Microorganism testing method and pretreatment container for microorganism testing

Abstract

The present disclosure pertains to a microorganism testing method includes an adsorption step for adsorbing microorganisms in a liquid sample to particles, a seeding step for seeding the particles to which the microorganisms are adsorbed in a culture medium, and a culture step for culturing the microorganisms on the culture medium.

Description

Microbiological testing method and pretreatment container for microbiological testing

[0001] The present invention relates to a microbiological testing method and a pretreatment container for microbiological testing.

[0002] In infectious disease diagnosis, environmental testing, and food microbiology testing, there is a need for a rapid and simple method to detect, identify, and / or enumerate pathogens or harmful microorganisms that impair quality in samples. For example, in infectious disease diagnosis, point-of-care (POCT) tests are used to identify pathogenic bacteria or viruses so that doctors and nurses can make appropriate clinical decisions at the patient's side. In environmental testing and food microbiology testing, tests are conducted to detect environmental indicator bacteria such as common bacteria and coliform bacteria, as well as harmful microorganisms such as food poisoning bacteria and viruses, for employee or environmental hygiene management and quality control of raw materials, intermediate products, and final products. Small-scale businesses and on-site testing require simple operating methods that do not require specialized equipment or knowledge of microbiology.

[0003] Testing methods for these microorganisms can be broadly divided into culture methods, which identify the microorganisms based on their biochemical properties after isolation and culture; genetic methods, which amplify and detect the microorganism's specific genes using PCR or LAMP (Likely amplified polymorphism); and immunoassays, which detect the microorganisms by utilizing the specific reaction between antigen markers specific to the microorganism and antibodies. Among these methods, culture methods are widely used due to their sensitivity and simplicity. Culture methods include standard methods, which use agar media or other media optimized for each target microorganism to form colonies and then count them; and rapid microbial culture testing methods, which use sheet or film media developed specifically for microbial testing. Each test method utilizes a small sample volume, approximately 0.01 to 1 mL, which limits the amount of detectable material (bacteria or viruses themselves, or their surface or internal components) contained in the approximately 0.01 to 1 L of patient specimens (e.g., urine or body fluids), food samples (e.g., liquid samples or bacterial or viral extracts obtained using a stomacher), or environmental samples (e.g., water quality). This limits the detection accuracy and sensitivity of microbial testing. To solve this problem and ensure sufficient detection material without using large-scale equipment, concentration is performed by adsorbing, separating, and recovering microorganisms using an adsorption carrier.

[0004] For example, in Patent Document 1 below, E. coli is adsorbed by shaking inorganic microparticles (amorphous metal silicate) and a sample for 1 to 20 minutes. However, the particles described in Patent Document 1 require shaking for 10 minutes or more to achieve an adsorption rate of 50% or more, making rapid concentration difficult. Furthermore, a large amount of inorganic microparticles (10 mg) is required for 10 mL of E. coli sample containing 1e3 cfu / mL. Furthermore, the detection method described in Patent Document 1 destroys the adsorbed microorganisms by contacting them with an extract, releasing the analyte inside the microorganisms, which is then detected to indirectly quantify the amount of microorganisms. This method requires the released analyte to be separated from the microparticles, which leads to problems such as reduced release efficiency and reduced sensitivity due to nonspecific adsorption.

[0005] Furthermore, Patent Document 2 below describes that porous cellulose microparticles modified with polycations can be used industrially as microcarriers for culturing bacteria, yeast, and animal and plant cells, but this is based on the premise of cell suspension culture, and it is difficult to use it directly for microbial testing.

[0006] Patent No. 5972174 Publication JP-A-4-91142

[0007] In view of the current state of the prior art described above, the problem to be solved by the present invention is to provide a simple and highly sensitive microorganism testing method and a pretreatment container for microorganism testing suitable for the method.

[0008] As a result of extensive research and experiments to solve this problem, the inventors unexpectedly discovered that the problem could be solved by adsorbing microorganisms in a sample onto particles, and then seeding and culturing the particles with the adsorbed microorganisms in a culture medium, which led to the completion of the present invention.

[0009] That is, the present invention is as follows. [1] A microbiological testing method comprising the following steps: an adsorption step of adsorbing microorganisms in a liquid sample to particles; a seeding step of seeding the particles with adsorbed microorganisms onto a culture medium; and a culture step of culturing the microorganisms on the culture medium. [2] The microbiological testing method according to [1], further comprising a counting step of counting colonies after the culture step. [3] The microbiological testing method according to [1] or [2], wherein the culture medium is a flat membrane culture medium, a sheet culture medium, or a film culture medium. [4] The microbiological testing method according to any one of [1] to [3], wherein in the seeding step, particles are seeded onto the culture medium using a liquid. [5] The microbiological testing method according to [4], wherein the amount of liquid used to seed the particles in the seeding step is less than the amount of liquid sample in the adsorption step. [6] The microbiological testing method according to any one of [1] to [5], wherein the microorganism concentration in the liquid sample is 100 cfu / mL or less. [7] The microbiological testing method according to any one of [1] to [6], wherein the particles are made of cellulose. [8] The microorganism testing method according to any one of [1] to [7], wherein the particles are porous particles. [9] The microorganism testing method according to [8], wherein the porous particles have a large number of pores therein and have a continuous pore structure in which adjacent pores are connected to each other by openings in a membrane that separates them.

[10] The microorganism testing method according to [9], wherein the porous particles have an average pore size of 5 μm or more and 100 μm or less.

[11] The microorganism testing method according to any one of [1] to

[10] , wherein the particles have an average particle size of 1000 μm or less.

[12] The microorganism testing method according to any one of [1] to

[10] , wherein the particles have an average particle size of 0.3 m ２ / g or more 4.4m ２ / g or less.

[13] The microorganism testing method according to any one of [1] to

[12] , wherein the surface of the particles is modified with a cationic substituent.

[14] The microorganism testing method according to any one of [1] to

[13] , wherein the charge capacity of the particles is 0.5 mmol / g or more.

[15] The microorganism testing method according to any one of [1] to

[14] , wherein in the adsorption step, a liquid sample containing microorganisms is passed through a column containing particles.

[16] A cylindrical pretreatment container for microorganism testing, filled with particles and having an inlet opening and an outlet opening, wherein both the inlet opening and the outlet opening are liquid-passable, but a filter is provided near the outlet opening to prevent the passage of the particles, and the space above the filter is filled with the particles.

[17] The microorganism testing pretreatment container according to

[16] , wherein the particles are made of cellulose.

[18] A pretreatment container for microbiological testing according to

[16] or

[17] , wherein the particles are porous particles.

[19] A pretreatment container for microbiological testing according to

[18] , wherein the porous particles have a large number of pores therein and have a continuous pore structure in which adjacent pores are connected to each other by openings in a membrane that separates them.

[20] A pretreatment container for microbiological testing according to

[19] , wherein the porous particles have an average pore size of 5 μm or more and 100 μm or less.

[21] A pretreatment container for microbiological testing according to any one of

[16] to

[20] , wherein the particles have an average particle size of 100 μm or more and 1000 μm or less.

[22] A pretreatment container for microbiological testing according to

[18] , wherein the particles have a specific surface area of ​​0.3 m when dry. ２ / g or more 4.4m ２

[23] The pretreatment container for microorganism testing according to any one of

[16] to

[22] , wherein the surface of the particles is modified with a cationic substituent.

[24] The pretreatment container for microorganism testing according to any one of

[16] to

[23] , wherein the charge capacity of the particles is 0.5 mmol / g or more.

[0010] The microorganism testing method according to the present invention does not require the recovery of microorganisms or the destruction of microorganisms to extract antigens, and therefore allows for simple and highly sensitive testing of microorganisms. Furthermore, the pretreatment container for microorganism testing according to the present invention can be suitably used in such a microorganism testing method.

[0011] 2 is an explanatory diagram of a microorganism testing method according to the present embodiment. FIG. 3 is a schematic diagram (on the right hand side of FIG. 2) of a pretreatment container for microorganism testing (reference numeral 1) according to the present embodiment.

[0012] The first embodiment of the present invention is a microorganism testing method including the following steps: an adsorption step of adsorbing microorganisms in a liquid sample onto particles, a seeding step of seeding the particles with the adsorbed microorganisms onto a culture medium, and a culture step of culturing the microorganisms on the culture medium.

[0013] The adsorption step in the microorganism testing method of this embodiment can be performed by any method as long as the particles and the sample containing microorganisms come into contact with each other. For example, it can be performed by passing the sample containing microorganisms through a column packed with particles. Alternatively, it can be performed by adding the sample containing microorganisms to a container packed with particles. In either case, the sample containing microorganisms can be passed through, filled, and then allowed to stand or stirred. Furthermore, these adsorption steps can be repeated multiple times to further increase sensitivity. By adsorbing microorganisms in a liquid sample to particles through these adsorption steps, it is possible to efficiently concentrate microorganisms dispersed in a large amount of sample liquid on the particle surface.

[0014] The seeding step of the microorganism testing method of this embodiment is carried out by seeding particles with adsorbed microorganisms into a medium. At this time, there is no need to detach the microorganisms from the particles; the particles with adsorbed microorganisms can be directly seeded into the medium. There are no particular limitations on the seeding method, but one example is a method in which particles with adsorbed microorganisms are extruded from a column filled with particles with adsorbed microorganisms using physiological saline or the like to seed the particles into the medium. When seeding is carried out using this method, a filter such as a frit is placed only at the outlet of the column, and no filter is placed on the inlet side, and physiological saline or the like is passed through the outlet to extrude the particles with adsorbed microorganisms from the inlet. For example, a cylindrical microorganism testing pretreatment container having an inlet opening and an outlet opening and filled with particles, both of which allow liquid to pass through, is characterized by having a filter near the outlet opening that prevents the particles from passing through, and the particles being filled in the space above the filter. A liquid sample containing microorganisms can be passed through the inlet opening side (from above), and then the container can be turned upside down to allow the particles to fall through the now-down inlet opening. Alternatively, the container containing the particles with adsorbed microorganisms and the sample liquid can be left to stand or centrifuged to precipitate the particles, and then the particles alone can be removed using a pipette or the like for seeding.

[0015] The particles with adsorbed microorganisms may be seeded in a culture medium using a liquid. As described above, particles in a column may be seeded by pushing them out by passing a liquid such as physiological saline through the column, and particles that have settled in a container may be removed together with the liquid and seeded. The amount of liquid used to seed the particles is preferably less than the amount of liquid sample used in the adsorption step. For example, when the amount of liquid sample used in the adsorption step is 10 mL to 1000 mL, 1 mL of liquid can be used for seeding in the seeding step. By performing the seeding step with a liquid volume smaller than the amount of sample liquid (amount of liquid sample) before adsorption, the amount of seeded microorganisms in the seeding liquid can be suitably increased, which is likely to be advantageous from the perspective of simple and highly sensitive microorganism testing. The concentration ratio (volume of sample liquid before adsorption / volume of seeding liquid) may be more than 1x, 10x or more, 100x or more, or 1000x or more. However, a concentration ratio that allows easy counting of colonies after the culture step is preferably a concentration ratio that results in the amount of seeded microorganisms in the seeding liquid being 100 cfu / mL or less.

[0016] In the culture step of the microorganism testing method of this embodiment, microorganisms associated with particles are cultured on a medium. Microorganisms are concentrated on particles in the adsorption step, and then the microorganisms associated with the particles are seeded and cultured in the subsequent seeding and culture steps, enabling testing with higher sensitivity than conventional methods. Furthermore, post-concentration processing, specifically, steps such as recovering microorganisms from the concentration medium or destroying the microorganisms to extract antigens, is not required, and microorganisms can be tested simply and with high sensitivity while reducing the influence of culture inhibitors in the sample solution.

[0017] In the microorganism testing method of this embodiment, a draining step of removing the remaining solution can be included after the adsorption step. The draining step can be performed by any method that can remove the remaining liquid, for example, by aerating the column after passing the microorganism-containing sample through it. It is also possible to remove the supernatant from a container filled with particles to which microorganisms are adsorbed by filtration or suction of the liquid. By performing the draining step, it is possible to reduce the influence of the remaining solution, which is likely to be advantageous from the viewpoint of highly sensitive microorganism testing.

[0018] The microbial testing method of this embodiment can include a washing step for separating inhibitory substances and the like after the adsorption step. The washing step can be performed by any method capable of separating the particles with adsorbed microorganisms from the inhibitory substances. For example, the washing step can be performed by passing a liquid that does not inhibit microbial activity, such as physiological saline, through the column after passing the microorganism-containing sample through the column. Alternatively, the washing step can be performed by removing the supernatant from a container filled with particles with adsorbed microorganisms, adding physiological saline, stirring, and removing the supernatant. Inhibitory substances include any substance that affects culture, including, but not limited to, salts, sugars, acids and bases, catechins, parabens, and other preservatives. Performing the washing step can remove inhibitory substances from culture, which is advantageous in terms of highly sensitive testing of microorganisms while reducing the influence of inhibitory substances.

[0019] The upper limit of the microbial concentration in the liquid sample tested by the microbial testing method of this embodiment is preferably 100 cfu / mL or less, more preferably 50 cfu / mL or less, and even more preferably 10 cfu / mL or less. Samples of the above concentrations tend to have large variations in evaluation and are affected by sampling when used with a typical sheet culture medium. However, the microbial testing method of this embodiment is advantageous in terms of high sensitivity even at the above concentrations, and the effect of sampling can be easily reduced. When the seeding step is performed with a liquid volume smaller than the sample liquid volume (liquid sample volume) before adsorption, the upper limit of the microbial concentration may be 5 cfu / mL or less, 1 cfu / mL or less, 0.1 cfu / mL or less, or 0.01 cfu / mL or less. The lower limit of the microbial concentration in the liquid sample tested by the microbial testing method of this embodiment may be greater than 0 cfu / mL.

[0020] Although there are no particular limitations on the type of particles used in this embodiment, polysaccharides such as cellulose are preferred. Cellulose does not inhibit culture for microbial testing. Furthermore, surface modification is easy, allowing for the formation of a surface suitable for uniform microbial adsorption. In the present specification, the term "surface" includes, in the case of porous particles, both the outer surface and the inner surfaces of the pores within the porous particles.

[0021] The type of cellulose is not particularly limited, but known celluloses such as cuprammonium regenerated cellulose, viscose regenerated cellulose, cotton, pulp, and polynosic are used, preferably cuprammonium regenerated cellulose and viscose regenerated cellulose, and more preferably cuprammonium regenerated cellulose. Cuprammonium regenerated cellulose has a low degree of crystallinity, so it is highly reactive when modified with various functional groups, and even porous particles can be homogeneously modified all the way to the inside of the particles, and the particles also have excellent morphological stability.

[0022] The particles used in the microorganism testing method of this embodiment have a shape that allows them to be suitably dispersed in a sample liquid, such as spheres, ellipsoids, rods, fibers, pulverized particles, etc. These structures allow for efficient contact with the sample liquid and efficient adsorption of microorganisms.

[0023] The particle shape of this embodiment is preferably a porous particle, which has a large number of pores inside and a continuous pore structure in which adjacent pores are connected to each other by openings in the membrane that separate them. Porous particles have a large surface area and can efficiently adsorb microorganisms in a sample. Furthermore, when the particles are packed into a column, the packing density within the column is increased, making it possible to suppress a decrease in adsorption efficiency due to a short path. Furthermore, particles with a continuous pore structure allow the sample to easily diffuse into the interior of the particles, making it possible to efficiently adsorb microorganisms even to the interior of the particles, and furthermore, when the particles are packed into a column, pressure loss during liquid passage can be reduced.

[0024] When the particles used in the microorganism testing method of this embodiment are porous particles, the lower limit of the average pore size of the pores is preferably 5 μm or more, more preferably 10 μm or more. By setting the average pore size to 5 μm or more, the degree of freedom of movement of microorganisms, sample solutions, and culture solutions for microorganism testing within the particles is increased, allowing microorganisms to enter the interior of the particles and efficiently adsorb and grow the microorganisms. The upper limit of the average pore size is preferably 100 μm or less, more preferably 80 μm or less, and even more preferably 50 μm or less. When the average pore size is less than 100 μm, a sufficient surface area for microbial growth can be ensured, allowing the microorganisms to grow efficiently. In this specification, the term "average pore size" refers to the maximum value of the pore size distribution determined by mercury intrusion porosimeter after solvent substitution of porous particles with water, ethanol, and t-butanol in that order, followed by freeze-drying to remove the remaining excess solvent.

[0025] The upper limit of the average particle size of the particles used in the microorganism testing method of this embodiment is preferably 1000 μm or less, more preferably 500 μm or less, and even more preferably 400 μm or less. An average particle size smaller than 1000 μm allows efficient adsorption of microorganisms to the center of the particles. Furthermore, since the gaps between particles can be minimized when packed into a column, a decrease in adsorption efficiency due to short paths can be suppressed, and further, microorganisms can be uniformly seeded on the surface of the medium during culture. As used herein, the term "average particle size" refers to the average particle size measured by swelling porous particles with water, removing excess water, and measuring 20 or more particles using an optical microscope at an appropriate magnification. A specific swelling process for porous particles involves immersing 1 mg of porous particles in 10 ml of physiological saline for half a day to allow swelling, and then filtering and removing excess water using an empty column (BIO-RAD, 7311550) until the particles fall under their own weight.

[0026] The lower limit of the surface area (specific surface area) per 1 g of particles used in the microorganism testing method of this embodiment when dried is 0.3 m ２ / g or more, more preferably 0.6m ２ / g or more, and more preferably 0.8m ２The surface area required for culturing microorganisms is 0.3 m / g or more from the viewpoint of efficient adsorption of microorganisms. ２ The upper limit of the specific surface area is preferably 4.4 m / g or more. ２ / g or less, and ２ / g or less is more preferable, and 2.0m ２ / g or less is more preferable, and 1.7m ２ From the viewpoint of preventing a decrease in the adhesion rate of microorganisms without decreasing the charge amount per unit area, the specific surface area is preferably 4.4 m ２ In this specification, the term "specific surface area" refers to the surface area per 1 g of dry weight measured by the BET method.

[0027] The particles used in the microorganism testing method of this embodiment are preferably modified with a cationic substituent, which is preferably at least one selected from the group consisting of a primary amino group, a secondary amino group, and a tertiary amino group, more preferably a primary amino group or a tertiary amino group, and even more preferably a diethylaminoethyl group.

[0028] The charge capacity of the particles used in the microbial testing method of this embodiment must take into account the influence of functional groups such as hydroxyl groups of the substrate itself, and the fact that the optimal charge range for good adhesion of microorganisms and proliferation in the microbial testing medium varies. The lower limit of the charge capacity of the particles used in the microbial testing method of this embodiment is preferably 0.1 mmol / g or more, more preferably 0.5 mmol / g or more. A charge capacity of more than 0.1 mmol / g enables efficient adsorption and concentration of microorganisms with short contact with the sample liquid, enabling rapid and highly sensitive microbial testing. The upper limit of the charge capacity of the particles is preferably 3.5 mmol / g or less, more preferably 3.0 mmol / g or less. A charge capacity of 3.5 mmol / g or less allows microorganisms to form colonies favorably in the medium, thereby preventing colony counting and reducing false negatives during microbial counting. In this specification, the term "charge capacity" refers to an evaluation method in which chloride ions are adsorbed to the amine in the porous particles using hydrochloric acid, and the adsorbed chloride ions are then washed away with a sodium sulfate solution, and the chloride ion concentration of the resulting solution is titrated with silver nitrate using potassium chromate as an indicator, and the average value of measurements taken on three or more samples is taken as the charge capacity.

[0029] [Charge Capacity Measurement] 5.0 g of porous particles were weighed and washed twice with 30 mL of distilled water using a funnel and suction filter, followed by water replacement. The particles were then washed twice with 30 mL of 0.3 N hydrochloric acid and four times with 50 mL of 10-4 N hydrochloric acid. Then, using a funnel and suction filter, the particles were washed four times with 50 mL of 10% sodium sulfate aqueous solution, and the filtrate was recovered. The recovered filtrate was then added with 2 mL of 0.1 N potassium chromate aqueous solution and stirred. The sample was then titrated with a 1 N silver nitrate aqueous solution, and the titer A (mL) at the moment when the reddish-brown precipitate of silver chromate began to form was determined. Separately, a sample that had been washed with 10% sodium sulfate aqueous solution in the above-mentioned process was washed four times with 50 mL of pure water, dried at 105°C for at least 15 hours, and weighed on a balance to determine the dry weight B (g). The charge capacity is calculated using the titer A (mL) measured above and the dry weight B (g) of the sample according to the following formula (1) (N=3): Charge capacity (mmol / g) = 1 (N) (mol / L) × A (mL) / B (g) (1)

[0030] The particles used in the microorganism testing method of this embodiment can be produced, for example, by a method in which a solution in which cellulose has been dissolved is formed into a desired shape while being cooled to a temperature below the solidification temperature of the solution to freeze it, and then the solvent is extracted and removed or the dissolving ability is lost. In general, the freezing temperature is preferably not set to be more than 40°C lower than the freezing temperature of the solvent, etc., and is usually selected in the range of 0 to 20°C lower than the freezing temperature.

[0031] The frozen cellulose solution or cellulose derivative solution is then subjected to extraction and removal of the solvent dissolving the cellulose or cellulose derivative, or to reducing its dissolving power (hereinafter, these processes are collectively referred to as "solvent removal, etc."), resulting in a solidified porous cellulose body. The conditions for solvent removal, etc., are not particularly limited. Typically, the frozen body can be quickly placed in a coagulation bath or regeneration bath, but it is preferable and recommended that the coagulation bath or regeneration bath be kept below the freezing temperature of the solution. However, in the case of cellulose derivatives, a cellulose regeneration process is required, and this regeneration is carried out simultaneously with or sequentially (i.e., after solvent removal, etc.). The regeneration itself can be carried out by conventional methods. Using the above production method, there is no need to add foreign substances such as porosifying agents to the polymer solution during production, making it easy to produce droplets of uniform diameter, and particle size can be freely controlled. The size and shape of the pores are essentially determined by the size and shape of the solvent crystals formed when the solvent in the solution freezes and solidifies. Therefore, the shape and size of the pores can be adjusted by changing the type of polymer solution, temperature, and other freeze-solidification conditions. When the above-mentioned production method is used, the porous cellulose microparticles obtained have a continuous pore structure in which the pores communicate with each other via openings in the membrane that separate the pores.

[0032] As the porous cellulose microparticles used in the microorganism testing method of this embodiment, granulated cellulose particles can be used as they are, but it is preferable to crosslink them before use. There are no limitations on the crosslinking method, but any crosslinking agent having two or more functional groups capable of reactively bonding with the hydroxyl groups of cellulose can be used for intermolecular crosslinking of porous cellulose. Examples include bifunctional organic substances. Examples include X-R-Z type compounds (where R represents an aliphatic residue containing a carbon atom, and X and Z are various halogens, epoxy, etc., bonded to the carbon atoms of the aliphatic residue) that react relatively easily in the presence of an alkaline reactant. Examples of bifunctional compounds suitable for the above reaction include epichlorohydrin, dichlorohydrin, 1,2- or 3,4-diepoxybutane, bisepoxypropyl ether, ethylene glycol-bis-epoxypropyl ether, 1,4-butanediol-bis-epoxypropyl ether, and compounds closely related to these, but are not limited to these. Crosslinking allows the crystalline structure of cellulose to be maintained even when modified with many cationic substituents, thereby keeping the particle structure stable.

[0033] The method for modifying the cellulose constituting the particles used in the microbial testing method of this embodiment with cationic substituents is not particularly limited, but multiple methods are possible, such as direct modification to hydroxyl groups or modification via a functional group having an epoxy group. A preferred method is to directly modify the hydroxyl groups of cellulose using 2-(diethylamino)ethyl chloride hydrochloride or the like. Because cellulose has many hydroxyl groups, direct modification of hydroxyl groups makes it possible to modify a large amount of cationic substituents uniformly, even to the interior of the particles. On the other hand, when introducing the cationic substituents via a third substituent such as an epoxy group, the amount and variation of the introduced cationic substituents depend on the amount of the third substituent introduced, making it difficult to control the amount introduced into the interior of the particles. Furthermore, the reaction conditions for introducing cationic substituents using 2-(diethylamino)ethyl chloride hydrochloride are not particularly limited, but the charge density inside the pores can be controlled by setting an optimal pH taking into account changes in the pH of the solution caused by the 2-(diethylamino)ethyl chloride hydrochloride itself, and by adding an additive such as Glauber's salt to minimize the effect of the charge on the hydroxyl groups of the cellulose.

[0034] The medium used in the microorganism testing method of this embodiment broadly includes media capable of growing microorganisms, such as LB medium, M9 medium, TB medium, SOB medium, SOC medium, 2X YT medium, NZCYM liquid medium, yeast medium, yeast nitrogen base medium, phage medium, and NZ Amine (registered trademark) liquid medium. Furthermore, the medium is preferably in the form of a flat membrane medium, sheet medium, or film medium. These medium forms result in low sensitivity when testing samples with low microbial concentrations, and are prone to false negatives. However, by seeding particles with adsorbed microorganisms to form colonies, high sensitivity is achieved and false negatives are eliminated.

[0035] A second embodiment of the present invention is a cylindrical pretreatment container for microbiological testing, having an inlet opening and an outlet opening, filled with particles, wherein both the inlet opening and the outlet opening are capable of passing liquid, but a filter is provided near the outlet opening to prevent the particles from passing through, and the particles are filled in the space above the filter. The particles used in this second embodiment can be the same as those used in the first embodiment described above.

[0036] The pretreatment container for microorganism testing has a structure on the inlet opening side that allows particles to pass through, and liquid can be passed through either the inlet or outlet opening. This structure allows microorganisms to be adsorbed onto particles by discharging a sample containing microorganisms from the inlet opening. Furthermore, after the adsorption process, the column can be removed and physiological saline or the like can be discharged from the outlet opening, allowing the particles with adsorbed microorganisms to be easily removed from the column, facilitating seeding on a culture medium.

[0037] As used herein, the term "microorganism" is not particularly limited and broadly encompasses various bacteria, such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella, various viruses, fungi, protozoa, and rickettsia. In this embodiment, these microorganisms are the subject of concentration. Furthermore, sample liquids containing bacteria, viruses, and the like are not particularly limited and broadly encompass patient specimens (e.g., urine and body fluids), food samples (e.g., liquid samples and bacterial or viral extracts obtained by stomachers), and environmental samples (e.g., water quality).

[0038] EXAMPLES The present invention will be specifically explained below with reference to Examples and Comparative Examples, but the present invention is not limited to the Examples alone.

[0039] [Charge Capacity Measurement] 5.0 g of a sample was weighed out and washed twice with 30 mL of distilled water using a funnel and a suction filter, and then substituted with water. －４The sample is washed four times with 50 mL of 10% sodium sulfate solution. Then, using a funnel and a suction filter, the sample is washed four times with 50 mL of 10% sodium sulfate solution, and the filtrate is recovered. The recovered filtrate is then added with 2 mL of 0.1 N potassium chromate solution and stirred. The sample is then titrated with 1 N silver nitrate solution, and the titer A (mL) is determined at the moment when the reddish-brown precipitate of silver chromate begins to form. Separately, the sample that was washed with 10% sodium sulfate solution in the above process is washed four times with 50 mL of pure water, dried at 105°C for at least 15 hours, and weighed on a balance to determine the dry weight B (g). The charge capacity is calculated using the measured titer A (mL) and the dry weight B (g) of the sample according to the following formula (1) (N = 3): Charge capacity (mmol / g) = 1 (N) (mol / L) x A (mL) / B (g)... formula (1)

[0040] [Preparation of E. coli solution] 15 μL of E. coli strain (ATCC No. 25922) was inoculated into 1 mL of autoclave-sterilized 3% Trypticase Soy Broth liquid medium (211768, manufactured by BD) and cultured with shaking at 37°C under aerobic conditions for 16 hours. Centrifuged at 4000 × g for 15 minutes, the supernatant was removed, and 1 mL of distilled water was added to obtain a bacterial solution with an OD (600 nm) of 1 (approximately 1e9 cfu / mL). The resulting E. coli solution was diluted with saline to approximately 1 cfu / mL for use.

[0041] [Preparation of Cellulose Particles] 500 mL of a cellulose-ammonium copper solution with a viscosity of 2 poise at 5°C, a cellulose concentration of 2%, a copper concentration of 1.2%, and an ammonia concentration of 4% was prepared using purified linters as a raw material. The solution was sprayed with nitrogen gas using a two-fluid nozzle and stirred into 10 L of silicone oil (SH200, 1.5 cs, Toray Industries, Inc.) cooled to -40°C. The silicone oil was heated to -20°C and stirred for 20 minutes, after which 10 L of 40% sulfuric acid cooled to -35°C was added. After adding the sulfuric acid, stirring was continued for 8 hours, and the mixture was heated to 20°C. The silicone oil was removed, and the remainder was extracted and washed with water to obtain porous cellulose particles. Using a classification sieve, porous cellulose particles with an average particle size of 240 μm and an average pore size of 30 μm were obtained. The cellulose particles before cationization modification were designated as Particle 1.

[0042] [Cationization of Cellulose Particles] 6.66 g of Particles 1 was weighed out, and pure water, anhydrous sodium sulfate, sodium dodecyl sulfate, and a 50% aqueous solution of 2-(diethylamino)ethyl chloride hydrochloride were added in the amounts shown in Table 1 below and stirred. A reaction initiation liquid (48% aqueous sodium hydroxide solution) was immediately added, and the reaction was carried out with stirring for 1 hour in a thermostatic chamber at 70°C. After 1 hour, 36% hydrochloric acid was added to neutralize the mixture until the pH reached 7, and the sample was washed three times with 8,000 mL of pure water by suction filtration. The above process was repeated once or twice to obtain Particles 2 to 7, which are cationic porous cellulose particles. The charge capacity of the dried particles was measured.

[0043]

[0044] [Preparation of evaluation columns (pretreatment containers for microbiological testing)] The included frit was pushed into a Luer-type cartridge column (Tomoe Manufacturing Co., Ltd. CL0311). Particles 1 to 8 or Cytodex 1 (Cytiva) were weighed and suspended in pure water to a concentration of 10 mg / mL. 200 μL or 800 μL of the particle solution suspended at 10 mg / mL was passed through the column. The column was placed in a sterilization bag and sterilized in an autoclave at 121°C for 20 minutes or more to obtain columns 1 to 10, as shown in Table 2 below.

[0045]

[0046] Comparative Example 1: Sheet Culture Evaluation The surface film of an MC-Media Pad EC (for E. coli and coliform bacteria) (JNC Corporation) was peeled off, 1 mL of E. coli solution adjusted to approximately 1 cfu / mL was dropped, and the film was reattached. The plate was cultured at 35°C for 24 hours, and the number of colonies was counted. The counting results are shown in Table 3 below.

[0047] Examples 1 to 12: Evaluation of Sheet Culture by Seeding Particles After Column Concentration As shown in Table 3, 100 mL of E. coli solution adjusted to approximately 1 cfu / mL using physiological saline, Job's Tears lotion (Naturie) (containing methylparaben), or 2.0% saline was measured into a beaker, and columns 1 to 10 were passed through at 15 mL / min using a peristaltic pump with the fritted side facing downward. Physiological saline was then measured into a 1 mL syringe and attached with the fritted side facing upward. With the surface film of an MC-Media Pad EC (for E. coli and coliform bacteria) (JNC Corporation) peeled off, physiological saline was passed through the column, and the solution passed through the column together with the particles in the medium was dripped onto the medium. The passed solution was spread evenly on the medium, and then the film was reattached. After 24 hours of culture at 35°C, the number of colonies was counted, and the average value of three samples was recorded as the colony count. Colony size was also evaluated according to the following criteria. (Evaluation criteria) Large: Less than 50% of colonies were smaller than when no enrichment was performed Medium: Between 50% and 80% of colonies were smaller than when no enrichment was performed Small: More than 80% of colonies were smaller than when no enrichment was performed The results of counting the number of colonies and evaluating the colony size are shown in Table 3 below.

[0048] Example 13: Evaluation of Sheet Culture by Seeding Particles After Column Concentration of Low-Concentration Bacterial Solution. E. coli solution adjusted to approximately 1 cfu / mL was further diluted to approximately 0.01 cfu / mL using physiological saline. 1000 mL of E. coli solution adjusted to approximately 0.01 cfu / mL was measured into a beaker, and using a peristaltic pump, the solution was passed through Column 6 at 15 mL / min with the frit facing downwards. Then, physiological saline was measured into a 1 mL syringe and attached with the frit facing upwards. With the surface film of an MC-Media Pad EC (for E. coli and coliform bacteria) (JNC Corporation) peeled off, physiological saline was passed through, and the solution passed through the column together with the particles in the column was dripped onto the medium. The passed solution was spread evenly on the medium, and then the film was reattached. After culturing at 35°C for 24 hours, the number of colonies was counted, and the average of the three samples was recorded as the colony count. The colony size was determined according to the same criteria as above. The results of counting the number of colonies and evaluating the colony size are shown in Table 3 below.

[0049] Comparative Examples 2 and 3: Evaluation of Sheet Culture by Microbial Extraction after Column Concentration. 100 mL of E. coli solution adjusted to approximately 1 cfu / mL using physiological saline was measured into a beaker, and the solution was passed through Column 1 with the frit facing downwards using a peristaltic pump at 15 mL / min. Subsequently, magnesium chloride solutions adjusted to 0.01 and 1 M were measured into 1 mL syringes and attached with the frit facing downwards. With the surface film removed from an MC-Media Pad EC (for E. coli and coliform bacteria) (JNC Corporation), the magnesium chloride solution was passed through, and the filtrate was dripped onto the medium. The passed solution was spread evenly on the medium, and the film was then reattached. After incubation at 35°C for 24 hours, the number of colonies was counted, and the average value of three samples was recorded as the colony count. Colony size was determined according to the same criteria as above. The results of the colony count and colony size evaluation are shown in Table 3 below.

[0050] Comparative Examples 4 and 5: Sheet Culture Evaluation The surface film of an MC-Media Pad EC (for Escherichia coli and coliform bacteria) (JNC Corporation) was peeled off, and 1 mL of Escherichia coli solution adjusted to approximately 1 cfu / mL using Job's Tears lotion (Naturie) (containing methylparaben) or 2.0% saline was dropped onto the pad, and the film was then reattached. The pad was cultured at 35°C for 24 hours, and the number of colonies was counted. The counting results are shown in Table 3 below.

[0051]

[0052] As shown in Table 3, by using the microorganism testing method and pretreatment container for microorganism testing of this embodiment, particles with adsorbed microorganisms were directly seeded on a culture medium and cultured on a sheet culture medium, and it was possible to test for bacteria with sensitivity 10 to 200 times higher than by directly forming colonies with microorganisms obtained without using the particles or obtained by using the particles and then separating them from the particles. As shown in Example 13, by increasing the concentration ratio (volume of sample liquid before adsorption / volume of seed liquid) to 1000 times, it was possible to test for bacteria with high sensitivity even in samples with a low microbial concentration of about 0.01 cfu / mL.

[0053] Furthermore, as shown in Examples 11 and 12, it was possible to test bacteria with high sensitivity while reducing the influence of culture inhibitors such as parabens and high concentrations of salt.

[0054] The microorganism testing method and pretreatment container for microorganism testing according to the present invention enable rapid and simple concentration of microorganisms, and furthermore, since the particles with adsorbed microorganisms can be directly seeded on a culture medium and cultured for testing, there is no need to recover the microorganisms or destroy them to extract antigens, and microorganisms can be directly tested with high sensitivity. Therefore, the microorganism testing method and pretreatment container for microorganism testing according to the present invention can be suitably used in microorganism testing, etc.

[0055] 1 Microbiological test pretreatment container 2 Inlet opening 3 Outlet opening 4 Filter 5 Particles

Claims

1. A microorganism testing method comprising the following steps: an adsorption step of adsorbing microorganisms in a liquid sample to particles; a seeding step of seeding the particles to which the microorganisms are adsorbed onto a culture medium; and a culture step of culturing the microorganisms on the culture medium.

2. The microorganism testing method according to claim 1, further comprising a step of counting colonies after the culturing step.

3. A microorganism testing method according to claim 1 or 2, wherein the medium is a flat membrane medium, a sheet medium or a film medium.

4. A microorganism testing method according to claim 1 or 2, wherein in the seeding step, the particles are seeded in the medium using a liquid.

5. The microorganism testing method according to claim 4, wherein the amount of liquid used for seeding particles in the seeding step is less than the amount of liquid sample in the adsorption step.

6. A microorganism testing method as described in claim 1 or 2, wherein the microbial concentration in the liquid sample is 100 cfu / mL or less.

7. A microbiological testing method according to claim 1 or 2, wherein the particles are made of cellulose.

8. A microorganism testing method according to claim 1 or 2, wherein the particles are porous particles.

9. A microorganism testing method according to claim 8, wherein the porous particle has a number of pores therein and has a continuous pore structure in which adjacent pores communicate with each other via openings in the membrane that separate the pores.

10. The microorganism testing method according to claim 9, wherein the porous particles have an average pore size of 5 μm or more and 100 μm or less.

11. A microorganism testing method according to claim 1 or 2, wherein the particles have an average particle size of 1000 μm or less.

12. The specific surface area of ​​the particles when dried is 0.3 m ２ / g or more 4.4m ２ The microorganism testing method according to claim 1 or 2, wherein the concentration of the microbial component is 0.01 to 0.1 μg or less.

13. The microorganism testing method according to claim 1 or 2, wherein the surface of the particles is modified with a cationic substituent.

14. A microorganism testing method according to claim 1 or 2, wherein the charge capacity of the particles is 0.5 mmol / g or more.

15. A microorganism testing method according to claim 1 or 2, wherein in the adsorption step, a liquid sample containing microorganisms is passed through a column containing particles.

16. A cylindrical pretreatment container for microbiological testing having an inlet opening and an outlet opening and filled with particles therein, wherein both the inlet opening and the outlet opening allow liquid to pass through, but there is a filter near the outlet opening that prevents the particles from passing through, and the space above the filter is filled with the particles.

17. The pretreatment container for a microorganism test according to claim 16, wherein the particles are made of cellulose.

18. A pretreatment container for a microorganism test according to claim 16 or 17, wherein the particles are porous particles.

19. A pretreatment container for a microorganism test as described in claim 18, wherein the porous particle has a large number of pores therein and has a continuous pore structure in which adjacent pores are connected to each other by openings in a membrane that separate the pores.

20. A pretreatment container for a microorganism test according to claim 19, wherein the average pore size of the porous particles is 5 μm or more and 100 μm or less.

21. A pretreatment container for a microorganism test as described in claim 16 or 17, wherein the average particle size of the particles is 100 μm or more and 1000 μm or less.

22. The specific surface area of ​​the particles when dry is 0.3 m ２ / g or more 4.4m ２ The pretreatment container for a microorganism test according to claim 16 or 17, wherein the concentration of the ion exchange resin is 0.1 to 0.5% by mass.

23. A pretreatment container for a microorganism test according to claim 16 or 17, wherein the surface of the particles is modified with a cationic substituent.

24. A pretreatment container for a microorganism test as described in claim 16 or 17, wherein the particles have a charge capacity of 0.5 mmol / g or more.

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