Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M1/00—Apparatus for enzymology or microbiology
  • C12M1/26—Inoculator or sampler
  • C12M1/28—Inoculator or sampler being part of container
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
  • C12Q1/06—Quantitative determination

Definitions

• the present invention relates to a microbiological testing method and a pretreatment container for microbiological testing.
• infectious disease diagnosis or in environmental and food microbiological testing, there is a demand for a rapid and simple method for detecting, identifying, and/or enumerating pathogens or harmful microorganisms that impair quality in a sample.
• pathogenic bacteria or viruses are identified by point-of-care tests (POCT) so that doctors and nurses can make appropriate clinical decisions at an early stage alongside the subjects.
• POCT point-of-care tests
• environmental testing and food microbiology testing tests are conducted for environmental indicator bacteria such as general bacteria and coliform bacteria, as well as harmful microorganisms such as food poisoning bacteria and viruses, as part of employee or environmental hygiene management and quality control of raw materials, intermediate products, and final products.
• environmental indicator bacteria such as general bacteria and coliform bacteria
• harmful microorganisms such as food poisoning bacteria and viruses
• Testing methods for these microorganisms can be broadly divided into culture methods, in which the microorganisms are identified based on their biochemical properties after isolation and cultivation; genetic methods, in which genes specific to the microorganisms are amplified and detected by PCR, LAMP, or the like; and immunological methods, in which detection is performed by utilizing a specific reaction between an antigen marker specific to the microorganism and an antibody.
• culture methods in which the microorganisms are identified based on their biochemical properties after isolation and cultivation
• genetic methods in which genes specific to the microorganisms are amplified and detected by PCR, LAMP, or the like
• immunological methods in which detection is performed by utilizing a specific reaction between an antigen marker specific to the microorganism and an antibody.
• the culture method is widely used due to its sensitivity and simplicity of procedure.
• each test method uses a small amount of sample, about 0.01 to 1 mL, it is only possible to use a very small portion of the content of the detection substance (bacteria or viruses themselves, or their surface or internal components) contained in about 0.01 to 1 L that can be collected from a patient specimen (e.g., urine or body fluids), food sample (e.g., liquid sample or bacterial or viral extract using a stomacher), or environmental sample (e.g., water quality).
• a patient specimen e.g., urine or body fluids
• food sample e.g., liquid sample or bacterial or viral extract using a stomacher
• environmental sample e.g., water quality
• E. coli is adsorbed by shaking inorganic microparticles (amorphous metal silicate) and a sample for 1 to 20 minutes.
• 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.
• a large amount of inorganic microparticles, as much as 10 mg, is required for 10 mL of E. coli sample of 1e3 cfu/mL.
• the adsorbed microorganisms are destroyed by contacting them with an extraction liquid, releasing the specimen inside the microorganisms, which are then detected to indirectly quantify the amount of microorganisms. With this method, it is necessary to separate the released specimen from the microparticles, which causes problems such as a decrease in release efficiency and a decrease in sensitivity due to non-specific adsorption.
• Patent Document 2 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.
• Patent No. 5972174 Japanese Patent Application Publication No. 4-91142
• the problem that the present invention aims to solve is to provide a simple and highly sensitive microorganism testing method and a pretreatment container for microorganism testing suitable for said method.
• the inventors unexpectedly discovered that the problem could be solved by adsorbing microorganisms in a sample onto particles, and then seeding the particles with the adsorbed microorganisms onto a culture medium for culturing, which led to the completion of the present invention.
• the present invention is as follows. [1] the steps of: an adsorption step of adsorbing microorganisms in a liquid sample onto particles; A seeding step of seeding the particles to which the microorganisms are adsorbed onto a medium, and a culturing step of culturing the microorganisms on the medium.
• Microbiological testing methods including: [2] The microorganism testing method described in [1], further comprising a counting step of counting colonies after the culturing step. [3] The microorganism testing method described in [1] or [2], wherein the culture medium is a flat membrane culture medium, a sheet culture medium or a film culture medium.
• [4] The microorganism testing method according to any one of [1] to [3], wherein in the seeding step, particles are seeded in the medium using a liquid.
• [5] The microorganism testing method described in [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] The microorganism testing method according to any one of [1] to [5], wherein the microbial concentration in the liquid sample is 100 cfu/mL or less.
• [7] The microorganism testing method according to any one of [1] to [6], wherein the particles are made of cellulose.
• the shape of the particles in 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 inside the column is high, making it possible to suppress a decrease in adsorption efficiency due to a short path. Furthermore, if the particles have a continuous pore structure, the sample can easily diffuse into the inside of the particles, making it possible to efficiently adsorb microorganisms even to the inside of the particles, and furthermore, when the particles are packed into a column, pressure loss during liquid passage can be reduced.
• the lower limit of the average pore size of the pores is preferably 5 ⁇ m or more, more preferably 10 ⁇ m or more.
• the upper limit of the average pore size is preferably 100 ⁇ m or less, more preferably 80 ⁇ m or less, and even more preferably 50 ⁇ m or less.
• the upper limit of the average particle size of the particles used in the microorganism testing method of this embodiment is preferably 1000 ⁇ m or less, more preferably 500 ⁇ m or less, and even more preferably 400 ⁇ m or less. If the average particle size is smaller than 1000 ⁇ m, microorganisms can be efficiently adsorbed to the center of the particles. In addition, since the gaps between the particles can be minimized when packed in a column, the decrease in adsorption efficiency due to short paths can be suppressed, and further, it is possible to uniformly seed the surface of the medium during microbial culture.
• the specific surface area is preferably 4.4 m 2 /g or less.
• the term "specific surface area” refers to the surface area per gram of dry weight measured by the BET method.
• the particles used in the microorganism testing method of this embodiment are preferably modified with a cationic substituent.
• the cationic substituent 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.
• the particles used in the microorganism testing method of this embodiment can be manufactured, for example, by forming a solution in which cellulose has been dissolved into the desired shape while cooling it below the solidification temperature of the solution to freeze it, and then extracting and removing the solvent or causing it to lose its dissolving ability.
• the freezing temperature is not set 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.
• 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 ability (hereinafter, these processes are collectively referred to as "solvent removal, etc.") to form a solidified cellulose porous body.
• solvent removal, etc. are not particularly limited. Usually, it is sufficient to quickly put the frozen body into any coagulation bath or regeneration bath, but it is preferable and recommended that the coagulation bath or regeneration bath is also at or 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 the solvent removal, etc. or sequentially (i.e., after the solvent removal, etc.).
• the regeneration itself can be carried out by a conventional method. If the above-mentioned production method is used, it is not necessary to add foreign matter such as a porosifying material to the polymer solution during production, so that minute droplets of uniform diameter can be easily produced and the particle size can be controlled arbitrarily.
• the diameter and shape of the pores are basically determined by the size and shape of the crystals of the solvent, etc. formed when the solvent in the solution is frozen and solidified. Therefore, the shape and diameter of the pores can be adjusted by changing the type of polymer solution, the freezing and solidifying conditions such as temperature, etc.
• the resulting porous cellulose microparticles have a continuous pore structure in which the pores communicate with each other via openings in the membrane that separate the pores.
• 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.
• crosslinking method for intermolecular crosslinking of the cellulose porous body, any crosslinking agent having two or more functional groups capable of reacting and bonding with the hydroxyl groups of cellulose can be used.
• An example is a bifunctional organic substance.
• An example of this is an X-R-Z type (wherein R represents an aliphatic residue containing a carbon atom, and X and Z are various halogens, epoxy, etc., which are bonded to the carbon of the aliphatic residue.) which reacts relatively easily in the presence of an alkaline reactant.
• bifunctional compounds suitable for the above reaction include epichlorohydrin, dichlorohydrin, 1,2- or 3,4-diepoxybutane, bisepoxy propyl ether, ethylene glycol-bis-epoxy propyl ether, 1,4-butanediol-bis-epoxy propyl ether, and compounds closely related to these, but are not particularly limited to these.
• Cross-linking allows the cellulose crystal structure to be maintained even when it is modified with many cationic substituents, so the particle structure can be kept stable.
• the method of modifying the cellulose constituting the particles used in the microorganism testing method of this embodiment with cationic substituents is not particularly limited, but it can be introduced by several methods, such as a method of directly modifying the hydroxyl groups or a method of modifying via a functional group having an epoxy group.
• a method of directly modifying the hydroxyl groups of cellulose using 2-(diethylamino)ethyl chloride hydrochloride or the like is preferable. Since cellulose has many hydroxyl groups, it is possible to modify a large amount of cationic substituents uniformly even inside the particles by directly modifying the hydroxyl groups.
• the amount and variation of the cationic substituent introduced depends on the amount of the third substituent introduced, making it difficult to control the amount introduced inside the particles.
• the charge density inside the pores can be controlled by setting an optimal pH taking into account the change in pH of the solution caused by 2-(diethylamino)ethyl chloride hydrochloride itself, and by adding an additive such as Glauber's salt to minimize the effect of the charge of the hydroxyl groups of cellulose.
• 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 source base medium, phage medium, NZ Amine (registered trademark) liquid medium, etc.
• 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 to which microorganisms are adsorbed to form colonies, high sensitivity is achieved and false negatives are eliminated.
• the 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 inside, both of which allow liquid to pass through, but which is characterized in that there is a filter near the outlet opening that prevents the passage of the particles, and the space above the filter is filled with the particles.
• the particles used in this second embodiment can be the same as those used in the first embodiment described above.
• 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 makes it possible to discharge a sample containing microorganisms from the inlet opening, thereby allowing the microorganisms to be adsorbed onto the particles.
• the column can be removed once and saline solution or the like can be discharged from the outlet opening, allowing the particles with adsorbed microorganisms to be easily discharged from within the column, making it easy to sow the medium.
• microorganism is not particularly limited and broadly includes 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 to be concentrated.
• sample liquids containing bacteria, viruses, etc. are not particularly limited and broadly include patient specimens (e.g., urine and body fluids), food samples (e.g., liquid samples and bacterial or viral extracts obtained by a stomacher), or environmental samples (e.g., water quality).
• Examples 1 to 12 Evaluation of sheet culture by particle seeding after column concentration
• 100 mL of E. coli solution adjusted to about 1 cfu/mL using physiological saline, Job's tears lotion (Naturie) (containing methylparaben), or 2.0% saline was measured into a beaker, and the solution was passed through columns 1 to 10 at 15 mL/min using a peristaltic pump with the fritted side facing down. Then, physiological saline was measured into a 1 mL syringe and attached with the fritted side facing up.
• the surface film of MC-Media Pad EC for E.
• Example 13 Evaluation of sheet culture by particle seeding after column concentration of low-concentration bacterial liquid
• the E. coli solution adjusted to about 1 cfu/mL was further diluted to about 0.01 cfu/mL using physiological saline.
• 1000 mL of the E. coli solution adjusted to about 0.01 cfu/mL was measured into a beaker, and the column 6 was passed through at 15 mL/min using a peristaltic pump with the frit facing down. Then, physiological saline was measured into a 1 mL syringe, and the column was attached with the frit facing up.
• the surface film of MC-Media Pad EC for E.
• microorganisms can be concentrated quickly and easily, and furthermore, particles to which microorganisms are adsorbed can be directly sown in a culture medium, cultured, and tested, so 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.

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Molecular Biology (AREA)
• Immunology (AREA)
• Analytical Chemistry (AREA)
• Physics & Mathematics (AREA)
• Toxicology (AREA)
• Medicinal Chemistry (AREA)
• Biomedical Technology (AREA)
• Sustainable Development (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Priority Applications (1)

Applications Claiming Priority (2)

Publications (1)

Family

ID=93658087

Family Applications (1)

Country Status (2)

Citations (4)

• 2024
  • 2024-05-30 JP JP2025524876A patent/JPWO2024248091A1/ja active Pending
  • 2024-05-30 WO PCT/JP2024/019884 patent/WO2024248091A1/ja not_active Ceased

Patent Citations (4)

Also Published As

Similar Documents

Legal Events