A lactic acid phenol base composition, a lactic acid phenol cotton blue dyeing solution and a preparation method and application thereof

Abstract

This invention, entitled "A Lactophenol Matrix Composition, a Lactophenol Cotton Blue Staining Solution, its Preparation Method, and its Application," belongs to the field of microbiological detection technology. The technical problem it aims to solve is the presence of numerous air bubbles in existing lactophenol cotton blue staining solutions during microscopic observation. This invention proposes a lactophenol cotton blue staining solution that reduces the amount of air bubbles in the microscopic field of view. The key technical solution is a lactophenol matrix composition, comprising: lactic acid, phenol, monosaccharides and their derivatives, and polyols.

Description

Technical Field

[0001] This invention relates to the field of microbiological detection technology, specifically to a lactic acid phenol matrix composition, a lactic acid phenol cotton blue staining solution, its preparation method, and its application. Background Technology

[0002] Fungi are eukaryotic organisms. Pathogenic and opportunistic pathogenic fungi can cause infectious, hypersensitivity, and toxic diseases in humans after invading the body. Some fungi can also cause plant diseases, human allergic diseases, and fungal poisoning.

[0003] In the medical field, the staining and identification of fungal smears are often required to study the structure of fungi. The principle of lactic acid cotton blue (LPCB) staining is based on the components of the fungal cell wall, and its staining of fungi is not a single mechanism, but the result of the combined action of physical adsorption, chemical affinity, and the osmotic fixation and clearing effects of the staining solution components.

[0004] Fungal cell walls differ significantly from bacterial cell walls, typically containing chitin, β-glucan, mannan, and proteins. Chitin and proteins are the primary sites for binding with staining agents. Chitin is a polysaccharide composed of N-acetylglucosamine linked by β-1,4 glycosidic bonds, containing numerous free amino (-NH2) and hydroxyl (-OH) groups that can form hydrogen or ionic bonds. Basic amino acid residues in proteins (such as lysine and arginine) impart a positive charge to the cell wall. Lactophenol-cotton blue staining solution typically contains phenol, lactic acid, and water-soluble aniline blue. On one hand, the synergistic effect of lactic acid and phenol softens the fibrous structure of the fungal cell wall, disrupting the internal hydrogen bond network and significantly increasing cell wall permeability. Simultaneously, the acidic staining solution protonates basic protein residues in the cell wall, further exposing positively charged sites and creating conditions for their binding with the staining agent. On the other hand, water-soluble aniline blue, as an acidic staining agent, dissociates into negatively charged anions in aqueous solution, which can form stable ionic bonds with positively charged sites in the fungal cell wall. At the same time, water-soluble aniline blue can also combine with free amino and hydroxyl groups in chitin and dextran through hydrogen bonds, ultimately causing the staining agent to remain on the cell wall and exhibit a bright blue color.

[0005] When using existing lactic acid cotton blue staining solution to stain some fungi, it often produces a lot of air bubbles due to improper mounting techniques, excessively thick or uneven samples, and the high viscosity and surface tension of the lactic acid cotton blue staining solution. These factors affect microscopic observation and thus the identification of fungi.

[0006] Relevant patent documents retrieved:

[0007] This document, published in China (CN106947799A) on July 14, 2017, discloses a lactic acid phenol blue staining solution and its preparation method. The lactic acid phenol blue staining solution comprises 10-15 parts PVA, 20-40 parts lactic acid, 20-40 parts phenol, and 0.05-0.1 parts aniline blue. The preparation method includes the following steps: adding 10-15 parts PVA powder to a container filled with water, heating the container while stirring with a magnetic rod; then sequentially adding 20-40 parts lactic acid, 20-40 parts phenol, and 0.05-0.1 parts aniline blue; continuously heating the solution and stirring with a magnetic rod until the temperature reaches 70-90°C, then stopping heating to obtain the lactic acid phenol blue staining solution. This invention can effectively and permanently preserve fungal wet slides or glass slide cultures, with a wider range of applications and longer preservation time.

[0008] Relevant non-patent literature retrieved:

[0009] The document, titled "Lactic Acid Phenol Solution," is from the website *Microbial Encyclopedia*, published on March 31, 2023. It discloses lactic acid phenol solution, also known as lactic phenol oil, as a flocculating agent used to immerse fungal slides to improve microscopic observation. Two formulations are disclosed: Formulation 1: 20g phenol (carbolic acid) crystals, 20mL lactic acid, 40mL glycerol, and 20mL distilled water. If necessary, 0.05%-0.1% aniline blue (cotton blue) can be added, along with small amounts of safranin, brilliant green, or trypan blue dyes; Formulation 2: 10g phenol, 10g lactic acid, 20g glycerol, and 10mL distilled water. If necessary, 0.025g aniline blue dye can be added.

[0010] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects:

[0011] The problem of air bubbles in the microscopic field of view is prominent, which affects the observation of fungal morphology. The relevant evidence is: Publication No. CN106947799A. The lactic acid cotton blue staining solution disclosed in this literature has a combined effect of overall viscosity, permeability and surface tension, which affects the penetration and wetting of fungal structures by the staining solution, and is prone to causing air bubbles, thus affecting the observation of fungal morphology.

[0012] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles:

[0013] Before finalizing this formulation, various experiments were conducted, such as adding different amounts of polyether defoamer and surfactants like Tween 80 to the lactic acid matrix in an attempt to reduce the surface tension of the matrix. However, the results not only failed to reduce bubbles during microscopic examination, but also affected the uniformity of the matrix and staining solution. Summary of the Invention

[0014] The purpose of this invention is to provide:

[0015] A lactic acid phenol matrix composition, a lactic acid phenol cotton blue staining solution, and related technologies, to solve technical problems such as reducing air bubbles that obscure bacterial structures in the microscopic field of view during fungal auxiliary identification, or a combination thereof.

[0016] Terminology Explanation:

[0017] Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0018] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0019] Definitions of standard chemical terms can be found in the references Pocket Guide to Mycological Diagnosis (2019 edition), Clinical Laboratory Procedures (4th edition), and Introductory Microbiology (1996 edition).

[0020] Unless otherwise stated, conventional methods within the scope of the art, such as direct microscopic examination and culture staining, shall be used.

[0021] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0022] The term "lactic acid phenol matrix composition" used in this article refers to a basic system formed by using lactic acid and phenol as core components, combined with solvents (such as water and polyols). This system combines the functions of keratin dissolution, bacterial fixation, and antiseptic and moisturizing properties, serving as the core carrier for fungal staining solutions. Its mechanism of action is as follows: lactic acid dissolves the keratin layer of the sample and facilitates the penetration of the staining agent; phenol fixes the fungal cell structure and inhibits microbial contamination. Together, they provide a stable environment for subsequent dye staining. This is commonly found in the basic formulations of staining solutions such as lactic acid cotton blue and lactic acid aniline blue.

[0023] The term "lactic acid cotton blue staining solution" used in this article refers to a classic staining solution for fungal morphological identification, composed of a lactic acid matrix and a dye. Staining is achieved through "lactic acid matrix clearing the sample + specific binding to fungal cell wall polysaccharides." During microscopic examination, fungal hyphae and spores appear blue against a transparent background, allowing for clear observation of fungal morphology (such as hyphal septa and spore arrangement). It is widely used for clinical fungal specimen screening (dander, sputum) and scientific research species identification.

[0024] The term "lactic acid" used in this article refers to α-hydroxypropionic acid (molecular formula C3H6O3), a water-soluble organic acid (density 1.21 g / cm³ at 25℃), commonly used in pharmaceutical-grade form at a concentration of 85%. Its main functions in staining solutions include: dissolving the stratum corneum of the sample (such as skin scales and nail clippings), lowering the pH of the system to enhance the staining stability of acidic dyes (such as cotton blue and aniline blue), and assisting the staining solution in penetrating into the bacterial cells. It is a key functional component of lactic acid-phenolic staining solutions.

[0025] The term "aniline blue" as used in this article refers to a class of triphenylmethane dyes synthesized from aniline. Based on solubility, they are classified into acidic aniline blue (water-soluble, such as aniline blue WS, CI Acid Blue 22) and basic aniline blue (alcohol-soluble, such as CI Basic Blue 26). The acidic type contains a sulfonyl group (-SO3H), has excellent water solubility, and binds to fungal cell wall polysaccharides through electrostatic attraction, producing a deep blue stain. The basic type contains an amino group (-NH2), has strong alcohol solubility, is suitable for staining cell nuclei, and is a commonly used specific dye for fungal staining solutions.

[0026] The term "phenol" used in this article refers to carbolic acid (molecular formula C6H6O, molecular weight 94.11), an aromatic compound containing a benzene ring and a hydroxyl group (-OH). It is a colorless needle-like crystal at room temperature (purity ≥98% reagent grade), with a density of 1.07 g / cm³ at 25℃, a melting point of 43℃, and a boiling point of 181.7℃. It is readily soluble in solvents such as water, ethanol, and glycerol, has a special pungent odor, and possesses weak acidity and bactericidal and preservative properties.

[0027] The term "monosaccharides and their derivatives" as used in this article refers to: the simplest sugars that cannot be hydrolyzed (such as glucose, fructose, and galactose), containing 3-6 carbon atoms and multiple hydroxyl groups (-OH); derivatives refer to products of monosaccharides modified by oxidation, reduction, or substitution (carboxylation, amination, phosphorylation, etc.) (such as glucuronic acid, glucosamine, and sorbitol). In staining solutions, these derivatives can form hydrogen bonds through multiple hydroxyl groups, thereby regulating viscosity, stabilizing osmotic pressure, and enhancing dye dispersibility. For example, glucuronic acid increases the consistency of the system, while sorbitol maintains the integrity of fungal morphology.

[0028] The term "polyol" used in this article refers to alcohol compounds (such as glycerol, sorbitol, mannitol, and xylitol) containing two or more hydroxyl groups (-OH) in their molecules. These compounds are highly water-soluble and chemically stable. Their core functions in staining solutions are to adjust viscosity, wet the sample, prevent drying, and regulate osmotic pressure. Glycerol increases the system's viscosity and delays drying through hydrogen bonding, while sorbitol balances osmotic pressure to protect bacterial cell morphology. It is a key excipient in lactic acid-phenol staining solutions for optimizing formulation stability.

[0029] The term "mg / mL" as used in this article refers to a unit of mass-volume concentration, which represents the mass (milligrams, mg) of solute contained in 1 milliliter (mL) of solution. It is commonly used to indicate the concentration of components such as dyes and monosaccharides in staining solutions. This unit directly reflects the content of solute in the solution and is the most commonly used concentration expression method in laboratory reagent preparation and patent formulation writing, ensuring the reproducibility of the formulation.

[0030] The term "mixing" as used in this article refers to the operation of uniformly dispersing two or more components (solid or liquid) to form a homogeneous system through physical means (stirring, oscillation, grinding, ultrasound, etc.).

[0031] The term "room temperature" as used in this article refers to the standard operating temperature in a laboratory, with a standard range of 20-25℃ (which can be extended to 18-28℃ in some scenarios). This is the commonly used environmental condition for the preparation, preservation, and microscopic examination of staining solutions. Operating at room temperature avoids the volatilization of phenol and the fading of dyes caused by high temperatures (e.g., cotton indigo is sensitive to light and heat), while also ensuring the compatibility of various components. This must be clearly stated in the patent draft to ensure consistency of experimental conditions.

[0032] The term "microscopic identification" used in this article refers to the method of classifying and identifying biological samples (such as fungi) by observing their morphological characteristics under a microscope (optical microscope, fluorescence microscope). For fungi, the core basis for microscopic identification is hyphae (presence or absence of septa, morphology), spores (type, size, arrangement), and special structures (rhizoids, cleistothecia). Combined with the staining effects of staining solutions such as lactic acid cotton blue, preliminary classification of fungal genera and species (such as Aspergillus and Candida) can be achieved. This is a core technology for rapid clinical diagnosis and scientific screening of fungal species.

[0033] In a first aspect, the present invention provides: a lactic acid phenol matrix composition, wherein the components of the lactic acid phenol matrix composition include: lactic acid, phenol, monosaccharides and their derivatives and polyols.

[0034] The technical features include: lactic acid, phenol, monosaccharides and their derivatives, and polyols.

[0035] The monosaccharides and their derivatives include, but are not limited to, at least one of glucose, sucrose, fructose, maltose, lactose, mannose, galactose, rhamnose, sorbitol and glucuronic acid;

[0036] The monosaccharide and its derivatives are preferably at least one of glucose, sucrose, fructose, galactose and rhamnose;

[0037] The monosaccharide and its derivatives are further preferably glucose;

[0038] The monosaccharide and its derivatives are more preferably glucose monohydrate;

[0039] Preferably, the monosaccharide and its derivatives are solutions of monosaccharides and their derivatives;

[0040] More preferably, the solvent for the monosaccharide and its derivatives is water;

[0041] More preferably, the solvent of the monosaccharide and its derivative solution includes, but is not limited to, at least one of deionized water, purified water, ultrapure water, mineral water and purified water;

[0042] The polyols include, but are not limited to, at least one of glycerol, ethylene glycol, polyethylene glycol, 1,2-propanediol, mannitol, xylitol and maltitol;

[0043] The polyol is preferably at least one selected from glycerol, ethylene glycol, polyethylene glycol, and 1,2-propanediol;

[0044] The polyol is further preferably glycerol;

[0045] Preferably, the content of monosaccharides and their derivatives in the lactic acid phenol matrix composition is 100-320 mg / mL;

[0046] More preferably, the content of monosaccharides and their derivatives in the lactic acid phenol matrix composition is 100-227 mg / mL;

[0047] More preferably, the content of monosaccharides and their derivatives in the lactic acid phenol matrix composition is 190 mg / mL;

[0048] Preferably, the lactic acid phenol matrix composition comprises, by volume, 20 parts lactic acid, 20 parts phenol, 36-56 parts monosaccharide and its derivative solution, and 4-24 parts polyol;

[0049] For example, the lactic acid phenol matrix composition, by volume parts, comprises:

[0050] 20 parts lactic acid;

[0051] 20 parts phenol;

[0052] 36-50 parts, 40-56 parts, 45-55 parts, 37-55 parts, 41-49 parts, 40-50 parts, 45-53 parts, 38-52 parts, 47-51 parts or 50-56 parts of monosaccharide and its derivative solution;

[0053] And 4-20 parts, 15-20 parts, 20-24 parts, 4-10 parts, 5-15 parts, 15-23 parts, 10-20 parts, 12-24 parts, 11-23 parts, 9-24 parts, 8-23 parts or 22-24 parts of polyols;

[0054] As a further example, the lactic acid phenol matrix composition, by volume parts, comprises:

[0055] 20 parts lactic acid;

[0056] 20 parts phenol;

[0057] 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50 or 56 parts of monosaccharide and its derivative solution;

[0058] And 4, 5, 6, 7, 8, 9, 10, 15, 20, 21, 22, 23 or 24 parts of polyol.

[0059] More preferably, the lactic acid phenol matrix composition comprises, by volume, 20 parts lactic acid, 20 parts phenol, 50-56 parts monosaccharide and its derivative solution, and 4-10 parts polyol;

[0060] More preferably, the lactic acid phenol matrix composition comprises, by volume, 20 parts lactic acid, 20 parts phenol, 52-56 parts monosaccharide and its derivative solution, and 4-8 parts polyol;

[0061] Most preferably, the lactic acid phenol matrix composition comprises, by volume, 20 parts lactic acid, 20 parts phenol, 56 parts monosaccharide and its derivative solution, and 4 parts polyol.

[0062] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes:

[0063] The first preferred embodiment: the monosaccharide and its derivatives include, but are not limited to, at least one of glucose, sucrose, fructose, galactose, rhamnose, sorbitol, and glucuronic acid; preferably, at least one of glucose, sucrose, fructose, galactose, and rhamnose; further preferably, glucose; and even more preferably, glucose monohydrate. This technical solution, while addressing the technical problem of "reducing the number of air bubbles in the microscopic field of view," further addresses the technical problem of "further reducing the number of air bubbles in the microscopic field of view."

[0064] The second preferred embodiment: the polyol includes, but is not limited to, at least one of glycerol, ethylene glycol, polyethylene glycol, 1,2-propanediol, mannitol, xylitol, and maltitol; preferably, at least one of glycerol, ethylene glycol, polyethylene glycol, and 1,2-propanediol; more preferably, glycerol. This technical solution, while addressing the technical problem of "reducing the number of air bubbles in the microscopic field of view," further addresses the technical problem of "further reducing the number of air bubbles in the microscopic field of view."

[0065] The third preferred embodiment is that the content of monosaccharides and their derivatives in the lactic acid phenol matrix composition is 100-320 mg / mL; preferably 100-227 mg / mL; and more preferably 190 mg / mL. This technical solution, while addressing the technical problem of "reducing the number of air bubbles in the microscopic field of view," further addresses the technical problem of "further reducing the number of air bubbles in the microscopic field of view."

[0066] The fourth preferred embodiment: the lactic acid phenol matrix composition, by volume, comprises 20 parts lactic acid, 20 parts phenol, 36-56 parts monosaccharide and its derivative solution, and 4-24 parts polyol; preferably, the lactic acid phenol matrix composition, by volume, comprises 20 parts lactic acid, 20 parts phenol, 50-56 parts monosaccharide and its derivative solution, and 4-10 parts polyol; more preferably, the lactic acid phenol matrix composition, by volume, comprises 20 parts lactic acid, 20 parts phenol, 52-56 parts monosaccharide and its derivative solution, and 4-8 parts polyol; most preferably, the lactic acid phenol matrix composition, by volume, comprises 20 parts lactic acid, 20 parts phenol, 56 parts monosaccharide and its derivative solution, and 4 parts polyol. This technical solution, based on solving the technical problem of "reducing the number of air bubbles in the microscopic field of view," further solves the technical problem of "further reducing the number of air bubbles in the microscopic field of view."

[0067] In a second aspect, the present invention provides the use of the lactic acid phenol matrix composition in the preparation of lactic acid phenol cotton blue dyeing solution.

[0068] This includes technical features: lactic acid phenol cotton blue staining solution and its application.

[0069] Preferably, the application is as follows: providing a core base system for lactic acid cotton blue staining solution, which contains lactic acid (dissolving sample keratin and assisting staining solution penetration), phenol (fixing fungal cells and acting as a preservative), monosaccharides and their derivatives (increasing viscosity and regulating permeability), and polyols (regulating system viscosity and moisturizing to prevent drying). These components constitute the key functional matrix of the lactic acid cotton blue staining solution. During preparation, this system can be used as a base, with aniline blue dye added in proportion and mixed evenly. No additional adjustment to the core component ratio is required to form a lactic acid cotton blue staining solution that combines sample transparency, cell morphology preservation, and specific staining functions, simplifying the preparation process while ensuring the stability of the staining effect.

[0070] Thirdly, the present invention provides: a lactic acid cotton blue staining solution, wherein the components of the lactic acid cotton blue staining solution include: the lactic acid matrix composition and aniline blue.

[0071] The technical features include: lactic acid phenol matrix composition and aniline blue.

[0072] Preferably, the aniline blue is water-soluble aniline blue;

[0073] The aniline blue content in the lactic acid cotton blue staining solution is 1-5 mg / mL.

[0074] The preferred content of aniline blue in the lactic acid cotton blue staining solution is 3-5 mg / mL.

[0075] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the third aspect of the present invention includes:

[0076] The first preferred embodiment is that the aniline blue content in the lactophenol cotton blue staining solution is 1-5 mg / mL; preferably 3-5 mg / mL. This technical solution, while addressing the technical problem of "reducing the number of air bubbles in the microscopic field of view," further solves the technical problem of "further reducing the number of air bubbles in the microscopic field of view."

[0077] Fourthly, the present invention provides a method for preparing the lactophenol cotton blue staining solution, comprising the following steps:

[0078] (1) A lactic acid-phenol matrix composition is obtained by mixing a solution of lactic acid, phenol, monosaccharide and its derivatives and a polyol;

[0079] (2) The lactic acid phenol matrix composition obtained in step (1) and aniline blue are mixed by heating to obtain the lactic acid phenol cotton blue staining solution.

[0080] Among them are the technical features: mixing and heating.

[0081] The mixing method described in step (1) includes, but is not limited to, at least one of mechanical stirring, oscillating mixing, ultrasonic dispersion mixing, rotary mixing, and homogenizer mixing;

[0082] The preferred mixing method in step (1) is mechanical stirring.

[0083] The mixing method described in step (2) includes, but is not limited to, at least one of mechanical stirring, oscillating mixing, ultrasonic dispersion mixing, rotary mixing, and homogenizer mixing;

[0084] The preferred mixing method in step (2) is ultrasonic dispersion mixing;

[0085] The heating temperature in step (2) is 70-90℃;

[0086] The heating time in step (2) is 10 minutes;

[0087] Preferably, the heating and mixing in step (2) are performed simultaneously;

[0088] Preferably, step (2) further includes a cooling step after heating and mixing;

[0089] More preferably, the cooling temperature is cooled to room temperature.

[0090] Fifthly, the present invention provides the application of the lactic acid cotton blue staining solution in the microscopic identification of fungi.

[0091] This includes technical characteristics: fungi, microscopic identification, and applications.

[0092] The fungi mentioned include, but are not limited to, cocci, such as Candida and Cryptococcus; and filamentous fungi, such as molds, which are further subdivided into Mucor, Penicillium, Aspergillus, etc.

[0093] Furthermore, the fungus may be at least one of the following: Aspergillus nidus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Candida albicans, Candida tropicalis, Candida glabrata, Penicillium chrysogenum, Penicillium citrinum, Trichophyton rubrum, Trichophyton mentagrophytes, Disyllium Amsterdam, Microsporum canis, Microsporum gypseum, Malassezia spheroides, Malassezia furfur, Auricularia auricula-judae, Mucor racemosa, Rhizopus oryzae, Rhizopus aureus, Rhizopus oryzae, Rhizopus spp., Otocinus, Fusarium moniliforme, Paecilomyces lilacinus, and Cladosporium multiflorum.

[0094] Preferably, the application is as follows: by adjusting the volume fraction of polyols and the content of monosaccharides and their derivatives in the lactic acid cotton blue staining solution within a certain range, while maintaining or improving the auxiliary identification staining effect of the lactic acid cotton blue staining solution on fungi, the amount of air bubbles in the microscopic field of view can also be reduced.

[0095] In this invention, Examples 1-5 at least support the protection scope of "the lactic acid phenol matrix composition, by volume, includes 20 parts lactic acid, 20 parts phenol, 36-56 parts monosaccharide and its derivative solution and 4-24 parts polyol".

[0096] The statement that "the lactic acid phenol matrix composition, by volume, comprises 20 parts lactic acid, 20 parts phenol, 36-56 parts monosaccharide and its derivative solution, and 4-24 parts polyol" is summarized from the foregoing explanation and / or the corresponding technical features in Examples 1-5, such as "the lactic acid phenol matrix composition, by volume, comprises 20 parts lactic acid, 20 parts phenol, 50-56 parts monosaccharide and its derivative solution, and 4-10 parts polyol," "the lactic acid phenol matrix composition, by volume, comprises 20 parts lactic acid, 20 parts phenol, 52-56 parts monosaccharide and its derivative solution, and 4-8 parts polyol," and "the lactic acid phenol matrix composition, by volume, comprises 20 parts lactic acid, 20 parts phenol, 56 parts monosaccharide and its derivative solution, and 4 parts polyol." Therefore, based on reasonable presumption, those skilled in the art can determine that the technical feature "the lactic acid phenol matrix composition, by volume, comprises 20 parts lactic acid, 20 parts phenol, 36-56 parts monosaccharide and its derivative solution, and 4-24 parts polyol", its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of the technical feature "the lactic acid phenol matrix composition, by volume, comprises 20 parts lactic acid, 20 parts phenol, 36-56 parts monosaccharide and its derivative solution, and 4-24 parts polyol".

[0097] Examples 1-5 of this invention at least support the protection scope of "the application of lactic acid phenol matrix composition in the preparation of lactic acid phenol cotton blue dyeing solution".

[0098] The application of the lactic acid phenol matrix composition in the preparation of lactic acid cotton blue staining solution is summarized from the foregoing explanation and / or the corresponding technical features in Examples 1-5, which "provide the core basic system of lactic acid cotton blue staining solution, the lactic acid (dissolving sample keratin and assisting staining solution penetration), phenol (fixing fungal cells and preservative), monosaccharides and their derivatives (increasing viscosity and regulating permeability), and polyols (regulating system viscosity and moisturizing and preventing drying) constitute the key functional matrix of lactic acid cotton blue staining solution. In the preparation, this system can be directly used as the basis, and aniline blue dye can be added in proportion and mixed evenly without additional adjustment of the core component ratio, so as to form a lactic acid cotton blue staining solution that has the functions of sample transparency, fungal morphology preservation and specific staining, simplifying the preparation process while ensuring the stability of staining effect." Therefore, based on reasonable presumption, those skilled in the art can determine that "the application of lactic acid phenol matrix composition in the preparation of lactic acid phenol cotton blue dyeing solution", its subordinate concept, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "the application of lactic acid phenol matrix composition in the preparation of lactic acid phenol cotton blue dyeing solution".

[0099] Examples 1-5 of this invention at least support the protection scope of "aniline blue".

[0100] "Aniline blue" is defined by the foregoing explanation and / or the corresponding technical features in Examples 1-5, such as "the aniline blue is water-soluble aniline blue" and "the content of aniline blue in the lactic acid cotton blue dyeing solution is 1-5 mg / mL; the content of aniline blue in the lactic acid cotton blue dyeing solution is preferably 3-5 mg / mL," which are summarized by the common feature "a class of triphenylmethane dyes synthesized from aniline." Therefore, those skilled in the art can reasonably infer that "aniline blue," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace "aniline blue" within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of the technical feature "aniline blue." For example, replacing "aniline blue" with "cotton blue" while keeping other technical features unchanged still falls within the protection scope of this invention.

[0101] Examples 1-5 of this invention at least support a “hybrid” scope of protection.

[0102] The term "mixing" is derived from the foregoing explanation and / or the corresponding technical features in Examples 1-5, such as "mixing methods include, but are not limited to, at least one of mechanical stirring, oscillating mixing, ultrasonic dispersion mixing, rotary mixing, and homogenizing," and is summarized by the common feature "an operation that uses physical means (stirring, oscillation, grinding, ultrasound, etc.) to uniformly disperse two or more components (solid, liquid) to form a homogeneous system." Therefore, those skilled in the art can reasonably presume that "mixing," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace "mixing" within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "mixing."

[0103] Examples 1-5 of this invention at least support the protection scope of "the application of lactic acid cotton blue staining solution in the microscopic identification of fungi".

[0104] The technical feature "Application of lactic acid cotton blue staining solution in the microscopic identification of fungi" is summarized from the foregoing explanation and / or the corresponding technical feature in Examples 1-5, which states that "by adjusting the volume fraction of polyols and the content of monosaccharides and their derivatives in the lactic acid cotton blue staining solution within a certain range, while maintaining or improving the auxiliary identification staining effect of lactic acid cotton blue staining solution on fungi, the amount of air bubbles in the microscopic field of view can also be reduced." Therefore, those skilled in the art can reasonably presume that the technical feature "application," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of the technical feature "Application of lactic acid cotton blue staining solution in the microscopic identification of fungi."

[0105] The beneficial effects of this invention are as follows:

[0106] The present invention has at least the following beneficial effects:

[0107] 1. Compared with the prior art, the present invention provides a technical solution with a different technical concept, and its technical effect is equivalent to or slightly improved with the prior art. The difference between the technical concept of the present invention and the prior art includes, but is not limited to, "keeping the volume fractions of lactic acid and phenol constant, keeping the range of water-soluble aniline blue content constant, replacing water with glucose solutions of different concentrations, and adjusting the volume fractions of glycerol and glucose solutions to control the volume fraction of glycerol and glucose content in the staining solution within a certain range, maintaining or improving the auxiliary identification staining effect of the staining solution on fungi, and reducing the amount of air bubbles in the microscopic field of view," etc. Attached Figure Description

[0108] Figure 1 Image of commercially available lactic acid cotton blue dyeing solution.

[0109] Figure 2 Image of the lactic acid phenol cotton blue staining solution from Example 2. Detailed Implementation

[0110] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0111] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0112] Example 1

[0113] A lactic acid phenol cotton blue staining solution has a lactic acid volume fraction of 20%, a phenol volume fraction of 20%, and a glycerol volume fraction of 10%; a glucose solution with a volume fraction of 455 mg / mL is 50% (the glucose content in the staining solution is 227 mg / mL), and a water-soluble aniline blue content of 1 mg / mL.

[0114] Correspondingly, the preparation method of the lactophenol cotton blue staining solution in this embodiment is as follows:

[0115] Add 50 g of glucose monohydrate to a beaker containing 50 mL of water. While stirring with a magnetic rod, heat the beaker to dissolve the glucose. After the glucose is completely dissolved, cool the solution to room temperature and dilute it to 100 mL with a 100 mL volumetric flask to obtain a 455 mg / mL glucose solution for later use.

[0116] Add 50 mL of 455 mg / mL glucose solution to a beaker, then add 20 mL of lactic acid, 20 mL of phenol, and 10 mL of glycerol in sequence. Stir with a magnetic rod to mix the components evenly to prepare the matrix for later use.

[0117] Add 0.1 g of water-soluble aniline blue to the above matrix, heat and sonicate until the temperature reaches 70-90℃, maintain sonication for 10 minutes to mix the components evenly, stop heating and sonication, and cool to room temperature to obtain lactic acid phenol cotton blue staining solution.

[0118] Example 2

[0119] A lactic acid phenol cotton blue staining solution has a lactic acid volume fraction of 20%, a phenol volume fraction of 20%, and a glycerol volume fraction of 8%; a glucose solution with a volume fraction of 364 mg / mL is 52% (the glucose content in the staining solution is 190 mg / mL), and a water-soluble aniline blue content of 2 mg / mL.

[0120] Correspondingly, the preparation method of the lactophenol cotton blue staining solution in this embodiment is as follows:

[0121] Add 40 g of glucose monohydrate to a beaker containing 40 mL of water. While stirring with a magnetic rod, heat the beaker to dissolve the glucose. After the glucose is completely dissolved, cool the solution to room temperature and dilute it to 100 mL with a 100 mL volumetric flask to obtain a glucose solution of 364 mg / mL for later use.

[0122] Add 52 mL of 364 mg / mL glucose solution to a beaker, then add 20 mL of lactic acid, 20 mL of phenol, and 8 mL of glycerol in sequence. Stir with a magnetic rod to mix the components evenly to prepare the matrix for later use.

[0123] Add 0.2 g of water-soluble aniline blue to the above matrix, heat and sonicate until the temperature reaches 70-90℃, maintain sonication for 10 minutes to mix the components evenly, stop heating and sonication, and cool to room temperature to obtain lactic acid phenol cotton blue staining solution.

[0124] Example 3

[0125] A lactic acid phenol cotton blue staining solution has a lactic acid volume fraction of 20%, a phenol volume fraction of 20%, and a glycerol volume fraction of 4%; a glucose solution with a volume fraction of 337 mg / mL (the glucose content in the staining solution is 190 mg / mL), and a water-soluble aniline blue content of 3 mg / mL.

[0126] Correspondingly, the preparation method of the lactophenol cotton blue staining solution in this embodiment is as follows:

[0127] Add 37.1 g of glucose monohydrate to a beaker containing 45 mL of water. While stirring with a magnetic rod, heat the beaker to dissolve the glucose. After the glucose is completely dissolved, cool the solution to room temperature and dilute it to 100 mL with a 100 mL volumetric flask to obtain a 337 mg / mL glucose solution for later use.

[0128] Add 56 mL of 337 mg / mL glucose solution to a beaker, then add 20 mL of lactic acid, 20 mL of phenol, and 4 mL of glycerol in sequence. Stir with a magnetic rod to mix all components evenly to prepare the matrix for later use.

[0129] Add 0.3 g of water-soluble aniline blue to the above matrix, heat and sonicate until the temperature reaches 70-90℃, maintain sonication for 10 minutes to mix the components evenly, stop heating and sonication, and cool to room temperature to obtain lactic acid phenol cotton blue staining solution.

[0130] Example 4

[0131] A lactic acid phenol cotton blue staining solution has a lactic acid volume fraction of 20%, a phenol volume fraction of 20%, and a glycerol volume fraction of 4%; a glucose solution with a volume fraction of 337 mg / mL is 56% (the glucose content in the staining solution is 190 mg / mL), and a water-soluble aniline blue content of 5 mg / mL.

[0132] Correspondingly, the preparation method of the lactophenol cotton blue staining solution in this embodiment is as follows:

[0133] Add 37.1 g of glucose monohydrate to a beaker containing 45 mL of water. While stirring with a magnetic rod, heat the beaker to dissolve the glucose. After the glucose is completely dissolved, cool the solution to room temperature and dilute it to 100 mL with a 100 mL volumetric flask to obtain a 337 mg / mL glucose solution for later use.

[0134] Add 56 mL of 337 mg / mL glucose solution to a beaker, then add 20 mL of lactic acid, 20 mL of phenol, and 4 mL of glycerol in sequence. Stir with a magnetic rod to mix all components evenly to prepare the matrix for later use.

[0135] Add 0.5 g of water-soluble aniline blue to the above matrix, heat and sonicate until the temperature reaches 70-90℃, maintain sonication for 10 minutes to mix the components evenly, stop heating and sonication, and cool to room temperature to obtain lactic acid phenol cotton blue staining solution.

[0136] Example 5

[0137] A lactic acid phenol cotton blue staining solution has a lactic acid volume fraction of 20%, a phenol volume fraction of 20%, and a glycerol volume fraction of 24%; a glucose solution with a volume fraction of 337 mg / mL (the glucose content in the staining solution is 121 mg / mL), and a water-soluble aniline blue content of 5 mg / mL.

[0138] Correspondingly, the preparation method of the lactophenol cotton blue staining solution in this embodiment is as follows:

[0139] Add 37.1g of glucose monohydrate to a beaker containing 45mL of water. While stirring with a magnetic rod, heat the beaker to dissolve the glucose. After the glucose is completely dissolved, cool the solution to room temperature and dilute it to 100mL with a 100mL volumetric flask to obtain a 337mg / mL glucose solution for later use.

[0140] Add 36 mL of 337 mg / mL glucose solution to a beaker, then add 20 mL of lactic acid, 20 mL of phenol, and 24 mL of glycerol in sequence. Stir with a magnetic rod to mix the components evenly to prepare the matrix for later use.

[0141] Add 0.5 g of water-soluble aniline blue to the above matrix, heat and sonicate until the temperature reaches 70-90℃, maintain sonication for 10 minutes to mix the components evenly, stop heating and sonication, and cool to room temperature to obtain lactic acid phenol cotton blue staining solution.

[0142] Evaluation of staining results in test case 1

[0143] Using commercially available lactic acid cotton blue staining solution as a control, the lactic acid cotton blue staining solutions prepared in Examples 1-5 of this invention were used to stain Aspergillus nidulans, which is most prone to producing bubbles, and the staining results under 40× objective microscopy were statistically analyzed.

[0144] The commercially available lactic acid cotton blue dyeing solution used was manufactured by Zhuhai Beso Biotechnology Co., Ltd., with production batch number C240601.

[0145] Commercially available lactic acid cotton blue dyeing solution contains 20% lactic acid and 20% phenol by volume.

[0146] The specific scoring criteria are as follows:

[0147] Table 1. Scoring Criteria

[0148]

[0149] Table 2. Results of Aspergillus nidulans staining

[0150]

[0151] The staining solutions in Examples 1-5 were used to stain Aspergillus nidulans, which is prone to producing bubbles on the slides during the staining process. The staining effect was comparable to or better than that of commercially available staining solutions. There were fewer bubbles in the microscopic field of view, and the important structures such as fungal cells and hyphae were almost not obscured.

[0152] Verification of technical effectiveness and / or analysis of technical problem solving

[0153] As can be seen from the above analysis, the preparation method provided by the present invention keeps the volume fractions of lactic acid and phenol constant, the content range of water-soluble aniline blue constant, and replaces water with glucose solutions of different concentrations when preparing lactic acid phenol cotton blue staining solution. It also adjusts the volume fractions of glycerol and glucose solutions to control the volume fraction of glycerol and the content of glucose in the staining solution within a certain range, thereby maintaining or improving the auxiliary identification staining effect of the staining solution on fungi and reducing the amount of air bubbles in the microscopic field of view.

[0154] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A lactic acid phenol matrix composition, characterized in that, By volume, it includes 20 parts lactic acid, 20 parts phenol, 52-56 parts monosaccharide and its derivative solution, and 4-8 parts polyol; The lactic acid phenol matrix composition can be used to prepare lactic acid phenol cotton blue dyeing solution; The lactic acid phenol cotton blue staining solution is used for microscopic identification of fungi.

2. The lactic acid phenol matrix composition according to claim 1, characterized in that, The monosaccharide and its derivatives are selected from at least one of glucose, sucrose, fructose, maltose, lactose, mannose, galactose, rhamnose, sorbitol and glucuronic acid.

3. The lactic acid phenol matrix composition according to claim 2, characterized in that, The monosaccharide and its derivatives are glucose.

4. The lactic acid phenol matrix composition according to claim 1, characterized in that, The polyol is selected from at least one of glycerol, ethylene glycol, polyethylene glycol, 1,2-propanediol, mannitol, xylitol and maltitol.

5. The lactic acid phenol matrix composition according to claim 4, characterized in that, The polyol is glycerol.

6. The lactic acid phenol matrix composition according to claim 1, characterized in that, The content of the monosaccharide and its derivatives is 100-320 mg / mL.

7. The lactic acid phenol matrix composition according to claim 1, characterized in that, The fungus is selected from at least one of cocciform fungi and filamentous fungi.

8. A lactic acid phenol cotton blue staining solution, characterized in that, The ingredients include: the lactic acid phenol matrix composition according to any one of claims 1-6 and aniline blue.

9. The lactic acid phenol cotton blue staining solution according to claim 8, characterized in that, The aniline blue is water-soluble aniline blue.

10. The lactophenol cotton blue staining solution according to any one of claims 8-9, characterized in that, The content of the aniline blue is 1-5 mg / mL.

11. The method for preparing the lactophenol cotton blue dyeing solution according to any one of claims 8-10, characterized in that, Including the following steps: (1) A lactic acid-phenol matrix composition is obtained by mixing a solution of lactic acid, phenol, monosaccharide and its derivatives and a polyol; (2) The lactic acid phenol matrix composition obtained in step (1) and aniline blue are mixed by heating to obtain the lactic acid phenol cotton blue staining solution.

12. The preparation method according to claim 11, characterized in that, The mixing methods described in steps (1) and (2) are selected from at least one of mechanical stirring, oscillating mixing, ultrasonic dispersion mixing, rotary mixing, and homogenizer mixing.

13. The preparation method according to claim 11 or 12, characterized in that, The heating temperature in step (2) is 70-90℃; the heating time in step (2) is 10min.

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