CN115683789A - Fungus fluorescent staining solution with double background removal reagents and preparation method and application thereof - Google Patents

Abstract

The invention discloses a preparation method and application of a fungus fluorescent staining solution which is a double background removal reagent and can effectively reduce background brightness of dandruff and nail filing and improve contrast of fungi. The composite material is characterized by being prepared from the following components in percentage by mass: fluorescent whitening agent, a first kind of blue background removing agent and a second kind of red background removing agent. The fluorescent whitening agent is mixed with the first class and the second class of background removing agents, so that only blue background removing agents are used in the market at present, and the red background removing agents are added on the basis of the blue background removing agents to remove green light, so that the background brightness of the dandruff nail chips is effectively reduced, the brightness contrast of the fungi is improved, the fungi can be detected quickly and easily, and the fluorescent whitening agent is suitable for accurate and quick judgment of clinical large-batch samples.

Description

Double background removal reagent fungal fluorescent staining solution and its configuration method and application

technical field

The invention belongs to the field of microbial fluorescent detection reagents, in particular to the rapid detection and identification of fungi; in particular, it relates to a double background removal reagent fungal fluorescent staining solution and its configuration method and application.

Background technique

Fungi (fungus) are heterotrophic organisms with a nucleus and a cell wall. They are a large group in the biological kingdom and are usually divided into three categories: yeasts, molds and mushrooms (large fungi). Yeast is a tiny single-celled microorganism invisible to the naked eye. It can ferment sugar into alcohol and carbon dioxide. It is a typical facultative anaerobic microorganism. Mold is a common name for filamentous fungi, which means "moldy fungus". They tend to form a heavily branched mycelium, but do not produce the large fruiting bodies of mushrooms; mushrooms are fungi that form large fleshy fruiting bodies. More than 10,000 fungal genera and more than 100,000 species have been described in the world. In recent years, the number and types of clinical fungal infections have increased year by year, and it has become one of the main causes of nosocomial infections. Clinical analysis found that the source of most clinical fungal infections in hospitals is Candida, of which Candida albicans accounts for about 60% of all infections, resulting in a mortality rate of nearly 40%. This infection can even cause organ transplantation patients, cancer patients and AIDS patients. death. Therefore, methods and techniques for efficiently identifying and monitoring fungi are of great significance to the clinical diagnosis of fungal infection diseases.

Hematoxylin-eosin staining (hematoxylin-eosin staining) method, hexamine silver staining (GMS) method, iodic acid Schiff staining (PAS) method, KOH wet film microscopy and fluorescent staining (CFW) method are the current detection methods. Fungal staining methods commonly used in histology. When the fungus is a colored fungus, HE staining does not cover the color of the fungus itself, and can display the structure of the tissue where the fungus is located. It is an essential routine staining method for fungal histopathological examination. However, when the fungus is colorless, the color of the tissue is quite similar to that of the fungus after HE staining, and can only be identified by its outline, which is not easy to distinguish. When the number of fungi in the tissue is small, it is even more difficult to be identified Discover. GMS staining makes the fungal cell wall stained brown-black, which has a large contrast with the background, and the outline and shape of the fungus can be clearly displayed; PAS staining makes the fungal staining layer distinct, the cell wall is dark red, the cytoplasm is light red, and the nucleus is blue. This makes the fine structure of the fungus clearly visible. However, these two staining methods have many inconveniences in operation: the hexamine silver staining method involves a wide variety of reagents, and because the dye solution cannot be stored for a long time, it needs to be repeatedly prepared for immediate application. During the staining process, it is necessary to dip the slide in the working solution at 56°C for one hour, and constantly observe when it is stained and whether the slide is off, and then deal with it in time. In addition, the false positive rate of hexamine silver staining is high, and a variety of bacteria can also be stained by silver. The operation method of PAS staining is relatively simple, but the preparation process of magenta staining solution is complicated and time-consuming, and co-staining with tissue cells in the section occurs. Although the KOH wet film microscopy method is simple, fast, low-cost, and easy to master, the background is relatively messy, especially when there is a very small amount of hyphae or spores, it is easy to miss. Fluorescence staining (CFW) method combines fluorescent whitening agent with fungal cell wall β-polysaccharides that may exist in the tissue, such as chitin and cellulose, etc., and the fungi can show fluorescence under the activation of ultraviolet light. Observed under a fluorescence microscope with a 250-400nm filter, the hyphae or spores are bright blue, with a clear contrast to the background and a clear structure. Compared with the KOH wet film method, it is easier to observe, but there is autofluorescence and background of the biological matrix itself. Light interference, the current background removal reagents on the market are mainly blue background removal reagents based on Evans blue. The autologous background fluorescence emitted by the background of dander and nail scraps is blue-white, and the blue light absorption band such as Evans blue is roughly in the range of 500nm-650nm, but the absorption in the range of 500-550nm green light is weak, so that the background fluorescence will not be effectively absorb. Therefore, for clinical samples of dandruff and onychomycosis, the autofluorescence is too strong, resulting in insufficient fungal imaging contrast, which may make it difficult to find fungi.

In order to solve the above problems, we introduce the second type of red background removal reagent. The purpose of the second type of background removal dye is to absorb the green band in the background fluorescence, so that the background color can be further blackened.

Contents of the invention

The purpose of the present invention is to introduce the second type of red background removal agent under the condition of the presence of the first type of blue background removal agent to absorb the green band in the background fluorescence, so that the background color can be further blackened. Improves the contrast between fungi and the background for easy and rapid fungal detection.

In order to achieve the above object, the technical scheme adopted in the present invention is:

The invention provides a fungal fluorescent staining liquid with double background removing agent, the fungal fluorescent staining liquid with double background removing reagent comprises the following components in mass concentration: fluorescent whitening agent 0.01-2%, the first type of blue background removing reagent 0.001-1%, the second type of red background reagent 0.002-1%, the solvent is deionized water; the fluorescent whitening agent is fluorescent whitening agent 28, fluorescent whitening agent 31, fluorescent whitening agent 71, fluorescent whitening agent Whitening agent 85, fluorescent whitening agent 113, fluorescent whitening agent 134, fluorescent whitening agent 220, fluorescent whitening agent 351 or a mixture of two or more; the first type of blue background removal agent It is one or a mixture of two or more of Evans blue, trypan blue, bromophenol blue, bromothymol blue, bromocresol blue, and crystal violet; the second type of red background removal reagent is Galla rose Red, algal red B, erythrosin B sodium salt, phenol saffron, algal red B (algal red B sodium salt), eosin B, carmine, neutral red, quinaldine red, saffron red T, nuclear solid Red, eosin, safranin, Congo red, Sudan red, or a mixture of two or more.

Further, the fungal fluorescent staining solution of the double background removal reagent also includes one or a mixture of two or more of the following components:

Anti-bleaching agent, anti-fluorescence quenching agent, softener, solubilizer, humectant, penetrating agent, sodium chloride. Anti-fluorescence quenching agents can slow down the fluorescence quenching of dyes, humectants can slow down the drying speed of dye solutions and prolong the effective time of samples, softeners can soften sample tissues, and penetrants and solubilizers can help dyes enter the interior of tissues for full staining. NaCl can darken the background a bit.

Furthermore, the fungal fluorescent staining solution of the double background removal reagent also includes one or more mixtures of the following mass concentrations of the components:

Anti-bleaching agent 0.2-2%, anti-fluorescence quenching agent 0.2-2%, softener 0.002-0.2%, solubilizer 1-10%, moisturizing agent 1-5%, penetrating agent 0.5-5%, sodium chloride 0.5-0.9%.

Preferably, the fungal fluorescent staining solution of the double background removal agent is composed of the following components in mass concentration: fluorescent whitening agent 0.01-2%, the first blue background removal agent 0.001-1%, the second Red-like background removal reagent 0.002-1%, auxiliary agent and deionized water, the auxiliary agent is one or a mixture of two or more of the following components:

Anti-bleaching agent, anti-fluorescence quenching agent, softener, solubilizer, humectant, penetrating agent, sodium chloride.

Further preferably, the fungal fluorescent staining solution of the double background removal agent is composed of the following components in mass concentration: 0.01-2% of the fluorescent whitening agent, 0.001-1% of the first type of blue background removal agent, and 0.001-1% of the second type Red background removal reagent 0.002-1%, anti-bleaching reagent 0.2-2%, anti-fluorescence quenching agent 0.2-2%, softener 0.002-0.2%, solubilizer 1-10%, moisturizing agent 1-5%, transparent Agent 0.5-5%, sodium chloride 0.5-0.9% (preferably 0.9%), solvent is deionized water.

Preferably, the fluorescent whitening agent is one or a mixture of the fluorescent whitening agent 28 and the fluorescent whitening agent 220 .

The mass concentration is further preferably: 0.01-1%, particularly preferably 0.3% of the fluorescent whitening agent 28 and 0.3% of the fluorescent whitening agent 220 .

Preferably, the first type of blue background removal reagent is Evans blue.

Its content is further preferably: 0.005-0.5%, especially preferably Evans blue with a mass concentration of 0.01%.

Preferably, the second type of red background removal agent is one or a mixture of two or more of safranin, quinaldine red, Sudan red, eosin, and safranin. Especially preferred is one of safranin, quinaldine red, and Sudan red, or a mixture of two or more of safranine, quinaldine red, Sudan red, eosin, and safranin. Particular preference is given to mixtures of safranin, quinaldine red and Sudan red.

The mass concentration is further preferably: 0.01-1%, particularly preferably a mixture of 0.008% phenol safranin, 0.008% quinaldine red and 0.004% Sudan red.

Further, the anti-bleaching agent is one of butyl hydroxyanisole (BHA), dibutyl hydroxytoluene (BHT), tert-butyl hydroquinone (TBHQ), propyl gallate (PG), and ascorbic acid. a mixture of two or more.

Preferably, the anti-bleaching agent is dibutylhydroxytoluene.

Its mass concentration is further preferably: 0.1-1%. Particular preference is given to dibutylhydroxytoluene at a mass concentration of 0.5%.

Further, the anti-fluorescence quenching agent is one or a mixture of two or more of potassium citrate, potassium stearate, potassium acetate, sodium citrate, sodium stearate, and sodium acetate.

Preferably, the anti-fluorescence quenching agent is sodium citrate.

Its mass concentration is further preferably: 0.1-1%. Particularly preferred is sodium citrate with a mass concentration of 0.3%.

Further, the softening agent is one or a mixture of two or more of salicylic acid, citric acid, glycolic acid, malic acid, glycolic acid, and 2,4,5-trimethoxybenzoic acid.

Preferably, the softener is salicylic acid.

Its mass concentration is further preferably: 0.005-0.1%. Particularly preferred is salicylic acid with a mass concentration of 0.01%.

Further, the solubilizer is one or a mixture of sodium hydroxide and potassium hydroxide.

Preferably, the solubilizer is potassium hydroxide.

Its mass concentration is further preferably: 2-10%. Especially preferred is potassium hydroxide with a mass concentration of 10%.

Further, the humectant includes one or a mixture of two or more of pentaerythritol, propylene glycol, glycerol, and sorbitol.

Preferably, the humectant is glycerol.

Its mass concentration is further preferably: 1-10%. Particularly preferred is glycerol with a mass concentration of 5%.

Further, the permeation agent includes one or a mixture of two or more of Tween 20, Tween 40, Tween 60, Tween 80, polyethylene glycol octylphenyl ether, and DSMO.

Preferably, the penetrating agent is DMSO.

Its mass concentration is further preferably: 1-5%. Particularly preferred is DSMO with a mass concentration of 2%.

The present invention especially recommends that the double background removal reagent fungal fluorescent staining solution is composed of the following components in mass concentration: fluorescent whitening agent 28 0.3%, fluorescent whitening agent 220 0.3%, Evans blue 0.01%, phenol saffron 0.008 %, quinaldine red 0.008%, Sudan red 0.004%, butylated hydroxytoluene 0.5%, sodium citrate 0.3%, salicylic acid 0.01%, potassium hydroxide 10%, glycerol 5%, DSMO 2%, Sodium chloride 0.9%.

On the other hand, the present invention provides a method for configuring the above-mentioned double background removal reagent fungal fluorescent staining solution: dissolving the fluorescent whitening agent and moisturizing agent in water to obtain liquid A; dissolving the solubilizing agent in water for Obtain liquid B; dissolve the anti-bleaching reagent in the amount of the formula in the penetrating agent of the amount in the formula to obtain liquid C; mix the first type of blue background removal reagent, the second type of red background removal reagent, and the anti-fluorescence Dissolve the quenching agent and softener in water to obtain liquid D; dissolve the prescription amount of sodium chloride in water to obtain liquid E; then slowly add the liquid B, liquid C, liquid D, and liquid E to the liquid A in sequence , stir evenly, and supplement the solvent to obtain the double background removal reagent fungal fluorescent staining solution.

When the corresponding additive is not needed, it can be omitted. The above methods are only dissolving and mixing steps set according to the properties of the relevant components, mixing in other order is predictable by those skilled in the art, and is also within the protection scope of the present invention.

Finally, the present invention also provides an application of the above-mentioned double background removal reagent fungal fluorescent staining solution in the detection of fungi, especially the application in the detection of fungi contained in human dander and nail scraps samples.

Specifically, the application is as follows: place the sample to be tested on a glass slide, add the fungal fluorescent staining solution of the double background removal reagent dropwise, stain for 1-10 seconds, cover with a cover glass, and absorb the excess staining solution. Observe under a fluorescence microscope.

Compared with the prior art, the beneficial effect of the present invention lies in the optimization of the background removal reagent, and the second type of red background removal reagent is added on the basis of the first type of blue background removal reagent for the purpose of reducing background fluorescence. The function of the first type of blue background removal reagent is to absorb the orange and red light in the range of 550-650nm in the background. However, the absorption is weak in the range of 500-550nm green light, so that the fluorescence emitted by the background will not be effectively absorbed. The purpose of the second type of red background removal dye in the staining solution is to absorb the green band in the background fluorescence, so that the background color can be further darkened, and the imaging contrast of fungi and background cells can be significantly improved.

Description of drawings

Figure 1 is the ultraviolet absorption spectrum of the first type of blue background removal reagent

Figure 2 is the ultraviolet absorption spectrum of the second type of red background reagent

Fig. 3 is a diagram of the results observed under a fluorescence microscope at 40 times the scale after the dander sample is stained with the fungal fluorescent staining solution prepared in Example 1 of the present invention;

Figure 4 is a diagram of the results observed under a fluorescence microscope at 40 times the scale after the dander sample was stained with the fungal fluorescent staining solution prepared in Example 4 of the present invention;

Fig. 5 is a diagram of the results observed under a fluorescence microscope at 10 times the scale after the dander sample is stained with the double background removal reagent fungal fluorescent staining solution prepared in Example 4 of the present invention;

Fig. 6 is a diagram of the results observed under a fluorescence microscope at 40 times the scale after the dander sample is stained with the fungal fluorescent staining liquid of the double background removal reagent prepared in Example 7 of the present invention;

Fig. 7 is a diagram of the results observed under a fluorescence microscope at 40 times the scale after the dander sample is stained with the fungal fluorescent staining solution prepared in Example 9 of the present invention;

Figure 8 is a diagram of the results observed under a fluorescence microscope at 40 times the scale after the dander sample was stained with the fungal fluorescent staining solution prepared in Example 10 of the present invention;

Fig. 9 is a diagram of the results observed under a fluorescence microscope at 40 times the scale after the dander sample is stained with the fungal fluorescent staining solution prepared in Example 13 of the present invention;

Fig. 10 is a diagram of the results observed under a fluorescence microscope at 40 times the scale after the dander sample is stained with the fungal fluorescent staining solution prepared by the double background removal reagent prepared in Example 14 of the present invention;

Fig. 11 is a picture of the results observed under a fluorescence microscope at 40 times the scale after the dander sample was stained with the fungal fluorescent staining solution of the comparative example.

Detailed ways

The specific steps of the present invention are illustrated below through the examples, but not limited by the examples.

Example 1

Double background removal reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.01%, Evans blue 0.005%, phenol saffron 0.01%, dibutylhydroxytoluene 0.1%, Sodium citrate 0.1%, salicylic acid 0.005%, potassium hydroxide 2%, glycerol 1%, DSMO 1%, sodium chloride 0.9%, and the rest is supplemented with deionized water.

The preparation method of the double background removal reagent fungal fluorescent staining solution comprises the following steps:

Dissolve fluorescent whitening agent 28 and glycerin in water to obtain liquid A;

Dissolve potassium hydroxide in water to obtain liquid B. Since there will be an exothermic reaction, wait for the solution to cool before performing the experiment;

Dissolve dibutyl hydroxytoluene in DMSO to obtain liquid C, because dibutyl hydroxytoluene is insoluble in water, so dissolve it in an organic solvent;

Then dissolve Evans blue, phenol saffron, sodium citrate and salicylic acid in water to obtain liquid D;

Finally, sodium chloride is dissolved in water to obtain liquid E;

Slowly add solution B, solution C, solution D, and solution E into solution A in sequence, and stir well to obtain the double background removal reagent fungal fluorescent staining solution.

The configuration steps of the following examples are the same as those in the examples, except that the dyes added in the D solution are different.

The staining method of the double background removal reagent fungal fluorescent staining solution comprises the steps:

1. Add double background removal reagent fungal fluorescent staining solution on the glass slide, and put in samples such as dandruff and nail scraps (the data in this example are all from the detection of dandruff on the feet of patients with fungal infection)

2. After staining for 1-10S, observe under ultraviolet light under a fluorescent microscope.

Example 2

Double background removal reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.01%, Evans blue 0.005%, quinaldine red 0.01%, dibutyl hydroxytoluene 0.1% , sodium citrate 0.1%, salicylic acid 0.005%, potassium hydroxide 2%, glycerol 1%, DSMO 1%, sodium chloride 0.9%, and the rest is supplemented with deionized water.

Example 3

Double background removal reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.01%, Evans blue 0.005%, Sudan red 0.01%, dibutyl hydroxytoluene 0.1%, lemon 0.1% sodium chloride, 0.005% salicylic acid, 2% potassium hydroxide, 1% glycerol, 1% DSMO, 0.9% sodium chloride, and the rest was supplemented with deionized water.

Example 4

Double background removal reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.3%, fluorescent whitening agent 220 0.3%, Evans blue 0.01%, phenol saffron 0.008%, Quinaldine Red 0.008%, Sudan Red 0.004%, Butylated Hydroxytoluene 0.5%, Sodium Citrate 0.3%, Salicylic Acid 0.01%, Potassium Hydroxide 10%, Glycerin 5%, DSMO 2%, Chloride Sodium 0.9%, the rest is made up with deionized water.

The preparation method and staining method of the double background removal reagent fungal fluorescent staining solution are the same as in Example 1.

Example 5

Double background removal reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 1%, Evans blue 0.5%, Sudan red 1%, dibutyl hydroxytoluene 1%, lemon Na2SO4 1%, salicylic acid 0.1%, potassium hydroxide 10%, glycerin 10%, DSMO 5%, sodium chloride 0.9%, and the rest is supplemented with deionized water.

The preparation method and staining method of the double background removal reagent fungal fluorescent staining solution are the same as in Example 1.

Example 6

Double background removal reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 1%, Evans blue 0.5%, quinaldine red 0.5%, phenol saffron 0.5%, di Butylated hydroxytoluene 1%, sodium citrate 1%, salicylic acid 0.1%, potassium hydroxide 10%, glycerol 10%, DSMO 5%, sodium chloride 0.9%, and the rest is supplemented with deionized water.

The preparation method and staining method of the double background removal reagent fungal fluorescent staining solution are the same as in Example 1.

Example 7

Double background removal reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.01%, fluorescent whitening agent 220 0.01%, Evans blue 0.01%, Sudan red 0.01%, phenol Saffron 0.01%, BHT 0.2%, Sodium Citrate 0.2%, Salicylic Acid 0.002%, Potassium Hydroxide 10%, Glycerol 1%, DSMO 3%, Sodium Chloride 0.9%, and the rest are used Ionized water tops up.

The preparation method and staining method of the double background removal reagent fungal fluorescent staining solution are the same as in Example 1.

Example 8

Double background reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.5%, fluorescent whitening agent 220 0.5%, Evans blue 0.5%, Sudan red 0.3%, quinine Nadine red 0.2%, 2-butyl hydroxytoluene 0.5%, sodium citrate 0.3%, salicylic acid 0.05%, potassium hydroxide 2%, glycerol 5%, DSMO 5%, sodium chloride 0.7%, the rest are used Top up with deionized water.

The preparation method and staining method of the double background removal reagent fungal fluorescent staining solution are the same as in Example 1.

Example 9

Double background removal reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.03%, fluorescent whitening agent 220 0.05%, Evans blue 0.008%, quinaldine red 0.008% , phenol saffron 0.008%, dibutyl hydroxytoluene 1%, sodium citrate 0.3%, salicylic acid 0.01%, potassium hydroxide 10%, glycerol 3%, DSMO 6%, sodium chloride 0.9%, the rest Make up with deionized water.

The preparation method and staining method of the double background removal reagent fungal fluorescent staining solution are the same as in Example 1.

Example 10

Double background removal reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.5%, fluorescent whitening agent 220 0.5%, Evans blue 0.01%, eosin 0.008%, tomato Red 0.008%, Dibutyl Hydroxytoluene 0.5%, Sodium Citrate 0.3%, Salicylic Acid 0.01%, Potassium Hydroxide 10%, Glycerin 5%, DSMO 2%, Sodium Chloride 0.9%, the rest is deionized Water top up.

The preparation method and staining method of the double background removal reagent fungal fluorescent staining solution are the same as in Example 1.

Example 11

Double background reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.5%, fluorescent whitening agent 220 0.5%, Evans blue 0.01%, safranin 0.02%, di Butylated hydroxytoluene 0.5%, sodium citrate 0.3%, salicylic acid 0.01%, potassium hydroxide 10%, glycerol 5%, DSMO 2%, sodium chloride 0.9%, and the rest was made up with deionized water.

The preparation method and staining method of the double background removal reagent fungal fluorescent staining solution are the same as in Example 1.

Example 12

Double background reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.5%, fluorescent whitening agent 220 0.5%, Evans blue 0.01%, eosin 0.02%, di Butylated hydroxytoluene 0.5%, sodium citrate 0.3%, salicylic acid 0.01%, potassium hydroxide 10%, glycerol 5%, DSMO 2%, sodium chloride 0.9%, and the rest was made up with deionized water.

The preparation method and staining method of the double background removal reagent fungal fluorescent staining solution are the same as in Example 1.

Example 13

Double background removal reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.5%, fluorescent whitening agent 220 0.5%, Evans blue 0.01%, saffron T 0.02%, 0.5% dibutyl hydroxytoluene, 0.3% sodium citrate, 0.01% salicylic acid, 10% potassium hydroxide, 5% glycerol, 2% DSMO, 0.9% sodium chloride, and make up the rest with deionized water.

The preparation method and staining method of the double background removal reagent fungal fluorescent staining solution are the same as in Example 1.

Example 14

Double background reagent fungal fluorescent staining solution, made of the following components by mass percentage: fluorescent whitening agent 28 0.5%, fluorescent whitening agent 220 0.5%, Evans blue 0.01%, Congo red 0.02%, di Butylated hydroxytoluene 0.5%, sodium citrate 0.3%, salicylic acid 0.01%, potassium hydroxide 10%, glycerol 5%, DSMO 2%, sodium chloride 0.9%, and the rest was made up with deionized water.

The preparation method and staining method of the double background removal reagent fungal fluorescent staining solution are the same as in Example 1.

comparative example

The fungal fluorescent staining solution that only contains the first type of blue background reagent is commonly used in the market. Its components are: fluorescent whitening agent 28 0.04%, glycerol 3%, DMSO 15%, potassium hydroxide 10%, Evans blue 0.08%.

Table 1 Example 1-14 and comparative example The dyeing effect of the foot dander samples of patients with fungal infection is as follows: (note: the more +, the better the effect)

Claims (10)

1. a double background reagent fungal fluorescent staining solution is characterized in that said double background reagent fungal fluorescent staining solution includes each component of the following mass concentrations: fluorescent whitening agent 0.01-2%, the first type of blue Background reagent 0.001-1%, the second type of red background reagent 0.002-1%, the solvent is deionized water; the fluorescent whitening agent is fluorescent whitening agent 28, fluorescent whitening agent 31, fluorescent whitening agent 71 , fluorescent whitening agent 85, fluorescent whitening agent 113, fluorescent whitening agent 134, fluorescent whitening agent 220, fluorescent whitening agent 351 or a mixture of two or more; The background reagent is one or a mixture of two or more of Evans blue, trypan blue, bromophenol blue, bromothymol blue, bromocresol blue, and crystal violet; the second type of red background reagent is added Rose red, algal red B, erythrosin B sodium salt, phenol saffron, algal red B, eosin B, carmine, neutral red, quinaldine red, saffron T, nuclear solid red, eosin, One or a mixture of two or more of saffron, Congo red, and Sudan red. 2. the double background reagent fungal fluorescent staining solution as claimed in claim 1, is characterized in that: described second class red background reagent is a kind of in phenol safflower red, quinaldine red, Sudan red, or phenol safari A mixture of two or more of flower red, quinaldine red, Sudan red, eosin, and safranin. 3. as claimed in claim 1, the double background reagent fungal fluorescent staining solution is characterized in that the double background reagent fungal fluorescent staining solution also includes one or more mixtures of the following components: anti-bleaching reagent , Anti-fluorescence quenching agent, softener, solubilizer, humectant, penetrating agent, sodium chloride. 4. as described in any one of claim 1-3 double background reagent fungal fluorescent staining liquid, it is characterized in that described double background reagent fungal fluorescent staining liquid is made up of each component of following mass concentration: fluorescent whitening agent 0.01-2%, the first type of blue background removal reagent 0.001-1%, the second type of red background removal reagent 0.002-1%, anti-bleaching reagent 0.2-2%, anti-fluorescence quencher 0.2-2%, softener 0.002-0.2%, solubilizer 1-10%, moisturizing agent 1-5%, penetrating agent 0.5-5%, sodium chloride 0.5-0.9%, solvent is deionized water. 5. double background reagent fungal fluorescent staining solution as claimed in claim 3, is characterized in that: described anti-bleaching reagent is butyl hydroxyanisole, dibutyl hydroxytoluene, tert-butyl hydroquinone, gall One or a mixture of two or more of propyl ester and ascorbic acid. 6. double background reagent fungal fluorescent staining liquid as claimed in claim 3 is characterized in that: described anti-fluorescence quencher is potassium citrate, potassium stearate, potassium acetate, sodium citrate, stearic acid One or more mixtures of sodium and sodium acetate. 7. double background reagent fungal fluorescent staining liquid as claimed in claim 3 is characterized in that: described softening agent is salicylic acid, citric acid, glycolic acid, malic acid, glycolic acid, 2,4,5 - One or a mixture of two or more of trimethoxybenzoic acid. 8 . The double background removal reagent fungal fluorescent staining solution according to claim 3 , wherein the solubilizing agent is one or a mixture of sodium hydroxide and potassium hydroxide. 9. The fungal fluorescent staining liquid with double background removal reagent according to claim 3, characterized in that: said moisturizing agent comprises one or a mixture of two or more of pentaerythritol, propylene glycol, glycerol, and sorbitol. 10. double background reagent fungal fluorescent staining solution as claimed in claim 3, is characterized in that: described penetrating agent comprises Tween 20, Tween 40, Tween 60, Tween 80, polyethylene glycol octane One or more mixtures of phenyl phenyl ether and DSMO.

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