeing of epidermal fungi, easy contamination of samples, difficult dyeing operations, easy loss of samples, and difficulty in distinguishing dander and fungi, the present invention provides a method for sampling and dyeing epidermal fungi.

In order to achieve the above-mentioned purpose of the invention, the technical scheme of the present invention is as follows:

A method for sampling and dyeing epidermal fungi, including the following steps:

Step S01. Provide a transparent detection card, the transparent detection card has a detection area, the detection area is attached with a layer of transparent viscous material, and the surface of the transparent viscous material layer is covered with a protective film;

Step S02. Use a sterilized sampling comb to comb several times along the direction of the hair of the subject to be tested, so that the dander sticks to the sampling comb;

Step S03. Remove the protective film on the transparent detection card, and repeatedly stick the tip of the sampling comb with dander on the surface of the transparent viscous material layer, so that a sufficient amount of dander sticks to the transparent On the viscous material layer;

Step S04. Drop A dye solution on the surface of the transparent viscous material layer, and then add the same amount of B dye solution as the A dye solution on the surface of the transparent viscous material layer, and use deionized water after standing still. rinse;

Step S05. Transfer the transparent inspection card to a microscope for microscopic examination to obtain information on dander and fungus.

The technical effects of the present invention are:

Compared with the prior art, the epidermal fungus sampling and dyeing method provided by the present invention directly transfers the sample obtained by the sampling comb to the transparent test card, which is convenient for sampling, easy to transfer and not easy to be contaminated, and easy to fix and not easy to dye and clean. The elution and dyeing time is short, which improves the success rate and efficiency of sampling and dyeing.

The beneficial effects of the invention

Brief description of the drawings

Description of the drawings

In order to explain the technical solutions in the embodiments of the present invention more clearly, the following will briefly introduce the drawings that need to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. A person of ordinary skill in the art can obtain other drawings based on these drawings without creative work.

Figure 1 is a schematic top view of a transparent detection card of the present invention;

Figure 2 is a schematic cross-sectional view taken along line A-A of Figure 1;

Figure 3 shows the effect of sampling and dyeing epidermal fungi using conventional sampling and dyeing methods;

Fig. 4 shows the dyeing effect of sampling and dyeing epidermal fungi in Example 1 of the present invention;

Fig. 5 shows the dyeing effect of sampling and dyeing epidermal fungi in Example 2 of the present invention;

Fig. 6 shows the dyeing effect of sampling and dyeing epidermal fungi in Example 3 of the present invention;

Among them, 100-transparent detection card, 1-transparent area, 2-label area; 11-detection area; 111-transparent adhesive material layer, 112-protective film.

Invention embodiment

Embodiments of the present invention

In order to make the objectives, technical solutions, and advantages of the present invention clearer, the following further describes the present invention in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not used to limit the present invention.

The invention provides a method for sampling and dyeing epidermal fungi.

See Figures 1 and 2 for details. The method for sampling and dyeing epidermal fungi needs to involve a transparent detection card 100. The transparent detection card 100 includes a transparent area 1 and a label area 2. The transparent area 1 is fixed with a detection area 11, and the detection area 11 is pasted with a transparent viscous material layer 111, and a protective film 112 is pasted directly above the transparent viscous material layer 111 to prevent the transparent viscous material layer 111 from being contaminated before use, and the protective film 112 should be removed during use; The label area 2 can be used to paste information of the person to be tested, and the operator can hold the label area 2 for easy operation. The above-mentioned transparent inspection card 100 is divided into a transparent area 1 and a label area 2. It does not mean that only the transparent area 1 is transparent. It is mainly used to distinguish two different areas. The entire transparent inspection card 100 is made of transparent material. Of course, the label area 2 can be a non-transparent material.

The above-mentioned epidermal fungus sampling and staining method includes the following steps:

Step S01. Provide a transparent detection card 100 as shown in Figures 1 and 2;

Step S03. Remove the protective film 112 on the transparent detection card 100, and repeatedly stick the tip of the sampling comb with dander on the surface of the transparent viscous material layer, so that a sufficient amount of dander sticks to the surface of the transparent viscous material layer. The transparent adhesive material layer;

The following is a further explanation of the above-mentioned epidermal fungus sampling and staining method.

In step S01, the viscous material used for the viscous material layer 111 is preferably acrylic. Acrylic is a transparent material that will not be dyed by the dye solution. Observe the morphology of the sample clearly under the microscope. Further preferably, the area of ^{the transparent detection area 11 is (70-350) mm 2} , and the transparent detection area 11 may be a square area, a circular area, or the like. More preferably, it is a circular area, and the area and the circular area are conducive to the adhesion of the epidermal sample, the uniform distribution and diffusion of the dye solution, and the use of the dye solution can be reduced.

Preferably, the thickness of the transparent viscous material layer 111 is (0.5～1.5) mm. After the thickness is not suitable, the adhesion of the epidermal sample will not be improved after the thickness exceeds 1.5 mm. Instead, acrylic acid will be wasted, which is too thin, such as thickness. If it is less than 0.5 mm, the sampling comb is likely to directly damage the transparent viscous material layer 111.

In step S02, the sampling comb is a plastic comb, that is, a common plastic comb. It is better to have special hand-held and non-hand-held parts for the sampling comb, so as not to interfere with the sampling by external factors such as hands during the sampling process or even contaminate the obtained skin . In the sampling process, the sampling comb is placed in different positions along the direction of the subject’s hair for 3 to 5 times. When combing, try to keep only one side of the comb and the tip of the comb during combing. To touch the scalp, but avoid excessive force to cause damage to the scalp. If the comb sticks to the hair, use tweezers to remove the hair on the comb surface, and avoid touching the tips of the teeth when removing the hair with tweezers to avoid sample loss.

After sampling, if you do not directly perform on-site testing, you can store the sampling comb in a clean sealed bag and store it in a cool and dry environment. The storage time should be less than one week as much as possible, and the test should be performed within one week.

In step S03, when transferring the obtained skin sample to the transparent detection card 100, the protective film 112 on the surface of the detection area is first removed. When removing the protective film 112, avoid touching the viscous material layer 111 to avoid contamination. Lightly touch the tooth tip of the sampling comb with the dander on the surface of the viscous material layer 111, and touch the sampling comb back and forth several times to transfer the dander sample to the surface of the viscous material layer 111. If the amount of dander sample is insufficient, other parts of the sampling comb can be flatly attached to the surface of the viscous material layer 111, or secondary sampling can be performed.

In step S04, the dye solution A is a mixed solution of eosin and potassium hydroxide, and the mass concentration of eosin in the mixed solution is 0.8% to 1.2%, and the mass concentration of potassium hydroxide is 8.5% to 12.5% . Specifically, in dye solution A, the mass concentration of eosin may be 1.0%, and the mass concentration of potassium hydroxide may be 10%.

The B dye solution is a three-mixed solution of gentian violet, lactic acid and glycerin, and the mass concentration of gentian violet in the three-mixed solution is 0.8% to 1.5%, the mass concentration of lactic acid is 18% to 25%, and glycerin The mass concentration of 15% to 25%. Specifically, the mass concentration of gentian violet in the B dye solution can be 1.0%, the mass concentration of lactic acid can be 20%, and the mass concentration of glycerin can be 20%.

Specifically, the A dye solution is dripped onto the surface of the viscous material layer 111, and then the B dye solution in the same amount as the A dye solution is dripped onto the surface of the viscous material layer 111, and the dyeing can be completed after standing for 10 to 15 seconds. Ionized water cleaning.

Preferably, the dripping amount of the A dye solution is (80-120) μL; the dripping amount of the B dye solution is (80-120) μL. The dripping amount is too small to be able to dye completely, and the dripping amount is too much, resulting in waste.

In step S05, microscope inspection, please refer to the microscope manual for specific operation methods.

For example, if the stained transparent inspection card 100 is stuck on the microscope stage with the inspection area 11 facing up, move the inspection area 11 to just below the lens, observe with a 10x objective lens, and locate the area where the dander is attached. Turn to 40x and fine-tune the focus until a clear picture is presented. Note: When adjusting the stage up and down, especially when using a 40x objective lens, be careful to avoid the detection area from contacting the objective lens.

In order to more effectively illustrate the technical solution of the present invention, a number of specific embodiments are used to illustrate the following.

Example 1

(1) Provide a transparent detection card made of glass, the transparent detection card has a detection area, the detection area is attached with a layer of acrylic, the acrylic layer is circular, the thickness is 1mm, and the area is 153.86mm ² , the The surface of the acrylic layer is covered with a protective film;

(2) Use a sterilized sampling comb to comb three times along the direction of the subject's hair, each time combing different parts of the hair, so that the dander sticks to the sampling comb;

(3). Remove the protective film on the glass material transparent detection card, stick the tip of the sampling comb with dander on the surface of the acrylic layer, and attach the sampling comb with other dander The parts stick to the surface of the acrylic layer, so that a sufficient amount of dander sticks to the acrylic layer;

(4). Drop A dye solution formed of eosin with a mass concentration of 1.0% and potassium hydroxide with a mass concentration of 10% on the surface of the acrylic layer, drop 100 μL, and then mix with the A dye solution. A quantity of B dye solution (in B dye solution, the mass concentration of gentian violet is 1.0%, the mass concentration of lactic acid is 20%, and the mass concentration of glycerin is 20%) are dropped on the surface of the acrylic layer, and after standing for 15 seconds Rinse with deionized water;

(5). Transfer the glass material transparent inspection card to a microscope for microscopic examination to obtain information on dander and fungus;

The test result is shown in Figure 4. Under the 10x objective lens, the dander can be seen, most of which are transparent, and a small part of it is dyed blue-purple. In the sample with Malassezia, small particles can be seen around the dander, zoomed in to 40 times, you can see that the particles dyed in blue-purple are rice grains, and some particles have small protrusions at one end, which is typical For Malassezia, part A represents free Malassezia, part B represents sterile dander, and part C represents a large number of fungi attached to the dander.

The conventional sampling and staining method as mentioned in the background art is used to sample and stain the epidermal fungus, and the staining result is shown in Figure 3. It can be seen from Figure 3 that the dander was also stained and was stained first, making it difficult to identify the fungus.

Example 2

(1) Provide a transparent detection card made of glass, the transparent detection card has a detection area, the detection area is attached with a layer of acrylic, the acrylic layer is circular, the thickness is 0.5mm, the area is 346.18mm ² , so The surface of the acrylic layer is covered with a protective film;

(3). Remove the protective film on the glass material transparent detection card, stick the tip of the sampling comb with dander on the surface of the acrylic layer, and put the sampling comb with other dander on the surface The parts stick to the surface of the acrylic layer, so that a sufficient amount of dander sticks to the acrylic layer;

(4). Drop A dye solution formed of eosin with a mass concentration of 1.2% and potassium hydroxide with a mass concentration of 10% on the surface of the acrylic layer, drop 100 μL, and then mix with the A dye solution. A quantity of B dye solution (in B dye solution, the mass concentration of gentian violet is 0.8%, the mass concentration of lactic acid is 18%, and the mass concentration of glycerin is 15%) is dropped on the surface of the acrylic layer, and after standing for 15s Rinse with deionized water;

(5). Transfer the glass material transparent inspection card to a microscope for microscopic examination to obtain information on dander and fungus.

The test results are shown in Figure 5. It can be seen from Figure 5 that under a 40x objective lens, the dander can be clearly seen as transparent or light blue, and there are a large number of spherical fungi dyed in blue around it, and signs of budding and reproduction can be seen. The morphology is preliminarily judged as Mala. Chromobacteria.

Example 3

A sampling and staining method for epidermal fungi. In this embodiment, the detection is performed by mailing sample combs.

(1). Obtaining samples: Because the subject is not in the local area, it is inconvenient to test on the spot. By sending a clean and disinfected sample comb, the subject himself uses the sample comb to comb the scalp according to the operating instructions of the present invention and then combs the sample. Put the comb into a sterile sealed bag, store it at room temperature, and send it to the testing laboratory for testing within one week by mail.

(2) Provide a transparent detection card made of glass, the transparent detection card has a detection area, the detection area is attached with a layer of acrylic, the acrylic layer is round, the thickness is 1.5mm, the area is 78.5mm ² , so The surface of the acrylic layer is covered with a protective film;

(4). Drop A dye solution formed of eosin with a mass concentration of 0.8% and potassium hydroxide with a mass concentration of 12.5% on the surface of the acrylic layer, drop 100 μL, and then mix with the A dye solution. A quantity of B dye solution (in the B dye solution, the mass concentration of gentian violet is 0.8%, the mass concentration of lactic acid is 25%, and the mass concentration of glycerin is 25%) are dropped on the surface of the acrylic layer, and after standing for 15 seconds Rinse with deionized water;

The test results are shown in Figure 6. It can be seen from Fig. 6 that at 40 times, a large number of spherical and rice-shaped fungi stained blue can be seen around transparent or light blue dander, which are preliminarily determined to be two fungi belonging to the genus Malassezia by morphology. At the same time, it was proved that the sampling comb was stored at room temperature for one week after sampling, and no impact on the test results was found.

The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention shall be included in the protection of the present invention. Within range.

Claims

A method for sampling and dyeing epidermal fungi, which is characterized in that it comprises the following steps:

Step S01. Provide a transparent detection card, the transparent detection card has a detection area, the detection area is attached with a layer of transparent viscous material, and the surface of the transparent viscous material layer is covered with a protective film;

Step S02. Use a sterilized sampling comb to comb several times along the direction of the hair of the subject to be tested, so that the dander sticks to the sampling comb;

Step S03. Remove the protective film on the transparent detection card, and repeatedly stick the tip of the sampling comb with dander on the surface of the transparent viscous material layer, so that a sufficient amount of dander sticks to the transparent On the viscous material layer;

Step S04. Drop A dye solution on the surface of the viscous material layer, and then add the same amount of B dye solution as the A dye solution on the surface of the transparent viscous material layer, and rinse with deionized water after standing. ；

Step S05. Transfer the transparent inspection card to a microscope for microscopic examination to obtain information on dander and fungus.
The epidermal fungus sampling and staining method according to claim 1, wherein the A dye solution is a mixed solution of eosin and potassium hydroxide, and the mass concentration of eosin in the mixed solution is 0.8% to 1.2% , The mass concentration of potassium hydroxide is 8.5% ~ 12.5%;

The B dye solution is a three-mixed solution of gentian violet, lactic acid and glycerin, and the mass concentration of gentian violet in the three-mixed solution is 0.8% to 1.5%, the mass concentration of lactic acid is 18% to 25%, and glycerin The mass concentration of 15% to 25%.
The method for sampling and staining epidermal fungi according to claim 1, wherein the dripping amount of the A dye solution is (80-120) μL; the dripping amount of the B dye solution is (80-120) μL .
The method for sampling and staining epidermal fungi according to claim 1, wherein the area of the detection area is (70-350) mm 2 .
The method for sampling and dyeing epidermal fungi according to claim 1, wherein the thickness of the transparent viscous material layer is (0.5-1.5) mm.
The method for sampling and dyeing epidermal fungi according to claim 1, wherein the viscous material of the transparent viscous material layer is acrylic.
The method for sampling and dyeing epidermal fungi according to claim 1, wherein the sampling comb is a clean and disinfected plastic comb.
The method for sampling and staining epidermal fungi according to claim 1, wherein the standing time is (10-15) s.
The method for sampling and staining epidermal fungi according to claim 1, wherein the transparent detection card further comprises a label area, and the label area is used to mark the information of the test subject.