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
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
• • 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
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/38—Caps; Covers; Plugs; Pouring means
• • 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
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings

Definitions

• the invention relates to culture equipment for simply counting the number of microorganisms in a sample.
• An agar medium used for culturing the microorganisms in the methods described above is a material prepared by solidifying a medium in which a nutritional ingredient and a selective ingredient are dissolved thereinto with agar, and needs to be pre-prepared prior to culturing and counting the microorganisms.
• the agar spread plate method upon applying a sample onto a plate medium, the sample is applied onto the medium while the sample is completely absorbed into the medium, and therefore the method also has a problem of needing time for operation.
• Patent literature No. 1 discloses a sheet-shaped culturing apparatus provided with an adhesive layer, a chilled water-soluble gelling agent powder layer containing a nutritional ingredient, and a cover sheet, on a top surface portion of a water-proof base.
• Patent literature No. 2 discloses a simple medium having a water-soluble gelling agent and a fibrous absorbent sheet having a mesh, on a top surface of a water-proof base.
• Patent literature No. 3 discloses sheet-shaped culture equipment in which an absorbent polymer layer and a porous matrix layer are sequentially laminated on a top surface of a water-proof base.
• Patent literature No. 4 discloses sheet-shaped culture equipment in which a frame on which a sample spreads is provided on a base sheet, and a medium solution containing an adhesive ingredient and a gelling agent is subjected to patter formation within the frame.
• Such a frame is composed of a hydrophobic resin having a contact angle set to a specific value, and a sample solution spreads only within the frame.
• Patent literature No. 1 JP H2-49705 B.
• Patent literature No. 2 JP 2000-325072 A.
• Patent literature No. 3 WO 97/24432 A.
• Patent literature No. 4 JP 2015-204845 A.
• Non-Patent literature No. 1 Manual to Practice Bacteriology, 2nd, p59, 4.3 Bacteria counting and culture method, edited by The Institute of Medical Science, The University of Tokyo, Maruzen Co., Ltd.
• a porous matrix including a nonwoven fabric, is used, and therefore a liquid sample added thereto is diffused by a capillary phenomenon to homogeneously spread wholly on the equipment, and therefore operability is high.
• Patent literature No. 2 a procedure of laminating the porous matrix layer and a medium layer is complicated
• Patent literature No. 3 a drawback in which colonies after culture become hard to observe by irregular reflection caused on a medium surface by irregularity on a surface of the porous matrix or opacity of the porous matrix itself.
• the frame is formed of a material set to a specific contact angle, but depending on a kind of the sample, for example, when food and drink or the like containing oil, protein or the like is applied as the sample, water repellency of the frame is assumed to change, and the art may be reasonably referred to as having a problem of versatility as the culture equipment.
• an object of the invention is to provide culture equipment of microorganisms, in which the culture equipment has high operability, and can be simply produced, and the number of microorganisms in the sample can be easily counted.
• the present inventors have diligently continued to conduct study in order to solve the problems as described above. As a result, the present inventors have arrived at a conception in which, if a medium ingredient is provided in a thin and flat space surrounded by a dish-shaped member having a recess and a lid member having a projection upon fitting both members, a sample can be homogeneously diffused upon application thereof without using a tool or a capillary phenomenon, and a medium can be formed in a significantly short period of time, and therefore culture and counting of microorganisms can be realized by simple operation. Then, the present inventors have found that one or more selected from guar gum and xanthan gum are preferred as a gelling agent forming the medium from a viewpoint of simplicity of the operation and high visibility from outside, and have completed the invention.
• the invention includes the items described below.
• Item 1 Culture equipment of microorganisms, including (a) an upper member; (b) a lower member having a recess; and (c) a medium ingredient, wherein (c) the medium ingredient contains (c1) one or more selected from guar gum and xanthan gum, and (c2) a nutritional ingredient.
• Item 2. The culture equipment according to item 1, wherein (a) the upper member includes a projection having a shape that can be mutually fitted to the recess of (b) the lower member through (c) the medium ingredient.
• the culture equipment according to item 2 wherein, in a state in which the projection of (a) the upper member is fitted to the recess of (b) the lower member, a distance between a top surface of the projection of (a) the upper member and a bottom surface of the recess of (b) the lower member is 0.01 to 1 millimeter.
• Item 4 The culture equipment according to item 2 or 3, wherein, in a state in which the projection of (a) the upper member is fitted to the recess of (b) the lower member, a volume surrounded by the top surface of the projection of (a) the upper member and the bottom surface and a side surface of the recess of (b) the lower member is 1.0 to 1.5 milliliters.
• Item 6. The culture equipment according to any one of items 1 to 5, wherein (a) the upper member and/or (b) the lower member is transparent.
• Item 7. A method, wherein microorganisms in a sample are cultured and the number of the microorganisms is counted by using the culture equipment according to any one of items 1 to 6.
• an upper member of the culture equipment includes a projection having a shape that can be mutually fitted to a recess of (b) a lower member through (c) a medium ingredient, including: a step of adding a sample to the recess of (b) the lower member; a step of fitting the projection of (a) the upper member to the recess of (b) the lower member; a step of culturing microorganisms contained in the sample; and a step of counting the number of colonies of the microorganisms.
• the sample herein is not particularly limited, but is ordinarily a liquid sample, and specific examples thereof include an aqueous liquid sample such as drinking water, soft drink, water for industrial use, water for pharmaceutical use, dialysis water and urine.
• the microorganisms herein ordinarily refer to Coliform bacteria, Staphylococcus species bacteria, Vibrio species bacteria, Enterococcus species bacteria, Fungi, Bacillus species bacteria and the like.
• microorganisms in a sample can be cultured by simple operation, and the number thereof can be easily counted. Moreover, culture equipment according to the invention does not have a complicated configuration, and therefore can be easily produced.
• Figure 1 is a diagram showing one aspect of culture equipment according to the invention, in which (i) shows an orthographic diagram of the culture equipment in a state in which an upper member and a lower member are not stacked, (ii) shows one example of a cross sectional view taken along line A-A’ in (i), (ii’) shows another example of the cross sectional view taken along line A-A’ in (i), (iii) shows one example of a cross sectional view taken along line B-B’ in (i), and (iii’) shows another example of the cross sectional view taken along line B-B’ in (i).
• Figure 2 is a diagram showing one aspect of culture equipment according to the invention, in which (i) shows an orthographic diagram of the culture equipment in a state in which an upper member is not fitted to a lower member, (ii) shows one example of a cross sectional view taken along line A-A’ in (i), (ii’) shows another example of the cross sectional view taken along line A-A’ in (i), (iii) shows one example of a cross sectional view taken along line B-B’ in (i), and (iii’) shows another example of the cross sectional view taken along line B-B’ in (i).
• Figure 3 is a diagram showing one aspect of culture equipment according to the invention, in which (i) shows an orthographic diagram of the culture equipment in a state in which an upper member is fitted to a lower member, (ii) shows one example of a cross sectional view taken along line C-C’ in (i), and (iii) shows another example of the cross sectional view taken along line C-C’ in (i).
• Figure 4 is an oblique projection diagram showing a use aspect example of culture equipment according to the invention.
• Figure 5 is a diagram showing one aspect of culture equipment according to the invention, in which (i) shows an orthographic diagram of the culture equipment in a state in which an upper member is fitted to a lower member, (ii) shows one example of a cross sectional view taken along line D-D’ in (i), (iii) shows another example of the cross sectional view taken along line E-E’ in (i), and (iv) shows a use aspect example in which the upper member is fitted to the lower member.
• Figure 6 shows a photograph of red colonies detected in culture equipment in Example.
• the culture equipment according to the invention has (a) upper member (30), (b) lower member (10) having a recess, and (c) medium ingredient (20) ( Figure 1).
• the culture equipment is ordinarily used by putting upper member (30) on the recess of lower member (10) in a manner of covering the recess of lower member (10). In a state of putting upper member (30) thereon, a proper space exists between the upper member and the recess of the lower member, and the medium ingredient exists therein.
• a space surrounded by the upper member, and a bottom surface and a side surface of the recess of the lower member serves as the space in which a medium formed of the medium ingredient and a sample exists (hereinafter, also described as “medium region”).
• upper member (30) has a projection having a shape that can be mutually fitted to the recess of lower member (10) through the medium ingredient (Figure 3).
• a columnar projection of the upper member is fitted to a columnar recess of the lower member, the recess having a diameter somewhat larger than a diameter of the projection.
• a top surface of the projection of the upper member and a bottom surface of the recess of the lower member need not be completely brought into close contact with each other, and the proper space exists between the projection of the upper member and the recess of the lower member, and the medium ingredient exists therein.
• the space surrounded by the top surface of the projection of the upper member, and the bottom surface and the side surface of the recess of the lower member serves as the medium region.
• a volume of the medium region can be arbitrarily designed depending on a kind of the sample to be counted or a scale of examination.
• the medium region is preferably designed to have a volume of about 1 milliliter to reduce a size of the culture equipment.
• the medium region is preferably designed in such a manner that the medium region is not excessively large (depth of the recess of the lower member is not excessively large) in comparison with an amount of the sample so that the sample can be spread wholly to the culture region by pressing or the like, upon putting the upper member on the lower member.
• the medium region is preferably designed in such a manner that, when the upper member has the projection, the medium region is not excessively large (height of the projection of the upper member is not excessively small) in comparison with the amount of the sample so that the projection can spread out the sample wholly to the medium region by fitting both.
• the above design is desirable in view of the fact that, if the amount of the sample is smaller than the medium region, a void is generated to hardly conduct correct observation and counting of colonies. Meanwhile, if the colonies are vertically stacked, the colonies become hard to be accurately observed and counted.
• the medium region is preferably designed in such a manner that the medium region is not excessively small in comparison with the amount of the sample (bottom area of the recess in a manner of being not excessively large in a thickness of the medium region).
• the above design is desirable also in view of the fact that a gel formed of guar gum or xanthan gum has transparency that is not very higher than a gel formed of polyacrylic acids, and therefore, if the medium is thick, correct observation and counting of colonies are hardly conducted.
• the thickness of the medium region is not particularly limited, but is preferably 0.01 to 1 millimeter, and further preferably 0.02 to 0.8 millimeter.
• the volume of the medium region is not particularly limited, but is preferably 1.0 to 1.5 milliliters, and further preferably 1.0 to 1.2 milliliters.
• the bottom area of the recess is preferably 10x to 100x cm 2 , and further preferably 20x to 50x cm 2 , although the bottom area is not particularly limited thereto.
• the bottom area of the recess can be designed to be 10 to 100 cm 2 , and when the recess is circular, a radius thereof only needs to be designed to be 1.8 to 5.6 centimeters.
• the projection of the upper member and the recess of the lower member may have an arbitrary shape as long as a fittable shape is applied.
• the top surface of the projection of the upper member and the bottom surface of the recess of the lower member may be either flat or curved, but from a viewpoint of operability, a flat surface is preferred.
• the medium ingredient is preferably coated onto a part of the upper member, the portion facing the recess of the lower member upon being put on the lower member, more specifically, at least a part of a portion forming the medium region and/or the recess of the lower member.
• the medium ingredient is preferably coated onto at least a part of the projection of the upper member and/or the recess of the lower member.
• a coated site is ordinarily a portion facing the medium region when the upper member is fitted to the lower member, and is preferably at least a part of the top surface of the projection of the upper member and/or the bottom surface of the recess of the lower member, and is further preferably a whole thereof.
• the medium ingredient is coated wholly onto the bottom surface of the recess of the lower member.
• the medium ingredient coated on the upper member and/or the lower member is preferably in a thin film form in such a manner that the sample applied to the culture equipment is smoothly wholly diffused to the medium region, and the sample and the medium ingredient are homogeneously gelled.
• a thickness of such a thin film is not particularly limited, but is 0.001 to 0.02 millimeter, and further preferably 0.005 to 0.01 millimeter.
• a material of the upper member and the lower member is not particularly limited, and a polyacrylic, polyvinyl-based, polyethylene-based or polyester-based polymer or the like can be adopted.
• rigidity of the material does not matter in particular, but when the upper member has no projection, the rigidity at a moderately deformable level is preferred so as to facilitate pressing after addition of a liquid sample.
• the upper member and/or the lower member is preferably transparent, and the upper member and the lower member are further preferably transparent.
• Specific examples of the material to be processed into the transparent upper member and/or lower member preferably include polyethylene, polypropylene, polyethylene terephthalate, vinyl chloride, polystyrene, polyester and polycarbonate.
• transparency herein may be at a degree at which an opposite side of the member can be visually observed through the member, and more specifically, visible light transmittance is preferably 70% or more, but not limited thereto.
• the upper member and the lower member may be separated, discretely, or may be united. A part of the upper member and a part of the lower member may be continuous by sharing one side as shown in Figure 4 and Figure 5, for example.
• the culture equipment can be used by folding the culture equipment, stacking the upper member with the lower member and putting the upper member on the lower member, and preferably by fitting the projection of the upper member to the recess of the lower member.
• the upper member and the lower member may have a plurality of projections and recesses, respectively.
• the culture equipment may have an aspect in which a plurality of medium regions are formed when in use, which is suitable for treating a plurality of samples in parallel at one time.
• the upper member and the lower member in addition to a portion forming the medium region, may have a projection or a recess and a recess or a projection having a shape that can be fitted to each other, respectively.
• a size and a shape of the upper member and the lower member are not particularly limited, but when the upper member is stacked with the lower member, the upper member and the lower member preferably have different sizes or shapes in such a manner that at least a part of an outer edge of a portion other than the portion forming the medium region is shifted.
• handling is easy when the upper member and the lower member stacked with each other are separated again.
• the medium ingredient according to the culture equipment of the invention contains (c1) one or more selected from guar gum and xanthan gum, and (c2) a nutritional ingredient.
• the medium ingredient is a material for preparing the medium for culturing the microorganisms. The preparation is ordinarily performed by adding, to the medium ingredient, a liquid sample containing the microorganisms to be counted, and permeating the sample into the medium ingredient, as a solvent of gel directly composing the medium.
• guar gum and xanthan gum play a role of a gelling agent composing the medium.
• Guar gum or xanthan gum is slower than polyacrylic acids in a rate of hydration (water absorbing), and therefore is suitable for homogeneously gelling the liquid sample while diffusing the liquid sample wholly to the medium region, and forming a thinly spread gel medium.
• xanthan gum is relatively stronger than guar gum in a gelling capacity (hydration capacity).
• the sample gelled by guar gum or xanthan gum ordinarily has no flowability, and the number of existing microorganisms can be accurately counted. Moreover, syneresis is hard to be caused from the gel, and therefore existence of the colonies of the microorganisms can be qualitatively counted, and also the number of existing microorganisms can be accurately counted. Moreover, guar gum or xanthan gum can form the gel without passing through dissolution by heating and without depending on cooling, and therefore operation of forming the medium is simple, and guar gum or xanthan gum does not inhibit growth of the target microorganisms.
• the gelling agent may be only guar gum or only xanthan gum, or a mixture of guar gum and xanthan gum for adjusting a gelling rate.
• the gelling agent may be further used in combination.
• the gelling agent such as agar, carragheenan and locust bean gum is generally used for the medium for microorganisms, or the like, but the agents described above require heating upon homogeneously solidifying the liquid sample, and therefore are unsuitable for directly solidifying the liquid sample containing the microorganisms, or a form of simple culture equipment. Moreover, the gel prepared by solidification using the gelling agent described above is also unsuitable therefor in view of low transparency. Moreover, polyvinyl alcohol is hard to be homogeneously mixed with the liquid sample, and also has a problem of easily causing syneresis. Carboxymethyl cellulose is unable to solidify the liquid sample to form flowable gel, and therefore is unsuitable for quantitative detection of the microorganisms.
• a concentration when in use of guar gum and/or xanthan gum in the invention is not particularly limited, but from a viewpoint of a solidifying capability, the total amount thereof is preferably 0.01 to 0.1 g/mL, and further preferably 0.01 to 0.05 g/mL. Therefore, the medium ingredient is preferably coated thereon so as to satisfy the range described above in the concentration when in use according to sample capacity targeted by the culture equipment.
• the nutritional ingredient contained in (c) the medium ingredient is applied for growing the target microorganisms.
• the nutritional ingredient is not particularly limited, and specific examples thereof preferably include peptone, an animal meat extract, a yeast extract and a fish meat extract.
• peptone an animal meat extract
• yeast extract a yeast extract
• a fish meat extract a standard agar medium
• an R2A agar medium is recommended. Therefore, when the medium is used for such an application, a bouillon medium in which agar is excluded from the agar culture media described above, or an ingredient equivalent thereto is preferably incorporated into the medium ingredient according to the invention.
• the medium ingredient further preferably contains (c3) a coloration reagent.
• a coloration reagent include a redox indicator including 2,3,5-triphenyltetrazolium chloride (TTC) and tetrazolium violet.
• TTC 2,3,5-triphenyltetrazolium chloride
• tetrazolium violet a redox indicator including 2,3,5-triphenyltetrazolium chloride (TTC) and tetrazolium violet.
• TTC 2,3,5-triphenyltetrazolium chloride
• tetrazolium violet tetrazolium violet
• the coloration reagent such a material may be used as a substrate (hereinafter, referred to as “enzyme substrate”) to an enzyme owned only by a specific microorganism species, and a compound that can release a pigment compound by being decomposed.
• enzyme substrate a substrate to an enzyme owned only by a specific microorganism species
• a compound that can release a pigment compound by being decomposed The material described above can be preferably used when the specific microorganisms are desirably counted.
• the pigment compound may be any of a compound colored under visible light and a fluorescent-colored compound.
• a functional group that can be released as the colored compound under visible light include a 5-bromo-4-chloro-3-indoxyl group, and 5-bromo-4-chloro-3-indole released is oxidized and fused into 5,5’-dibromo-4,4’-dichloro-indigo, and colored blue.
• a functional group that can be released as the fluorescent-colored compound include a 4-methyl umbelliferryl group, and 4-methylumbelliferone released emits fluorescence under irradiation with ultraviolet light.
• 5-bromo-4-chloro-3-indoxlyl-beta-D-galactopyranoside (X-GAL) or 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid can be preferably used, in a case of Staphylococcus aureus, 5-bromo-4-chloro-3-indoxyl phosphate (X-phos) can be preferably used, in a case of Enterococcus, 5-bromo-4-chloro-3-indoxlyl-beta-D-glucopyranoside (X-GLUC) can be preferably used, and in a case of Fungi, X-phos, 5-bromo-4-chloro-3-indoxyl acetate or 5-bromo-4-chloro-3-indoxyl butyrate can be preferably used, respectively.
• X-GAL 5-bromo-4-chloro-3-indoxlyl-beta-D-gal
• a concentration when in use of the above enzyme substrates is preferably 0.01 to 1.0 g/L, and further preferably 0.1 to 0.5 g/L, for example.
• the medium ingredient further contains (c4) a thickening agent.
• the thickening agent plays a role of an adhesive for stably coating the medium ingredient onto the upper member and/or the lower member.
• a thickening agent is not particularly limited, as long as the agent does not affect growth of the microorganisms, and specific examples thereof include hydroxypropyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, methyl cellulose, starch and a derivative thereof, polyether, hyaluronic acid and collagen.
• the medium ingredient arbitrarily may further contain a selective substance, an antibacterial substance, inorganic salts, saccharides, a thickening agent, a pH adjuster or the like, as long as the ingredient does not adversely affect advantageous effects of the invention.
• a selective substance include an antibiotic such as polymixin B and vancomycin, and a surfactant such as sodium lauryl sulfate (SDS), Tween 80 and a bile salt such as sodium cholate.
• SDS sodium lauryl sulfate
• Tween 80 a bile salt
• bile salt such as sodium cholate
• the antibacterial substance include polylysine, protamine sulfate, glycine and sorbic acid.
• the inorganic salts include an inorganic acid metal salt such as sodium chloride and sodium thiosulfate, and an organic acid metal salt such as sodium pyruvate, ferric ammonium citrate and sodium citrate.
• the saccharides include glucose, lactose, sucrose, xylose, cellobiose and maltose.
• the pH adjuster include sodium carbonate and sodium hydrogencarbonate.
• the composition according to the invention is such a composition to be preferably 6.0 to 8.0, and further preferably 6.5 to 7.5 in the pH when in use.
• the culture equipment according to the invention can be produced by an arbitrary method, and one example will be described.
• An acrylic plate or the like having a suitable size is used to be applied as an upper member and a lower member.
• a projection of the upper member and a recess of the lower member only need to be prepared by adhesion or hollowing of the acrylic plate, pressing using a mold or the like, molding by injection, or the like.
• a medium ingredient a material prepared by dissolving or a suspending the ingredient into a nonaqueous solvent is partially or wholly applied onto the projection of the upper member and/or the recess of the lower member, and then the resulting material coated is dried.
• the medium ingredient can be coated onto culture equipment.
• the nonaqueous solvent may be a solvent that can volatilize under ordinary temperature and ordinary pressure, and specific examples thereof preferably include lower alcohol such as ethanol, methanol, propanol and butanol. If the nonaqueous solvents described above are used, the medium ingredient can be coated thereon without gelling (c1) guar gum or xanthan gum during production, and therefore the culture equipment can be easily produced.
• the culture equipment according to the invention as described above can be preferably used in a method in which the microorganisms in the sample are cultured, and the number of the microorganisms is counted.
• the counting method preferably includes a step of adding a sample to a recess of (b) a lower member; a step of putting (a) an upper member on the recess of (b) the lower member; a step of culturing microorganisms contained in the sample; and a step of counting the number of colonies of the microorganisms.
• the recess is further preferably pressed from outside the equipment.
• the sample added to the recess of the lower member is homogeneously spread out wholly to the medium region.
• (c1) guar gum or xanthan gum in the medium ingredient is further quickly gelled by a moisture content of the sample, and thus the medium is easily formed.
• the counting method preferably includes a step of adding a sample to a recess of (b) a lower member of culture equipment; a step of fitting a projection of (a) an upper member to the recess of (b) the lower member; a step of culturing microorganisms contained in the sample; and a step of counting the number of colonies of the microorganisms.
• the sample added to the recess of the lower member is homogeneously spread out wholly to the medium region by fitting the projection of the upper member to the recess of the lower member.
• (c1) guar gum or xanthan gum in the medium ingredient is further quickly gelled by a moisture content of the sample, and thus the thin and flat medium having no flowability is easily formed.
• Culturing conditions of the microorganisms are not particularly limited, and are properly selected according to a kind of the target microorganisms, but are preferably 24 to 48 hours at 35 ⁇ 2°C, for example.
• the colonies formed by growth of the target microorganisms emerge in the medium after culture, and the number of the colonies is counted.
• the number of the colonies of the microorganisms can be counted without disassembling the culture equipment, and only needs to be counted by visually confirming the colonies from outside or analyzing an image picked up by a camera or the like by using image analysis software. According to the counting method of the invention, the number of the colonies can be accurately counted.
• the sample to which the counting method according to the invention can be applied is not particularly limited, and specific examples thereof preferably include a liquid sample such as drinking water, soft drink, water for industrial use, water for pharmaceutical use, dialysis water and urine.
• the sample may be a culture solution in which the sample described above is pre-cultured in tryptic soy broth or the like.
• the counting method according to the invention can also be applied to a diluted sample, and even when an amount of the microorganisms in the sample is 300 CFU/mL or less, for example, such a sample can be preferably provided for the counting method according to the invention.
• test strain Escherichia coli NBRC102203 was used, and the test strain was pre-cultured in a tryptic soy agar medium for 24 hours, and then was suspended into sterile physiological saline using a sterile swab to be a concentration corresponding to McFarland Turbidity Standard No. 1 (about 3.0 ⁇ 10 8 CFU/mL), and taken as a bacteria stock solution. Then, a bacteria diluted sample having a concentration of several ten CFU/1 mL was prepared by repeating 10-fold step dilution of each bacteria stock solution with the sterile physiological saline into a concentration of 10 -8 CFU/mL.
• a medium was formed by inoculating 1 mL of the bacteria diluted sample into the recess of the lower member of the culture equipment prepared in procedure (1), and immediately fitting the projection of the upper member thereto to homogeneously spread the bacteria diluted sample to the recess, and permeating the bacteria diluted sample into the medium ingredient.
• the sample was cultured at 35°C for 24 hours, and then presence or absence of growth was confirmed.
• Figure 6 shows colonies of Escherichia coli NBRC102203. If the culture equipment according to the invention was used, the liquid sample was able to be homogeneously spread wholly to the medium region only by fitting the upper member to the lower member without depending on a tool such as a spreader or a capillary phenomenon by a nonwoven fabric or the like. Moreover, the liquid sample was quickly homogeneously solidified by the gelling agent in the medium ingredient, and thus the thin and flat medium having no flowability was formed. After culture, as shown in Figure 6, the red colonies were able to be visually confirmed in substantially transparent gel, and the number thereof was able to be easily counted. Moreover, the culture equipment according to the invention did not have a complicated configuration, and therefore was able to be easily prepared.
• microorganisms in a sample can be cultured by simple operation, and the number thereof can be easily counted.
• culture equipment according to the invention does not have a complicated configuration, and therefore can be easily produced, and thus is industrially useful.

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• 2018
  • 2018-04-17 JP JP2018079079A patent/JP2019180369A/ja active Pending
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