Method for reducing background fluorescence and method for detecting microorganisms

Abstract

Provided are a method for reducing background fluorescence when detecting microorganisms on a membrane filter to which culture medium components are attached by fluorescent staining, and a method for detecting microorganisms using said method. [Solution] A method for reducing background fluorescence when detecting microorganisms on a membrane filter to which culture medium components are attached by fluorescent staining, wherein the fluorescent staining is performed by contacting the membrane filter with a solution containing a fluorescent reagent and having a pH of 5.0 or less; and a method for detecting microorganisms in a liquid, wherein the liquid is filtered through a membrane filter, the membrane filter is attached to the surface of a plate culture medium, cultured for a predetermined period of time, and the membrane filter, which has been peeled off from the plate culture medium, is contacted with a solution containing a fluorescent reagent and having a pH of 5.0 or less, thereby fluorescently staining the microorganisms on the membrane filter.

Description

[Technical Field]

[0001] The present invention relates to a method for fluorescently detecting microorganisms from a membrane filter that has captured the microorganisms on a plate medium. [Background technology]

[0002] Microbial detection tests have traditionally been performed using a culture method in which a sample is mixed and dispersed with dissolved agar medium in a petri dish, solidified on a plate medium, and cultured, and the presence or absence of colony formation is visually confirmed. However, this culture method requires a culture time of 2 to 5 days until colonies large enough to be visually confirmed are formed.

[0003] A method that requires less time for detection than the culture method is to stain microorganisms with a fluorescent reagent and detect bright spots derived from the microorganisms using a fluorescence detection device. For example, a method is known in which microorganisms are fluorescently stained by suspending them in a fluorescent reagent solution, and then the fluorescently stained microorganisms are detected using flow cytometry (Patent Document 1). Fluorescently staining microorganisms allows for detection of microorganisms with higher sensitivity than visual inspection, thereby shortening the culture time.

[0004] As a method for detecting microorganisms other than suspending them in a fluorescent reagent solution, a method is also known in which microorganisms are captured on a membrane filter and then penetrated into the cells with a fluorescent reagent while maintaining their viability (Patent Document 2). When detecting microorganisms in solutions such as beverages, a fluorescent detection method is used in which a sample is filtered and concentrated using a membrane filter, the filtration filter used in the filtration is attached to a plate culture medium for incubation, and the resulting tiny microcolonies are fluorescently stained and observed for fluorescence. Fluorescent images of the fluorescently stained membrane filter are captured using a fluorescent imaging device that combines a high-performance camera, a light source, and a fluorescent filter, and the microorganisms on the membrane filter are detected by image analysis of the obtained fluorescent images. To detect microorganisms with higher accuracy in this fluorescent detection method, a method is also known in which fluorescent images are analyzed using deep learning image processing software, and an algorithm generated by deep learning of the differences in feature values ​​between fluorescently stained microorganism-derived microcolonies and components other than microorganisms is used to improve the ability to distinguish between bright spots derived from microorganisms and bright spots derived from components other than microorganisms (Patent Document 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2000-69995 [Patent Document 2] Japanese Patent Application Laid-Open No. 2009-247352 [Patent Document 3] Japanese Patent Publication No. 2022-190404 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a method for reducing background fluorescence when detecting microorganisms on a membrane filter to which culture medium components are attached by fluorescent staining, and a method for detecting microorganisms using said method. [Means for solving the problem]

[0007] The inventors discovered that when microorganisms on a membrane filter that has been attached to the surface of a plate culture medium and cultured for a predetermined period of time are contacted with a fluorescent reagent solution for fluorescent staining, background fluorescence can be reduced by adjusting the pH of the fluorescent reagent solution to 5.0 or less, and completed the present invention.

[0008] The present invention includes the following aspects. [1] A method for reducing background fluorescence when detecting microorganisms on a membrane filter to which culture medium components are attached by fluorescent staining, comprising: The fluorescent staining is carried out by contacting the membrane filter with a solution containing a fluorescent reagent; the fluorescent reagent is CFDA, A method for reducing background fluorescence, wherein the pH of the solution containing the fluorescent reagent is 5.0 or less. [ 2 The pH of the solution containing the fluorescent reagent is 3.0 or higher. of method. [ 3 The membrane filter is made of one or more materials selected from the group consisting of cellulose acetate and nitrocellulose. or [2] How to do it. [ 4 ] A method for detecting microorganisms in a liquid, comprising: a filtering step of filtering the liquid through a membrane filter; a culturing step of attaching the membrane filter to the surface of a plate medium after the filtration step and culturing the medium for a predetermined period of time; a fluorescent staining step in which the membrane filter, which has been peeled off from the plate medium after the culturing step, is brought into contact with a solution containing a fluorescent reagent to fluorescently stain the microorganisms on the membrane filter; and the fluorescent reagent is CFDA, A method for detecting microorganisms, wherein the pH of the solution containing the fluorescent reagent is 5.0 or less. [ 5] The pH of the solution containing the fluorescent reagent is 3.0 or higher. 4 ] A method for detecting microorganisms. [ 6 The membrane filter is made of one or more materials selected from the group consisting of cellulose acetate and nitrocellulose. 4 ] or [5] Methods for detecting microorganisms. [ 7 ] the liquid is a beverage, 4 ]~[ 6 ] A method for detecting any of the microorganisms. [Effects of the Invention]

[0009] According to the present invention, microorganisms on a membrane filter to which medium components are attached can be detected with high accuracy by fluorescent staining. DETAILED DESCRIPTION OF THE INVENTION

[0010] <Methods for reducing background fluorescence> A background fluorescence reduction method according to an embodiment of the present disclosure (hereinafter, sometimes referred to as the "reduction method of this embodiment") is a method for reducing background fluorescence when detecting microorganisms on a membrane filter to which culture medium components are attached by fluorescent staining, wherein the fluorescent staining is performed by contacting the membrane filter with a fluorescent reagent solution (a solution containing a fluorescent reagent), and the pH of the fluorescent reagent solution is 5.0 or less. When a membrane filter to which culture medium components are attached is stained with a fluorescent reagent solution, the background fluorescence may be high, making it difficult to detect the fluorescence emitted from the target microorganisms. By adjusting the pH of the fluorescent reagent solution to 5.0 or less, background fluorescence can be reduced more than when the pH is closer to neutral.

[0011] The membrane filter used in the reduction method of this embodiment is a membrane filter to which microorganisms are attached together with medium components. Examples of such membrane filters include a membrane filter in which microorganisms are captured on the membrane filter through a filtration process and then attached to a plate medium for cultivation. Furthermore, the background fluorescence can also be reduced by the reduction method of this embodiment using a membrane filter in which microorganisms are cultured on a plate medium and then the membrane filter is attached to the surface of the plate medium, transferring the microorganisms on the plate medium together with the medium components.

[0012] The medium components adhering to the membrane filter used in the reduction method of this embodiment are components of a medium capable of culturing microorganisms. All or some of the components constituting the medium may adhere to the membrane filter. The medium is not particularly limited as long as it contains a carbon source and a nitrogen source necessary for the growth of microorganisms such as mold, yeast, and bacteria. Glucose is preferred as a carbon source because it can be assimilated by a wide variety of microorganisms, and peptone, yeast extract, plant extract, etc. are preferred as nitrogen sources. Peptones are not particularly limited, but casein peptone and meat peptone are preferred because they are commonly used in microbial culture. Examples of plant extracts include potato extract, malt extract, fruit juice, and vegetable juice. Potato extract is preferred as a plant extract contained in the plate medium because it contains trace metals and minerals such as copper and zinc necessary for the growth of fungi such as mold and yeast, and is commonly used as a component of fungal culture media.

[0013] The membrane filter to which microorganisms are attached together with the medium components by, for example, attaching it to a plate medium can be appropriately selected from membrane filters used in microbial experiments, biochemical experiments, and the like. For example, a membrane filter made of a porous material with a pore size of 0.1 to 1.0 μm, preferably 0.15 to 0.9 μm, and more preferably 0.45 to 0.8 μm, can be used. Preferred membrane filters used in the reduction method of this embodiment are cellulose acetate membrane filters, nitrocellulose membrane filters, and cellulose mixed ester membrane filters (membrane filters made of a mixture of cellulose acetate and nitrocellulose) because they are suitable for capturing microorganisms and have relatively low protein adsorption. Commercially available membrane filters can be used as appropriate.

[0014] The fluorescent reagent used in the reduction method of this embodiment is not particularly limited as long as it is a compound that can fluorescently label microorganisms. The reagent itself may be fluorescent, or it may be a reagent that is converted into a fluorescent substance within the living organism of the microorganism. An appropriate fluorescent reagent may be selected from various fluorescent reagents used for fluorescent staining of microorganisms such as bacteria, yeast, and filamentous fungi (mold). The fluorescent reagent may be a reagent that can stain living microorganisms, or a reagent that can stain dead microorganisms.

[0015] Fluorescent reagents capable of staining living microorganisms include non-fluorescent molecules that are decomposed by the esterase and oxidoreductase activities of living microorganisms to produce fluorescent molecules. Fluorescent reagents utilizing esterase activity include CFDA (6-carboxyfluorescein diacetate), CFDA-AM (Carboxyfluorescein diacetate-acetoxymethlester), FDA (Fluorescein diacetate), and Calcein-AM (Calcein-acetoxymethlester). Non-fluorescent CFDA taken up into the microbial cell is converted to green fluorescent 6-carboxyfluorescein by the esterase activity within the microorganism. Fluorescent reagents utilizing oxidoreductase activity include the monotetrazolium reduction dye CTC (5-cyano-2,3-ditolyl tetrazolium chloride). Non-fluorescent CTC taken up into the microbial cell is reduced in the electron transport system associated with respiration and converted to CTF (CTC formazan), which emits red fluorescence.

[0016] Furthermore, in the reduction method of this embodiment, by using a nucleic acid fluorescent stain as the fluorescent reagent, not only live microorganisms but also dead microorganisms can be fluorescently stained. Examples of nucleic acid fluorescent stains include acridine orange, DAPI (4',6-diamidono-2-phenylindole), ethidium bromide, PI (propidium iodine), Hoechst 33258, Hoechst 33342, and SYBR Green.

[0017] Many substances that emit autofluorescence, such as amino acids and dietary fiber, emit stronger fluorescence when exposed to short-wavelength light such as ultraviolet light. In order to suppress these effects, the fluorescent reagent used in the reduction method of this embodiment is preferably a fluorescent reagent that emits fluorescence when exposed to excitation light with a relatively long wavelength, in particular a fluorescent reagent that emits green to red fluorescence.

[0018] The fluorescent reagent solution used in the reduction method of this embodiment is a solution in which a fluorescent reagent is dissolved or diluted in an aqueous medium, and has a pH of 5.0 or less. A pH of 5.0 or less can reduce background fluorescence derived from medium components. The pH of the fluorescent reagent solution used in the reduction method of this embodiment is not particularly limited as long as it is 5.0 or less, and is preferably 3.0 to 5.0, more preferably 3.5 to 4.5, and even more preferably 4.0 to 4.5.

[0019] The fluorescent reagent solution used in the reduction method of this embodiment is preferably a solution in which the fluorescent reagent is dissolved or diluted in a buffer solution having a pH of 5.0 or less. The buffer solution is not particularly limited, and examples that can be used include phosphate buffer, acetate buffer, phthalate buffer, succinate buffer, citrate buffer, and glycine buffer.

[0020] In the reduction method of this embodiment, the membrane filter is contacted with a fluorescent reagent solution to fluorescently stain microorganisms present on the membrane filter. The contact between the membrane filter and the fluorescent reagent solution may be carried out for a period of time sufficient for the microorganisms on the membrane filter to be fluorescently stained. For example, the microorganisms on the membrane filter can be fluorescently stained by contacting the membrane filter with the fluorescent reagent solution and incubating at 4 to 38°C, preferably 30 to 37°C, for 5 minutes to 6 hours.

[0021] The fluorescently stained microorganisms on the membrane filter can be detected by irradiating the membrane filter with excitation light suitable for the fluorescent reagent used for the fluorescent staining and detecting the generated fluorescent signal. Irradiation with excitation light and detection of fluorescence can be performed by standard methods using a commonly used fluorescence microscope or the like.

[0022] <Microorganism detection method> A method for detecting microorganisms according to an embodiment of the present disclosure (hereinafter sometimes referred to as the "detection method of the present embodiment") is a method that uses the reduction method of the present embodiment when detecting the microorganisms to be detected using a fluorescent reagent in a state where the microorganisms are captured on a membrane filter together with culture components. Specifically, the method for detecting microorganisms according to an embodiment of the present disclosure is a method for detecting microorganisms in a liquid, and includes the following steps: a filtering step of filtering the liquid through a membrane filter; After the filtration step, the membrane filter is attached to the surface of the plate medium, and the medium is cultured for a predetermined period of time. a fluorescent staining step in which, after the culturing step, the membrane filter peeled from the plate medium is brought into contact with a fluorescent reagent solution having a pH of 5.0 or less, thereby fluorescently staining the microorganisms on the membrane filter;

[0023] The detection method of this embodiment detects microorganisms in a liquid. The liquid (liquid sample under test) used as a test sample for detecting microorganisms is not particularly limited. Examples of such liquids include beverages, liquid foods, liquid pharmaceuticals, liquid cosmetics, and liquid compositions that serve as raw materials for these. Furthermore, solids, such as foods, pharmaceuticals, cosmetics, and solids that serve as raw materials for these, can be crushed and dispersed or dissolved in a solvent to form solutions, or extracts of such solids or crushed solids, and these solids can also be used as test samples for detecting microorganisms using the detection method of this embodiment.

[0024] Beverages are particularly preferred as test samples in the detection method of this embodiment. Beverages may contain microorganisms such as lactic acid bacteria or yeast, or may contain autofluorescent components such as amino acids, proteins, or dietary fiber. They may also be non-alcoholic beverages such as soft drinks and dairy drinks, or alcoholic beverages. Examples of soft drinks include carbonated drinks such as ramune (ramune soda), cider, cola, and ginger ale; fruit and vegetable drinks such as concentrated fruit juices, fruit juice drinks, fruit juices, vegetable juices, mixed fruit and vegetable juices, and drinks containing fruit pulp or vegetable pieces; beverages such as coffee, black tea, green tea, oolong tea, barley tea, jasmine tea, herbal drinks, and cocoa; dairy beverages such as lactic acid bacteria drinks, fermented milk, milk, and milk coffee drinks containing dairy components; soy milk; sports drinks; mineral water; and non-alcoholic beverages with an alcoholic beverage flavor, such as non-alcoholic beer, non-alcoholic plum wine, and non-alcoholic cocktails. Examples of alcoholic beverages include beer, wine, sake, shochu, whiskey, brandy, liqueurs, and cocktails.

[0025] In the detection method of this embodiment, the microorganisms to be detected are not particularly limited, and examples thereof include bacteria, filamentous fungi (molds), yeasts, and the like.

[0026] In the detection method of this embodiment, microorganisms in a liquid are collected on a membrane filter and allowed to grow on the membrane filter to form colonies. The formed colonies are detected using fluorescent staining. While two to three days of culture is generally required to form colonies large enough to be visible to humans, the present invention utilizes fluorescence to enable detection even of very small colonies (microcolonies), thereby shortening the culture time and enabling more rapid detection of microorganisms.

[0027] In the filtration step, a liquid (a test liquid sample) that is a test sample for detecting microorganisms is filtered through a membrane filter. Specifically, the test liquid sample is poured into a funnel with a membrane at the bottom through the upper opening of the funnel and filtered. By suction filtering the beverage from the bottom of the funnel, a sufficient amount of microorganisms in the test liquid sample can be captured on the membrane in a short period of time. Suction filtration can be performed in the same manner as in a general membrane filter method. The membrane filter used can be the same as the membrane filters listed as being used in the reduction method of this embodiment.

[0028] In the culture step, the membrane filter that captured the microorganisms in the liquid in the filtration step is attached to the surface of a plate medium and cultured for a predetermined period of time. The plate medium used is not particularly limited as long as it contains a carbon source and a nitrogen source necessary for the growth of microorganisms such as mold, yeast, and bacteria, and the same medium as those listed as the medium attached to the membrane filter used in the reduction method of this embodiment can be used.

[0029] The membrane filter obtained in the filtration step is attached to the surface of a plate medium and cultured for a predetermined period of time, allowing the living microorganisms on the membrane filter to grow and form colonies. The culture time is preferably 3 to 72 hours, more preferably 3 to 48 hours, and even more preferably 12 to 24 hours. The culture temperature may be any temperature range that allows mold, yeast, and bacteria to grow, and can be, for example, within the range of 20 to 35°C.

[0030] After the culturing step, the membrane filter detached from the plate medium is contacted with a dye for fluorescently staining microorganisms to fluorescently stain the microorganisms on the membrane filter. The fluorescent reagent for fluorescently staining microorganisms is not particularly limited as long as it is capable of fluorescently staining live microorganisms. It may stain only live bacteria, or it may stain both live and dead bacteria. Specifically, among the fluorescent reagents listed as being used in the reduction method of this embodiment, fluorescent reagents similar to those capable of fluorescently staining live bacteria can be used. As fluorescent reagents used in the detection method of this embodiment, CFDA, CFDA-AM, and FDA are preferred, with CFDA being more preferred, as they are capable of accurately detecting live bacteria. Nucleic acid fluorescent stains such as acridine orange, DAPI, ethidium bromide, PI, Hoechst 33258, Hoechst 33342, and SYBR Green are also preferred.

[0031] Specifically, the membrane filter detached from the plate medium is brought into contact with a fluorescent dye solution, for example, by incubation at 4 to 38°C, preferably 30 to 37°C, for 5 minutes to 6 hours.

[0032] The fluorescent reagent solution used in the detection method of this embodiment is a solution in which a fluorescent reagent is dissolved or diluted in an aqueous medium, and has a pH of 5.0 or less, preferably 3.0 to 5.0, more preferably 3.5 to 4.5, and even more preferably 4.0 to 4.5. A pH of 5.0 or less can reduce background fluorescence derived from medium components. Specifically, the fluorescent reagent solution used in the detection method of this embodiment can be the same as the fluorescent reagent solution used in the reduction method of this embodiment.

[0033] The fluorescently stained microorganisms on the membrane filter can be detected by irradiating the membrane filter with excitation light suitable for the fluorescent reagent used for the fluorescent staining and detecting the generated fluorescent signal. Irradiation with excitation light and detection of fluorescence can be performed by standard methods using a commonly used fluorescence microscope or the like. [Example]

[0034] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0035] [Example 1] Methylobacterium aerolatum was collected on a membrane filter along with the culture medium components, and the membrane filter was fluorescently stained with a fluorescent reagent solution at pH 3.0 to 7.0 for detection. CFDA (commercially available: Dojindo Laboratories) was dissolved in MES (2-Morpholinoethanesulfonic acid, monohydrate) buffer adjusted to pH 3.0, 4.0, 5.0, 6.0, or 7.0 to the recommended concentration. This CFDA staining solution was used as the fluorescent reagent solution.

[0036] Microbial culture media included R2A agar (casein peptone 0.25 g, meat peptone 0.25 g, yeast extract 0.5 g, casamino acids 0.5 g, glucose 0.5 g, soluble starch 0.5 g, sodium pyruvate 0.3 g, magnesium sulfate heptahydrate 50 mg, dipotassium hydrogen phosphate 0.3 g, purified water 1000 mL, agar 15.0 g, pH 7.0-7.4), standard agar (casein peptone 5.0 g, yeast extract 2.5 g, glucose 1.0 g, agar 15.0 g, purified water 1000 mL, pH 6.9-7.3), and modified thioglycolic acid (TGC) agar (L-cystine 0.25 g, sodium chloride 2.5 g, glucose 6.0 g, yeast extract 15.0 g, pH 7.0-7.4). The following ingredients were used: 5.0 g of casein peptone, 17.0 g of casein peptone, 3.0 g of soybean peptone, 0.5 g of sodium thioglycolate, 0.1 g of sodium sulfite, 1000 mL of purified water, 15.7 g of agar, pH 6.9 to 7.3.

[0037] First, an aqueous solution containing microorganisms was filtered through a cellulose mixed ester membrane filter, and the microorganisms were captured on the membrane filter. The membrane filter containing the captured microorganisms was attached to R2A agar medium, standard agar medium, or modified TGC agar medium and cultured at 30°C for 48 hours using an incubator. After culture, the membrane filter was peeled from the medium, contacted with a CFDA staining solution, and fluorescently stained by incubating at 30°C for 10 minutes (n=3). The fluorescently stained membrane filter was photographed using a fluorescent imaging device (FLOX-AI, Asahi Soft Drinks Co., Ltd.). The average RGB values ​​of the microbial and background areas (areas without microorganisms) of the membrane filter in the photographed image were measured using image processing software (ImageJ, Wayne Rasband, NIH), and the brightness was calculated according to the following formula:

[0038] [Brightness value] = 0.299 × [R value] + 0.587 × [G value] + 0.114 × [B value]

[0039] [Table 1]

[0040] The measurement results (mean ± standard deviation) of the brightness values ​​of each membrane filter are shown in Table 1. As shown in Table 1, it was found that regardless of which agar medium was used, the background brightness was suppressed as the pH of the CFDA staining solution decreased. In particular, when the pH was 5.0 or less, regardless of the type of medium, the background brightness value was 100 or less, making it easier to distinguish between fluorescence from microorganisms and background fluorescence, and improving the accuracy of microbial detection.

Claims

1. A method for reducing background fluorescence when detecting microorganisms on a membrane filter to which culture medium components are attached by fluorescent staining, comprising: The fluorescent staining is carried out by contacting the membrane filter with a solution containing a fluorescent reagent; the fluorescent reagent is CFDA, A method for reducing background fluorescence, wherein the pH of the solution containing the fluorescent reagent is 5.0 or less.

2. The method of claim 1 , wherein the pH of the solution containing the fluorescent reagent is 3.0 or higher.

3. The method according to claim 1 or 2, wherein the membrane filter is made of one or more materials selected from the group consisting of cellulose acetate and nitrocellulose.

4. 1. A method for detecting microorganisms in a liquid, comprising: a filtering step of filtering the liquid through a membrane filter; a culturing step of attaching the membrane filter to the surface of a plate medium after the filtration step and culturing the medium for a predetermined period of time; a fluorescent staining step in which the membrane filter, which has been peeled off from the plate medium after the culturing step, is brought into contact with a solution containing a fluorescent reagent to fluorescently stain the microorganisms on the membrane filter; and the fluorescent reagent is CFDA, A method for detecting microorganisms, wherein the pH of the solution containing the fluorescent reagent is 5.0 or less.

5. 5. The method for detecting microorganisms according to claim 4, wherein the pH of the solution containing the fluorescent reagent is 3.0 or higher.

6. 5. The method for detecting microorganisms according to claim 4, wherein the membrane filter is made of at least one material selected from the group consisting of cellulose acetate and nitrocellulose.

7. The method for detecting microorganisms according to any one of claims 4 to 6, wherein the liquid is a beverage.

Images