Method for measuring microorganisms by using fluorescence

Abstract

Provided is a method for measuring microorganisms by using fluorescence, which is a method for measuring microorganisms in a sample containing rubber, evaluates the number of microorganisms in the sample through staining of the microorganisms with a fluorescent dye, and can exhibit excellent accuracy while improving the speed of measurement. By applying such a microorganism measurement method, the population of microorganisms contained in a sample can be counted in almost real-time, and thus microbial treatment such as the addition of preservatives can be performed not excessively, and therefore the method is economical and eco-friendly and can also improve the quality of final products.

Description

Microbial measurement method using fluorescence

[0001] This application claims the benefit of priority to Korean Patent Application No. 10-2024-0117308, filed August 30, 2024, Korean Patent Application No. 10-2024-0191096, filed December 19, 2024, and Korean Patent Application No. 10-2025-0120309, filed August 27, 2024, which are hereby incorporated herein by reference in their entirety.

[0002] The present invention relates to a method for measuring microorganisms using fluorescence. Specifically, the present invention relates to a method for measuring microorganisms in a sample containing rubber, wherein the microorganisms are stained with a fluorescent dye to assess the number of microorganisms in the sample.

[0003] Recently, the importance of the secondary industry, which processes products obtained from nature to produce goods necessary for daily life, has begun to receive renewed attention, and the importance of goods produced from the secondary industry is once again becoming important.

[0004] As for products produced from the secondary industry, especially products using latex, as mentioned above, they are mainly obtained by processing products obtained from nature, and therefore, the occurrence of microorganisms during the processing is inevitable due to their natural characteristics.

[0005] When microorganisms are included in the final product, the purity of the product is lowered, and as the purity is lowered, the properties essential to the final product, such as strength and ductility, change. Therefore, in the case of final products obtained by processing products obtained primarily from nature, the treatment of microorganisms during the manufacturing process is becoming increasingly important for quality control.

[0006] To handle such microorganisms, a method is being studied to determine the number of microorganisms in the sample for the production of the final product during the manufacturing process and to add a preservative proportional to the number of microorganisms to prevent the growth of microorganisms in the sample while simultaneously minimizing the number of microorganisms present in the final product.

[0007] However, in the case of products produced from the secondary industry, there was a problem that it was difficult to isolate microorganisms in the sample due to the inclusion of a large number of substances in the sample during the manufacturing process. To solve this problem, the method mainly used to count the number of microorganisms in the sample was to distribute the sample on a solid medium and culture the microorganisms for a certain period of time to count the number of microorganisms. However, in the case of the above method, it was difficult to immediately preservative the number of microorganisms because it was essential to secure a certain amount of time for microbial culture. In addition, since excessive preservatives were applied considering the number of microorganisms that could reproduce until the culture was completed, the economic feasibility of the process was lowered, and the amount of preservative included in the final product increased, which also caused a decrease in the quality of the final product.

[0008] Accordingly, there is a need for a method for measuring microorganisms that can quickly measure the number of microorganisms present in a sample while also ensuring reliable counting accuracy.

[0009] [Prior Art Literature]

[0010] [Patent Document]

[0011] (Patent Document 1) Japanese Patent Application Laid-Open No. 2009-247331

[0012] The purpose of the present invention is to provide a method for measuring microorganisms in a sample containing rubber, wherein the number of microorganisms in the sample is evaluated by staining the microorganisms with a fluorescent dye, thereby increasing the speed of the microorganism count, facilitating the treatment of microorganisms in a continuous process, and providing a method for measuring microorganisms using fluorescence, which also has excellent accuracy in the counting.

[0013] A first aspect of the present invention provides a method for measuring microorganisms in a sample containing rubber, wherein the method uses fluorescence to stain microorganisms with a fluorescent dye to evaluate the number of microorganisms in the sample.

[0014] In one specific example of the present invention, the rubber is a nitrile rubber.

[0015] In one specific embodiment of the present invention, the fluorescent dye is for staining living microorganisms.

[0016] In one specific example of the present invention, the staining includes (1) a step of introducing the fluorescent dye into the microorganism and (2) a step of culturing the microorganism to which the fluorescent dye has been introduced.

[0017] In one specific embodiment of the present invention, the culturing is performed for 0.1 to 3 hours.

[0018] In one specific embodiment of the present invention, the measuring method includes a step of washing the microorganism before staining.

[0019] In one specific embodiment of the present invention, the washing is performed by adding a dilution buffer containing sodium chloride to the sample.

[0020] In one specific embodiment of the present invention, the evaluation comprises the steps of (1) loading dyed microorganisms onto a sample tray and (2) evaluating the number of individuals of the loaded microorganisms.

[0021] In one specific embodiment of the present invention, the microorganism comprises a hydrolytic enzyme.

[0022] In one specific example of the present invention, the microorganism contains a hydrolytic enzyme, and the fluorescent dye reacts with the hydrolytic enzyme contained in the microorganism to exhibit fluorescence.

[0023] In one specific embodiment of the present invention, the microorganism is of the genus Alcaligenes.

[0024] In one specific embodiment of the present invention, the population assessment of the microorganism is performed using a fluorescence microscope.

[0025] In one specific example of the present invention, the evaluation of the number of objects is performed using a coefficient formula represented by the following [Formula 1].

[0026] [Formula 1]

[0027] Number of individuals = (Number of individuals showing Col1 fluorescence) - (Number of individuals showing Col2 fluorescence)

[0028] (Here, Col1 and Col2 are different from each other and are electromagnetic waves within the wavelength range of 1 nm to 1000 nm that appear independently due to fluorescence.)

[0029] In one specific example of the present invention, the time required from collecting the sample to evaluating the number of individuals is 0.1 to 6 hours.

[0030] The method for measuring microorganisms using fluorescence according to the present invention is a method for measuring microorganisms in a sample containing rubber, and by staining the microorganisms with a fluorescent dye to evaluate the number of microorganisms in the sample, not only is the accuracy of counting microorganisms excellent, but also the speed is significantly improved.

[0031] When these characteristics are applied to a process, the number of microorganisms in a solution used in an actual process can be counted in near real time, and accordingly, the amount of preservative to be added to the solution can be controlled so as not to be excessive, enabling a more environmentally friendly process and improving the quality of the final product.

[0032] [Figure 1] is an image of green fluorescence of an embodiment of the present invention taken using a fluorescence microscope.

[0033] [Figure 2] is an image of red fluorescence of an embodiment of the present invention taken using a fluorescence microscope.

[0034] [Figure 3] is an image of the green fluorescence of Comparative Example 2 of the present invention taken using a fluorescence microscope.

[0035] [Figure 4] is a graph showing the correlation between the number of microorganisms counted in Example 1 of the present invention and Comparative Example 1.

[0036] Hereinafter, the present invention will be described in more detail.

[0037] Prior to this, the terms and words used in this specification and claims should not be interpreted as limited to their conventional or dictionary meanings, and should be interpreted with meanings and concepts that conform to the technical spirit of the present invention based on the principle that the inventor can appropriately define the concept of the term to best explain his or her own invention. Therefore, the configurations described in the embodiments described in this specification are merely the most preferred embodiments of the present invention and do not represent all of the technical spirit of the present invention. Therefore, it should be understood that there may be various equivalents and modified examples that can replace them at the time of this application.

[0038] In this specification, when a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless specifically stated otherwise.

[0039] In this specification, singular expressions include plural expressions unless the context clearly indicates otherwise.

[0040] For the properties described in this specification, if the measurement conditions and methods are not specifically described, the properties are measured according to the measurement conditions and methods generally used by those skilled in the art.

[0041]

[0042] Hereinafter, the present invention will be described in detail.

[0043]

[0044] Microbial measurement method using fluorescence

[0045] The present invention provides a method for measuring microorganisms using fluorescence.

[0046] The above method of measuring microorganisms using fluorescence refers to a method of counting the number of microorganisms by determining whether or not fluorescence is emitted, and the sample is not particularly limited as long as it is necessary to count microorganisms in the sample.

[0047] If a sample is stored under harsh conditions or is not specially treated for preservation, it is common for microorganisms to grow naturally. Samples containing carbon-rich rubber, especially latex, which is rich in nutrients such as carbohydrates and lipids, provide an environment conducive to the growth of microorganisms. Therefore, in order to commercialize the final product manufactured from the sample, it is essential to treat the microorganisms present in the sample.

[0048] In the past, microorganisms were counted using the plate counting method using a solid medium for preservative treatment of microorganisms in the sample. The plate counting method is a counting method to solve the problem that the size of existing microorganisms is too small to be observed with the naked eye. In order to solve the problem, rather than observing the collected microorganisms immediately, the microorganisms are cultured for a certain period of time to form colonies, and then the colonies are counted when the colonies can be observed with the naked eye. In order to count microorganisms using the plate counting method, a minimum of 48 hours of culture time must be secured. Therefore, during the culture time, microorganisms in the sample proliferate, and an excessive amount of preservative must be treated compared to the counting result. Therefore, the purity and quality of the sample in the final product are lowered, and there is an uneconomical aspect.

[0049] Accordingly, in order to solve the above-mentioned problem, the present invention provides a method for measuring microorganisms using fluorescence, which enables accurate counting of microorganisms while performing the counting quickly.

[0050]

[0051] In one specific example of the present invention, the measuring method is a method for measuring microorganisms in a sample containing rubber.

[0052] The sample containing the above rubber may be a sample for manufacturing rubber products commonly used in daily life, and for example, may contain latex, etc. in the sample for manufacturing latex gloves, but is not limited thereto.

[0053] In one specific example of the present invention, the rubber is a nitrile rubber.

[0054] In one specific embodiment of the present invention, the rubber is nitrile latex.

[0055] In one specific embodiment of the present invention, the rubber is nitrile butadiene latex.

[0056] In one specific example of the present invention, the sample containing the rubber is a sample for manufacturing latex gloves.

[0057] When the microorganism measurement method using fluorescence of the present invention is applied to a sample containing rubber according to one specific example of the present invention, it is possible to count microorganisms with improved accuracy and speed compared to the measurement method used in the existing technical field.

[0058]

[0059] In one specific example of the present invention, the measuring method is to evaluate the number of microorganisms in a sample by staining the microorganisms with a fluorescent dye.

[0060] The above microorganisms can react with fluorescent dyes to exhibit fluorescence, and may be, but are not limited to, bacteria, fungi, viruses, protozoa, etc.

[0061] In one specific embodiment of the present invention, the microorganism is selected from the group consisting of Alcaligenes genus, Acinetobacter genus, Azospirillum genus, Azotobacter genus, Bacillus genus, Bdellovibrio genus, Caulobacter genus, Chromobacterium genus, Clostridium genus, Comamonas genus, Corynebacterium genus, Enterobacter genus, Escherichia genus, Halobacterium genus, Helicobacter genus, Klebsiella genus, Lactobacillus genus, Legionella, Listeria, Methylobacterium, Methylosinus, Micrococcus, Mycobacterium, Neisseria, Nitrobacter, Nocardia, Photobacterium, Proteus, Pseudomonas, Ralstonia, Rhizobium, Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Salmonella, Serratia, Shigella, Staphylococcus, Stenotrophomonas, Streptomyces,It is selected from the group consisting of the genus Thiobacillus and combinations thereof.

[0062] In one specific embodiment of the present invention, the microorganism is selected from the group consisting of bacteria, fungi, and combinations thereof.

[0063] The above bacteria can also react with fluorescent dyes to exhibit fluorescence, and include Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia, Pluralibacter gergoviae, Streptococcus mutans, Propionibacterium acnes, Staphylococcus epidermidis, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter gergoviae, Pseudomonas mendocina, It may be selected from the group consisting of, but is not limited to, Pseudomonas stutzeri, Pseudomonas fluorescens, Pseudomonas putida, Alcaligenes faecalis, Ochrobactrum tritici, Kocuria rhizophila, Rhizobium radiobacter, Comomonas aquatic, Sphingobium yanoikuyae, Thauera mechernichensis, and combinations thereof.

[0064] The above fungi include yeasts or molds, and likewise, can react with fluorescent dyes to exhibit fluorescence, such as Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Yarrowia lipolytica, Cryptococcus neoformans, Cryptococcus gattii, Rhodotorula mucilaginosa, and Saccharomyces. It can be a yeast such as Saccharomyces cerevisiae or a mold such as Aspergillus brasiliensis, Aspergillus flavus, Penicillium camemberti, Penicillium bilaiae, Penicillium chrysogenum, Penicillium claviforme, Penicillium crustosum, Penicillium roqueforti, Penicillium expansum, Penicillium puberulum, Penicillium verrucosum, or Penicillium digitatum. It is not limited.

[0065] In one specific embodiment of the present invention, the microorganism comprises a hydrolytic enzyme.

[0066] In one specific embodiment of the present invention, the microorganism comprises an esterase.

[0067] When the microorganism according to the present invention contains a hydrolytic enzyme such as an esterase, fluorescence may be exhibited by reacting with a specific fluorescent dye during the metabolic process of the microorganism, thereby enabling observation of fluorescence in living microorganisms, and thus enabling more rapid counting of microorganisms.

[0068] In one specific embodiment of the present invention, the microorganism is Alcaligenes faecalis, Bacillus amyloliquefaciens, Bacillus circulans, Bacillus coagulans, Bacillus lentus, Bacillus lichenformis, Bacillus megaterium, Bacillus mycoides, Bacillus pumulis, Bacillus sphaericus, Bacillus subtilis, Comamonas terrigena, Enterobacter aerogenes, Enterobacter cloaca. cloacae), Escherichia coli, Lactobacillus casei, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, Proteus mirabilis, Serratia marcescens, and combinations thereof. When the measuring method of the present invention is applied to a sample containing the above microorganisms, a more accurate counting is possible.

[0069] The fluorescent dye can exhibit fluorescence by reacting with microorganisms. For example, a dye that can exhibit fluorescence without accompanying a specific reaction, such as FITC (Fluorescein Isothiocyanate), Propidium Iodide, SYTO 9, Rhodamine green, Texas red, Alexa fluor, etc., or a dye that exhibits fluorescence by participating in a reaction during a metabolic process of microorganisms, such as Calcein AM, Carboxyfluorescein Diacetate (CFDA), etc., can be used. However, there is no particular limitation as long as it is commonly used in the relevant technical field.

[0070] In one specific embodiment of the present invention, the fluorescent dye is for staining living microorganisms.

[0071] In one specific example of the present invention, the fluorescent dye exhibits fluorescence through a reaction during a metabolic process of a microorganism.

[0072] In one specific example of the present invention, the fluorescent dye is Calcein AM, Fluorescein Diacetate (FDA), Carboxyfluorescein Diacetate (CFDA), Carboxyfluorescein Succinimidyl Ester, CMFDA (CellTracker Green), BCECF-AM (2',7'-Bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein), Calcein blue AM, AMCA-AM (7-Amino-4-Methylcoumarin Acetoxymethyl Ester), DAF-FM Diacetate (4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate), Calcein orange AM, Rhodamine It is selected from the group consisting of Rhodamine 110 Diacetate, Calcein red orange AM, Resazurin (AlamarBlue precursor), CMTPX (CellTracker Red), Rhodamine 123, TO-PRO-3 Iodide, Crystal Violet, DAPI (4',6-diamidino-2-phenylindole), and combinations thereof.

[0073] In one specific example of the present invention, the fluorescent dye reacts with a hydrolytic enzyme contained in the microorganism to exhibit fluorescence.

[0074] In one specific example of the present invention, the fluorescent dye reacts with an esterase contained in the microorganism to exhibit fluorescence.

[0075] When the fluorescent dye according to the present invention reacts with a hydrolytic enzyme such as an esterase contained in a microorganism and exhibits fluorescence, fluorescence observation in living microorganisms is also possible, so that the counting of microorganisms can be performed more quickly.

[0076] In one specific embodiment of the present invention, the fluorescent dye is capable of penetrating the cell membrane of a microorganism.

[0077] The above evaluation is to observe the fluorescence generated by staining the microorganism with a fluorescent dye and count the number of individuals exhibiting the fluorescence.

[0078] The above counting of the number of objects can be performed by directly observing and counting points that exhibit fluorescence, can be performed using a device capable of distinguishing light or color signals, can be performed through software capable of distinguishing light or color signals or a program capable of driving such an algorithm, but is not limited thereto as long as it is a method commonly used in the relevant technical field to distinguish light or color signals, and can be performed by hardware, software, or a combination thereof.

[0079] The above observation of fluorescence can be performed through a device that can clearly distinguish between a point where fluorescence appears and a point where fluorescence does not appear, and is not particularly limited as long as it is commonly used in the relevant technical field.

[0080] In one specific embodiment of the present invention, the population assessment of the microorganism is performed using a fluorescence microscope.

[0081] In one specific example of the present invention, the evaluation of the number of objects is performed using a coefficient formula represented by the following [Formula 1].

[0082] [Formula 1]

[0083] Number of individuals = (Number of individuals showing fluorescence in Col1) - (Number of individuals showing fluorescence in Col2)

[0084] (Here, Col1 and Col2 are different from each other and are electromagnetic waves within the wavelength range of 1 nm to 1000 nm that appear independently due to fluorescence.)

[0085] The above electromagnetic waves refer to those that appear due to fluorescence as described above, and may include, for example, ultraviolet rays, visible light such as green, red, and blue, infrared rays, and far-infrared rays, and the number of individuals exhibiting fluorescence of Col1 is greater than the number of individuals exhibiting fluorescence of Col2.

[0086] In one specific example of the present invention, the fluorescence of Col1 refers to fluorescence that appears when a fluorescent dye reacts with a microorganism, and the fluorescence of Col2 refers to fluorescence that is not fluorescence that appears by a reaction between a fluorescent dye and a microorganism, but rather fluorescence that occurs due to, for example, autofluorescence from another substance or an error that may occur during observation.

[0087] By evaluating the number of individuals using the coefficient expressed by the above [Formula 1], the accuracy of measuring the number of microorganisms can be improved by excluding from the counting factors other than fluorescence that appears due to staining with a fluorescent dye, autofluorescence that occurs from other substances, errors that may occur during observation, etc.

[0088] In one specific embodiment of the present invention, Col1 and Col2 are each independently selected from the group consisting of ultraviolet rays having a wavelength range of 400 nm or less, blue visible light having a wavelength range of 400 nm to 500 nm, green visible light having a wavelength range of 500 nm to 550 nm, yellow visible light having a wavelength range of 550 nm to 590 nm, orange visible light having a wavelength range of 590 nm to 620 nm, red visible light having a wavelength range of 620 nm to 700 nm, and infrared or far infrared light having a wavelength range of 700 nm to 1000 nm, and are electromagnetic waves having different wavelength ranges.

[0089] In one specific example of the present invention, the number of individuals exhibiting Col2 fluorescence, which is subtracted from the number of individuals exhibiting Col1 fluorescence, is the number of individuals observed at the same location as the individuals exhibiting Col1 fluorescence among the individuals exhibiting Col2 fluorescence. Even at the location where Col1 fluorescence appears, the fluorescence may be due to other factors such as autofluorescence rather than a reaction between a microorganism and a fluorescent dye. In this case, since the substance present at the location exhibits fluorescence having a different wavelength range as well as Col1 fluorescence, if fluorescence having a different wavelength range, here Col2 fluorescence, appears in the same manner at the location where Col1 fluorescence appears, it is excluded during counting.

[0090] In one specific example of the present invention, the object exhibiting fluorescence of Col2 is observed at the same location as the object exhibiting fluorescence of Col1.

[0091] In one specific embodiment of the present invention, the evaluation comprises the steps of (1) loading dyed microorganisms onto a sample tray and (2) evaluating the number of individuals of the loaded microorganisms.

[0092] In one specific embodiment of the present invention, the evaluation of the number of microorganisms includes the steps of (S1) capturing a fluorescent image of the loaded microorganisms and (S2) counting the number of microorganisms from the captured image.

[0093] The counting of the above microbial population is the same as described above, and the counting from the image can be performed using software such as Gwyddion software, Mountains SEM software, ImageJ, or an algorithm used for image analysis such as DX image processing, but is not limited thereto as long as it is commonly used in the relevant technical field, and can be performed by hardware, software, or a combination thereof.

[0094] In one specific embodiment of the present invention, the time required from the collection of the sample to the evaluation of the number of individuals is 0.1 to 6 hours. Specifically, the time required is 0.1 hour or more, 0.2 hours or more, 0.3 hours or more, 0.4 hours or more, 0.5 hours or more, 1 hour or more, or 1.5 hours or more, and 6 hours or less, 5.5 hours or less, 5 hours or less, 4.5 hours or less, 4 hours or less, 3.5 hours or less, 3 hours or less, 2.5 hours or less, or 2 hours or less, and may be 0.1 to 6 hours, 0.3 to 5 hours, or 0.5 to 3 hours. When the time required satisfies the above range, microbial measurement can be performed quickly while ensuring reliable accuracy.

[0095] The time required from the collection of the above sample to the evaluation of the number of individuals means the time required from the collection of the sample to the counting of the number of individuals after the staining of the microorganisms without delay due to factors other than the process that is essential for the measurement and the measurement.

[0096]

[0097] In one specific example of the present invention, the staining includes (1) a step of introducing the fluorescent dye into the microorganism and (2) a step of culturing the microorganism to which the fluorescent dye has been introduced.

[0098] The dye in the above step (1) can be added in an amount suitable for dyeing all microorganisms expected to exist in the sample, and a person skilled in the art can appropriately adjust the amount of the dye added according to the characteristics of the material in the sample, the storage environment, etc.

[0099] In one specific embodiment of the present invention, the volume ratio of the sample containing the microorganism and the fluorescent dye introduced is 1:1 to 20:1. Specifically, the volume ratio is 1:1 or more, 2:1 or more, 3:1 or more, 4:1 or more, or 5:1 or more, and 20:1 or less, 19:1 or less, 18:1 or less, 17:1 or less, 16:1 or less, 15:1 or less, 14:1 or less, 13:1 or less, 12:1 or less, 11:1 or less, 10:1 or less, 9:1 or less, 8:1 or less, 7:1 or less, or 6:1 or less, and may be 1:1 to 20:1, 2:1 to 15:1, or 5:1 to 10:1. When the volume ratio satisfies the above range, the dye may be introduced in an amount suitable for staining microorganisms present in the sample, particularly, the sample containing latex. In addition, the volume ratio of the sample containing the microorganism and the fluorescent dye injected means the volume ratio of the sample containing the microorganism immediately before the fluorescent dye is injected.

[0100] In one specific embodiment of the present invention, the culturing is performed for 0.1 to 3 hours. Specifically, the culturing is performed for 0.1 hours or more, 0.2 hours or more, 0.3 hours or more, or 0.4 hours or more, and is performed for 3 hours or less, 2.5 hours or less, 2 hours or less, 1.5 hours or less, 1 hour or less, or 0.5 hours or less, and may be performed for 0.1 to 3 hours, 0.2 to 2 hours, or 0.3 to 1 hour.

[0101] In one specific embodiment of the present invention, the culturing is performed at 5°C to 50°C. Specifically, the culturing is performed at 5°C or higher, 10°C or higher, 15°C or higher, 20°C or higher, 25°C or higher, 30°C or higher, or 35°C or higher, and 50°C or lower, 45°C or lower, or 30°C or lower, and at 5°C to 50°C, 20°C to 50°C, or 30°C to 40°C.

[0102] When the above-mentioned incubation time and incubation temperature satisfy the above-mentioned range, the microorganism can react with the fluorescent dye to exhibit fluorescence, and at the same time, the time required for counting microorganisms can be shortened so that rapid measurement of microorganisms can be performed.

[0103]

[0104] In one specific embodiment of the present invention, the measuring method includes a step of washing the microorganism before staining.

[0105] The above washing step is a step to reduce factors that may affect the staining of microorganisms and to increase the purity of microorganisms in the sample so that the staining of microorganisms can be performed well when a fluorescent dye is added to the sample. There are no particular limitations on the washing method as long as it is a washing method commonly used in the relevant technical field.

[0106] In one specific embodiment of the present invention, the washing is performed by adding a dilution buffer to the sample.

[0107] The above dilution buffer is a substance that serves to balance osmotic pressure to prevent cell death of microorganisms, adjust pH, or increase the permeability of the cell wall or cell membrane of microorganisms for the introduction of fluorescent dyes, and is not limited to those commonly used in the relevant technical field to achieve the above purpose.

[0108] In one specific embodiment of the present invention, the dilution buffer comprises one selected from phosphate buffered saline (PBS), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), sodium chloride (NaCl), potassium chloride (KCl), Tween-20, Triton X-100, bovine serum albumin, glucose, EDTA (Ethylenediaminetetraacetic acid), and combinations thereof.

[0109] In one specific embodiment of the present invention, the dilution buffer comprises sodium chloride.

[0110] In one specific embodiment of the present invention, the dilution buffer is added in an amount of 100% to 3000% by volume based on the sample volume present before the dilution buffer is added. Specifically, the dilution buffer is added in an amount of 100% to 3000% by volume, 500% to 1000% by volume, or 1000% to 1000% by volume, and is added in an amount of 3000% to 2500% to 2000% to 1500% by volume, and may be added in an amount of 100% to 3000% by volume, 100% to 2000% to 500% to 1500% by volume. When the amount of the dilution buffer added satisfies the above range, dilution can be performed more effectively.

[0111] When the above dilution buffer contains a substance according to one specific example of the present invention at a specific concentration, microorganisms in a sample, particularly a sample containing latex, can be easily separated from other substances, and staining can be effectively performed by adding a fluorescent dye, thereby improving the accuracy of microbial counting.

[0112] The above washing is intended to increase the purity or concentration of microorganisms in a sample for effective staining of microorganisms, and can be applied to any method commonly used in the relevant technical field to independently extract microorganisms in a sample, for example, centrifugation, freeze-drying, etc., but is not limited thereto.

[0113] In one specific embodiment of the present invention, the washing is performed through centrifugation.

[0114] In one specific embodiment of the present invention, the centrifugation for the washing is performed for 1 minute to 10 minutes. Specifically, the centrifugation is performed for 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, or 5 minutes or more, and for 10 minutes or less, 9 minutes or less, 8 minutes or less, 7 minutes or less, or 6 minutes or less, and may be performed for 1 minute to 10 minutes, 2 minutes to 10 minutes, or 2 minutes to 8 minutes.

[0115] In one specific embodiment of the present invention, the centrifugation for the washing may be performed 1 to 10 times. Specifically, the centrifugation may be performed 1 or more times, 2 or more times, or 3 or more times, and 10 or less times, 9 or less times, 8 or less times, 7 or less times, 6 or less times, 5 or less times, or 4 or less times, and may be performed 1 to 10 times, 1 to 8 times, or 2 to 4 times.

[0116] When the above centrifugation satisfies the above performance time and number of repetitions, microorganisms can be effectively separated from the sample, and the microorganisms can be effectively stained when stained with a fluorescent dye thereafter, thereby improving the speed and accuracy of microorganism measurement.

[0117] In one specific embodiment of the present invention, the evaluation comprises the steps of (1) loading dyed microorganisms onto a sample tray and (2) evaluating the number of individuals of the loaded microorganisms.

[0118] The above loading refers to the process of uniformly spreading the dyed microorganisms on the sample bed so that the fluorescence of the dyed microorganisms can be measured without omission across the entire sample.

[0119] In one specific embodiment of the present invention, the loading is performed by adding a loading buffer to the stained microorganism in the sample.

[0120] The above loading buffer is a material that prevents a change in the dye for a certain period of time for counting the number of microorganisms, stabilizes a fluorescent signal emitted due to a reaction between the microorganisms and the fluorescent dye, uniformly spreads the sample on the sample stage, or stabilizes the adsorption of the sample and the sample. The material is not limited to those commonly used in the relevant technical field to achieve the above purpose.

[0121] In one specific embodiment of the present invention, the loading buffer comprises a buffer selected from phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride (NaCl), potassium chloride (KCl), Tween-20, Triton X-100, poly-L-lysine, gelatin, agarose, ascorbic acid, Trolox, glycerol, sodium azide, and combinations thereof.

[0122] In one specific embodiment of the present invention, the loading buffer comprises one selected from glycerol, sodium azide, and combinations thereof.

[0123] In one specific embodiment of the present invention, the loading buffer is added in an amount of 10% to 150% by volume based on the sample volume present before the loading buffer is added. Specifically, the loading buffer is added in an amount of 10% to 150% by volume, 20% to 30% to 50% to 60% to 70% by volume, and is added in an amount of 150% to 140% to 130% to 120% to 110% to 100% to 90% to 80% by volume. When the amount of the loading buffer added satisfies the above range, loading can be performed more effectively.

[0124] Below, specific embodiments of the present invention are presented. However, the embodiments described below are intended solely to specifically illustrate or explain the present invention and are not intended to limit the scope of the invention. Furthermore, any details not described herein are technically feasible to those skilled in the art and thus are omitted.

[0125]

[0126] Example

[0127]

[0128] <Example>

[0129] (1) Washing

[0130] A sample was collected from the nitrile butadiene rubber raw material used in latex production, diluted in sterile water, and the diluted sample was separated using a centrifuge. The separation was performed three times for 5 minutes each, and then the supernatant was removed and a dilution buffer containing sodium chloride was added to the lower layer at 1000% by volume based on the sample volume present before adding the buffer, thereby washing the sample.

[0131] (2) Dyeing

[0132] To evaluate the number of living Alcaligenes faecalis in the washed solution, the fluorescent dye calcein acetoxymethyl (Calcein AM) was added at a volume ratio of the washed solution to the dye of 10:1, and then cultured at 36°C for 30 minutes.

[0133] (3) Loading

[0134] A loading buffer containing glycerol and sodium azide was added to the cultured solution at 80% by volume of the sample volume present before adding the buffer, and then a small amount was loaded into a slide chamber for evaluation of the number of individuals.

[0135] (4) Evaluation

[0136] The above slide chamber was placed on the stage of a fluorescence microscope to observe whether it fluoresced, and the number of microorganisms in the sample was evaluated using the following [Formula 1]. The images of green fluorescence and red fluorescence emission, respectively, are shown in [Figure 1] and [Figure 2].

[0137] [Formula 1]

[0138] Number of individuals = (Number of individuals showing Col1 fluorescence) - (Number of individuals showing Col2 fluorescence)

[0139] In the embodiment, the Col1 is a green visible light having a wavelength range of 500 nm to 550 nm among electromagnetic waves appearing due to fluorescence, and the Col2 is a red visible light having a wavelength range of 620 nm to 700 nm among electromagnetic waves appearing due to fluorescence.

[0140] The number of individuals exhibiting Col2 fluorescence in the above counting was calculated as the number of individuals exhibiting Col2 fluorescence at the same location as individuals exhibiting Col1 fluorescence, as shown in [Figure 1] and [Figure 2].

[0141] It took approximately 1.5 hours from sample collection to population assessment.

[0142]

[0143] <Comparative Example 1>

[0144] The number of individuals was measured using the conventional plate counting method. For this purpose, the sample was placed on a Tryptic Soy Agar (TSA) medium, cultured at 30°C for 48 to 72 hours, and the number of colonies observed was counted to evaluate the number of individuals.

[0145]

[0146] <Comparative Example 2>

[0147] Microorganisms were measured in the same manner as in Example 1, except that SYTO 9 was used as a fluorescent dye. The image of green fluorescence emission is shown in [Figure 3].

[0148]

[0149] Experimental example

[0150]

[0151] <Experimental Example: Accuracy Analysis>

[0152] Based on the measurement results of the above Comparative Example 1, the accuracy of the results of the microbial population measured by the Examples and Comparative Examples was analyzed. The measurement methods of the Examples and Comparative Examples were performed on a total of 47 samples, and the measurement results of Comparative Example 1 were subjected to linear regression analysis in units of log CFU / ml, and the results of the Examples were plotted against the analyzed baseline. The results of the comparison are shown in [Figure 4].

[0153]

[0154] As shown in [Figure 4], in the case of an example in which the number of microorganisms in a sample including rubber was evaluated by staining the microorganisms with a fluorescent dye as a method for measuring microorganisms, it can be confirmed that the results are almost similar to those of Comparative Example 1 in which the number of microorganisms was evaluated using a plate counting method conventionally used for counting microorganisms.

[0155] Through this, it can be seen that in the case of a measurement method for measuring the number of microorganisms using fluorescence, the microbial culture time, etc. can be shortened, enabling a much faster counting than the conventional plate counting method that requires a culture time of at least 48 hours, and at the same time, it can exhibit an accuracy level equivalent to that of the conventional plate counting method.

[0156] In addition, as shown in [Figure 1] and [Figure 3], in Comparative Example 2 using SYTO 9, a fluorescent dye that performs non-specific staining, it can be confirmed that even dead microorganisms in the sample are non-specifically stained when compared to the example using calcein AM, a fluorescent dye for staining living microorganisms.

[0157] Through this, it can be seen that when using fluorescent dyes to stain living microorganisms, there is a concern that the quality of the final product may be lowered, etc., and thus, the accuracy of microbial counting can be improved by enabling specific counting of microorganisms that require processing within the sample, and the ease and economy of processing can be increased.

[0158] Therefore, as a method of measuring microorganisms in a sample containing rubber, it can be seen that by staining the microorganisms with a fluorescent dye and evaluating the number of microorganisms in the sample, the measurement time for the number of microorganisms can be significantly shortened, allowing for a count of microorganisms that is close to real time and also highly accurate.

[0159] In addition, when using fluorescent dyes to stain living microorganisms with fluorescent dyes, specific counts of microbial individuals that may affect actual product quality can be made, which can further increase the accuracy of measurement and improve the efficiency of the processing process.

[0160]

[0161] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

Claims

1. A method for measuring microorganisms in a sample containing rubber, It is an evaluation of the number of microorganisms in a sample by staining the microorganisms with a fluorescent dye. Microbial measurement method using fluorescence.

2. In claim 1, The above rubber is a nitrile rubber, Microbial measurement method using fluorescence.

3. In claim 1, The above fluorescent dye is for staining living microorganisms. Microbial measurement method using fluorescence.

4. In claim 1, The above dyeing is, (1) a step of injecting the fluorescent dye into the microorganism; and (2) A step of culturing microorganisms to which the fluorescent dye has been added; which includes, Microbial measurement method using fluorescence.

5. In claim 4, The above culture is performed for 0.1 to 3 hours, Microbial measurement method using fluorescence.

6. In claim 1, The above measurement method includes a step of washing the microorganism before staining. Microbial measurement method using fluorescence.

7. In claim 6, The above washing is performed by adding a dilution buffer containing sodium chloride to the sample. Microbial measurement method using fluorescence.

8. In claim 1, The above evaluation is, (1) a step of loading the dyed microorganisms onto the sample tray; and (2) A step of evaluating the number of individuals of the loaded microorganisms; which includes, Microbial measurement method using fluorescence.

9. In claim 1, The above microorganism contains a hydrolytic enzyme, Microbial measurement method using fluorescence.

10. In claim 1, The above microorganism contains hydrolytic enzymes, The fluorescent dye reacts with the hydrolytic enzyme contained in the microorganism to exhibit fluorescence. Microbial measurement method using fluorescence.

11. In claim 1, The above microorganism belongs to the genus Alcaligenes. Microbial measurement method using fluorescence.

12. In claim 1, The above microbial population assessment is performed using a fluorescence microscope. Microbial measurement method using fluorescence.

13. In claim 1, The evaluation of the above number of objects is performed through a coefficient formula represented by the following [Formula 1]. Microbial measurement method using fluorescence. [Formula 1] Number of individuals = (Number of individuals showing fluorescence in Col1) - (Number of individuals showing fluorescence in Col2) (Here, Col1 and Col2 are different from each other and are electromagnetic waves within the wavelength range of 1 nm to 1000 nm that appear independently due to fluorescence.) 14. In claim 1, The time required from the collection of the above sample to the evaluation of the number of individuals is 0.1 to 6 hours. Microbial measurement method using fluorescence.

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