JP2019180369A - Microorganism culture equipment and method for measuring the number of microorganisms using the same - Google Patents

Abstract

To provide culture equipment that have high operability and can be produced easily, and further can measure the number of microorganisms in a sample readily.SOLUTION: Provided is a microorganism culture equipment having (a) an upper member, (b) a lower member having a recess, and (c) a medium component, which contains (c1) one or more selected from guar gum and xanthan gum and (c2) a nutritional ingredient. Preferably, (a) the upper member has a convex part with a shape that may be fitted to the recess of (b) the lower member each other through (c) the medium component.SELECTED DRAWING: Figure 5

Description

  The present invention relates to a culture device for simply measuring the number of microorganisms in a specimen.

Known methods for measuring the number of microorganisms include a pour culture method and an agar plate smear culture method (Non-Patent Document 1).
The agar medium used for culturing microorganisms in these methods is a solidified medium in which nutrient components and selective components are dissolved together with agar, and must be prepared in advance prior to culture and measurement. In addition, in the agar plate smearing method, there is also a problem that when the sample is applied to the plate medium, it takes time to operate because the sample is applied to the medium while completely absorbing the sample.

In recent years, various dry simple culture devices that do not require preparation of a culture medium in advance have been developed in order to more easily and efficiently detect and measure microorganisms. In such a culture device, when a liquid specimen is added at the time of use, a culture medium can be formed by the water and used for cultivation as it is.
For example, Patent Document 1 discloses a sheet-like culture apparatus that includes an adhesive layer, a cold water-soluble gelling agent powder layer containing a nutrient component, and a cover sheet on the upper surface of a waterproof substrate. Patent Document 2 discloses a simple medium comprising a water-soluble gelling agent and a fibrous water-absorbent sheet having a mesh on the upper surface of a waterproof base material. Patent Document 3 discloses a sheet-shaped culture device in which a water-absorbing polymer layer and a porous matrix layer are sequentially laminated on the upper surface of a waterproof substrate. Patent Document 4 discloses a sheet-like culture device in which a frame on which a specimen spreads is provided on a base material sheet, and a medium solution containing an adhesive component and a gelling agent is patterned in the frame. Such a frame is made of a hydrophobic resin whose contact angle is set to a specific value, and the sample liquid spreads only within the frame.

Japanese Examined Patent Publication No. 2-49705 JP 2000-325072 A International Publication No. 97/24432 Pamphlet Japanese Patent Laying-Open No. 2015-204845

The second edition of the microbiology training proposal 59 pages 4.3 Measurement and culture method of bacterial quantity Maruzen Co., Ltd.

In the culture device described in Patent Document 1, a specimen is applied between a waterproof substrate and a cover sheet, and an operation of spreading the sample liquid over a predetermined area by pressing the specimen from above the cover sheet with an instrument called a spreader is performed. This operation requires a flat surface, and if not carefully performed, the sample may flow out to the surroundings rather than spread uniformly, and the operability is difficult.
Since the culture device described in Patent Documents 2 and 3 uses a porous matrix including a non-woven fabric, the added liquid specimen diffuses by capillary action and spreads uniformly throughout the device, so that the operability is high. However, due to the use of a porous matrix, there are difficulties in controlling the dry state of the solvent during its production (Patent Document 2), and the procedure for laminating the porous matrix layer and the medium layer is complicated. (Patent Document 3). In addition, there is a problem that colonies after culturing are difficult to see due to irregular reflection generated on the surface of the culture medium due to irregularities on the surface of the porous matrix and the opacity of the porous matrix itself.
Although the culture device described in Patent Document 4 does not use a porous matrix, it requires a flat place or requires carefulness so that the sample does not spill out of the frame that becomes the bank when the liquid sample spreads. Or In addition, the frame is formed of a material set to a specific contact angle. However, depending on the type of specimen, for example, when a food or drink containing oil or protein is used as a specimen, the frame has water repellency. It can be said that there is a problem in versatility as a culture device.

  In view of such circumstances, an object of the present invention is to provide a microorganism culturing device that has high operability, can be easily manufactured, and can easily measure the number of microorganisms in a specimen. .

  As a result of intensive research to solve the above problems, the present inventor provides a medium component in a thin and flat space surrounded when a dish-shaped member having a concave portion and a lid member having a convex portion are fitted. Therefore, it is possible to achieve uniform culture and measurement of microorganisms by simple operation because the sample can be uniformly diffused without using instruments or capillaries, and the medium can be formed in a very short time. did. And as a gelling agent that forms a medium, one or more selected from guar gum and xanthan gum is found to be suitable from the viewpoint of easy operation and high visibility from the outside, and the present invention is completed. It was.

That is, the present invention is as follows.
[1] (a) an upper member, (b) a lower member having a recess, and (c) a culture medium component,
(C) The culture medium component comprises (c1) one or more selected from guar gum and xanthan gum, and (c2) a nutrient component.
[2] The culture device according to [1], wherein (a) the upper member has (b) a concave portion of the lower member and (c) a convex portion having a shape that can be fitted to each other via a medium component.
[3] The distance between (a) the upper surface of the convex portion of the upper member and (b) the bottom surface of the concave portion of the lower member in a state where the convex portion of the upper member and (b) the concave portion of the lower member are fitted. The culture equipment according to [2], which is 0.01 to 1 mm.
[4] In a state in which the convex portion of (a) the upper member and the concave portion of (b) the lower member are fitted, (a) the upper surface of the convex portion of the upper member, and (b) the bottom surface and side surfaces of the concave portion of the lower member, The culture equipment according to [2] or [3], wherein the volume of the space surrounded by is 1.0 to 1.5 mL.
[5] (c) The culture medium component is applied to (a) at least a part of the convex portion of the upper member and / or (b) the concave portion of the lower member, according to any one of [2] to [4] The culture equipment described.
[6] The culture device according to any one of [1] to [5], wherein (a) the upper member and / or (b) the lower member is transparent.
[7] A method of culturing microorganisms in a specimen using the culture device according to any one of [1] to [6] and measuring the number of microorganisms.
[8] (b) adding a specimen to the concave portion of the lower member;
(A) the step of fitting the convex portion of the upper member into the concave portion of (b) the lower member;
The method according to [7], comprising a step of culturing a microorganism contained in the specimen, and a step of measuring the number of colonies of the microorganism.
[9] The (a) upper member of the culture equipment has (b) a concave portion of the lower member and (c) a convex portion having a shape that can be fitted to each other via a medium component,
(B) adding a specimen to the recess of the lower member;
(A) the step of fitting the convex portion of the upper member into the concave portion of (b) the lower member;
The method according to [7], comprising a step of culturing a microorganism contained in the specimen, and a step of measuring the number of colonies of the microorganism.

In the present specification, the sample is not particularly limited, but is usually a liquid sample, specifically, an aqueous liquid sample such as drinking water, soft drink, industrial water, pharmaceutical water, dialysis water, urine and the like. Etc.
In the present specification, the microorganism usually refers to coliform bacteria, staphylococci, Vibrio bacteria, enterococci, fungi, Bacillus subtilis, and the like.

  According to the present invention, microorganisms in a specimen can be cultured by a simple operation, and the number thereof can be easily measured. Moreover, since the culture device of the present invention is not complicated, it is easy to manufacture.

The figure showing the one aspect | mode of the culture equipment of this invention. (A) is an orthographic view of the culture equipment in a state where the upper member and the lower member do not overlap, (B) is an example of a sectional view taken along line AA ′ in (A), (B ′) is ( (A) Another example of a sectional view taken along line AA ′, (C) is an example of a sectional view taken along line BB ′ in (A), and (C ′) is a sectional view taken along line BB ′ in (A). Another example of the figure is shown respectively. The figure showing the one aspect | mode of the culture equipment of this invention. (A) is an orthographic view of the culture equipment in a state where the upper member and the lower member are not fitted, (B) is an example of a sectional view taken along line AA ′ in (A), (B ′) (A) Another example of a cross-sectional view taken along line AA ′, (C) is an example of a cross-sectional view taken along line BB ′ in (A), and (C ′) is taken along line BB ′ in (A). Another example of a sectional view is shown respectively. The figure showing the one aspect | mode of the culture equipment of this invention. (B) is an example of a cross-sectional view taken along line CC ′ in (B), and (C) is (B). Another example of a sectional view taken along line CC ′ of FIG. The oblique projection figure which shows the usage example of the culture equipment of this invention. The figure showing the one aspect | mode of the culture equipment of this invention. (B) is an example of a cross-sectional view taken along line DD ′ in (B), and (C) is (B). Another example of a cross-sectional view taken along line EE ′ in FIG. 4A is a usage example in which the upper member and the lower member are fitted together. The photograph of the red colony detected with the culture equipment of the Example.

The culture equipment of the present invention will be described with reference to the drawings.
The culture device of the present invention has (a) an upper member (30), (b) a lower member (10) having a recess, and (c) a culture medium component (20) (FIG. 1). Usually, the upper member (30) is used so as to cover the concave portion of the lower member (10). In the covered state, an appropriate space exists between the upper member and the recess of the lower member, and a medium component exists. In such a state, a space surrounded by the bottom surface and side surfaces of the recesses of the upper member and the lower member is a space where a medium formed by the medium components and the specimen exists (hereinafter also referred to as “medium region”).

  In a preferred embodiment, the upper member (30) has a concave portion of the lower member (10) and a convex portion having a shape that can be fitted to each other via the culture medium component (20) (FIG. 3). In this aspect, as shown in FIG. 3, for example, the convex portion of the cylindrical upper member is fitted into the concave portion of the cylindrical lower member having a slightly larger diameter. In the fitted state, the upper surface of the convex portion of the upper member and the bottom surface of the concave portion of the lower member do not need to be in close contact with each other, and an appropriate space exists between the convex portion of the upper member and the concave portion of the lower member. However, there are medium components. In the fitted state, a space surrounded by the upper surface of the convex portion of the upper member and the bottom surface and side surfaces of the concave portion of the lower member is a culture medium region.

The volume of the culture medium region can be arbitrarily designed depending on the type of specimen to be measured and the scale of the test. For example, it is preferable to reduce the culture equipment to a volume of about 1 mL. In addition, when the upper member is placed on the lower member, the medium region should not be too large compared to the amount of the sample so that the sample can be spread over the entire medium region by pressing or the like (the depth of the recess of the lower member). It is preferable to design so that it does not become too large. In addition, when the upper member has a convex portion, the medium region does not become too large compared to the amount of the sample so that the convex portion can spread the sample over the entire medium region by fitting (the upper member). It is preferable to design so that the height of the convex portion of the projection does not become too small. This is also desirable from the viewpoint that if the amount of specimen is smaller than the medium region, voids are generated, making it difficult to accurately observe and measure colonies.
On the other hand, if the colonies overlap in the vertical direction, it is difficult to accurately observe and measure, so the medium area does not become too small compared to the amount of the specimen (recesses that do not make the medium area too thick). (Bottom area) is preferable. This is because gels made from guar gum or xanthan gum are not very transparent compared to gels made from polyacrylic acids, so it becomes difficult to accurately observe and measure colonies when the medium is thick. Is also desirable.

The thickness of the culture medium region, that is, the distance between the upper member and the bottom surface of the lower member concave portion, or the upper surface of the upper member convex portion and the lower member concave surface in a state in which the upper member is fitted in a form having the convex portion Is not particularly limited, but is preferably 0.01 to 1 mm, more preferably 0.02 to 0.8 mm.
The volume of the culture medium region, that is, the space surrounded by the bottom surface and the side surface of the concave portion of the upper member and the lower member, or the upper surface of the convex portion of the upper member and the lower member in a state where the upper member has a convex portion The volume of the space surrounded by the bottom and side surfaces of the recess is not particularly limited, but is preferably 1.0 to 1.5 mL, more preferably 1.0 to 1.2 mL.
When applying the analyte xmL the culture device of the present invention is not particularly limited, the bottom area of the recess is preferably 10X～100xcm ^2, more preferably 20x~50xcm ^2. For example, in the case of a culture device for applying a 1.0 mL specimen, the bottom area of the recess can be 10 to 100 cm ^2, and when the recess is circular, the radius is 1.8 to 5. What is necessary is just to design with 6 cm.

  The convex part of the upper member and the concave part of the lower member may be of any shape as long as they can be fitted. Further, the upper surface of the convex portion of the upper member and the bottom surface of the concave portion of the lower member may be flat or curved, but are preferably flat from the viewpoint of operability.

(C) The culture medium component is applied to at least a part of the upper member, the portion facing the concave portion of the lower member when the lower member is covered, that is, the portion forming the culture medium region and / or the concave portion of the lower member. It is preferable. In the aspect in which the upper member has a convex portion, the (c) medium component is preferably applied to at least a part of the convex portion of the upper member and / or the concave portion of the lower member. The application site is usually a portion facing the culture medium region when the upper member and the lower member are fitted, and at least a part of the upper surface of the convex portion of the upper member and / or the bottom surface of the concave portion of the lower member is formed. Preferably, all are more preferable. In a preferred embodiment of the present invention, the culture medium component is applied to the entire bottom surface of the recess of the lower member.
The medium component applied to the upper member and / or the lower member is in a thin film form so that the specimen applied to the culture equipment can be smoothly diffused throughout the medium area and the specimen and the medium component can be uniformly gelled. Is preferred. Although the thickness of this thin film is not specifically limited, Preferably it is 0.001-0.02 mm, More preferably, it is 0.005-0.01 mm.

  In the present invention, the material of the upper member and / or the lower member is not particularly limited, and polyacrylic, polyvinylic, polyethylene, and polyester polymers can be employed. Moreover, the rigidity of the material is not particularly limited. However, when the upper member does not have a convex portion, it is preferable that the rigidity is appropriately deformable so that pressing after addition of the liquid sample is facilitated.

In the present invention, the upper member and / or the lower member are preferably transparent, and more preferably the upper member and the lower member are transparent. Preferred examples of the material for forming the transparent upper member and / or lower member include polyethylene, polypropylene, polyethylene terephthalate, vinyl chloride, polystyrene, polyester, and polycarbonate. This makes it possible to easily observe and measure the colonies of microorganisms to be measured from the outside without decomposing the culture equipment.
Here, the term “transparent” may be such that the opposite side of the member can be seen through, and more specifically, the visible light transmittance is preferably 70% or more, but is not limited thereto.

In the culture device of the present invention, the upper member and the lower member may be separated separately or may be integrated.
For example, a part of the upper member and a part of the lower member may be continuous by sharing one side as shown in FIGS. 4 and 5. In such a case, it is preferable to fold the culture equipment, overlap the upper member and the lower member, and cover the upper member with the lower member, so that the convex portion of the upper member and the concave portion of the lower member are preferably fitted. It becomes possible to use it.
In the culture device of the present invention, the upper member and the lower member may each have a plurality of convex portions and concave portions. That is, it may be an embodiment in which a plurality of medium regions are formed at the time of use, and is suitable for processing a plurality of specimens in parallel.
In addition, in the culture equipment of the present invention, the upper member may have a convex portion or a concave portion, and the lower member may have a concave portion or a convex portion, which can be fitted to each other separately from the portion forming the culture medium region. Good. In the case of such an embodiment, the upper member and the lower member which are overlapped are hardly separated by fitting, so that formation of the medium region after application of the specimen and press for diffusion of the medium are easily maintained.
Further, in the culture device of the present invention, the size and shape of the upper member and the lower member are not particularly limited, but when the upper member and the lower member are overlapped, at least the outer edge of the portion other than the portion forming the culture medium region It is preferable that the upper member and the lower member have different sizes or shapes so that a part thereof is displaced. In the case of such an aspect, handling becomes easy when the upper member and the lower member which have been overlapped are separated again.

The (c) medium component in the culture device of the present invention contains (c1) one or more selected from guar gum and xanthan gum, and (c2) a nutrient component.
In the present invention, the (c) medium component is for preparing a medium for culturing microorganisms. The preparation is usually performed by adding and infiltrating a liquid specimen containing a measurement target microorganism as it is to a medium component as a solvent for a gel constituting the medium.

  Here, (c1) guar gum and xanthan gum play a role of a gelling agent constituting the medium. Guar gum and xanthan gum have a slower rate of water absorption (water absorption) than polyacrylic acids, so that the liquid specimen is uniformly gelled while diffusing the entire medium area to form a thinly spread gel medium. Suitable. Xanthan gum has a relatively strong gelling ability (water-holding ability) than guar gum.

Usually, a specimen gelled with guar gum or xanthan gum does not have fluidity, and the number of microorganisms can be accurately measured. In addition, since the water is hardly generated from the gel, the presence of microbial colonies can be measured not only qualitatively but also accurately.
Further, since guar gum and xanthan gum can form a gel without dissolving by heating and without cooling, the medium formation operation is simple and does not hinder the growth of the target microorganism.

When contained in the medium component, only guar gum or only xanthan gum may be used, or a mixture of guar gum and xanthan gum may be used to adjust the gelation rate. In the case of a mixture, gelation tends to be relatively fast when the guar gum amount ratio is high, and gelation tends to be relatively slow when the xanthan gum amount ratio is high.
Further, other gelling agents may be used in combination as long as the effects of the present invention are not hindered.

In general, gelling agents such as agar, carrageenan and locust bean gum are used for microbial media, etc., but these require heating when uniformly solidifying the liquid sample. It is not suitable for solidifying as it is or for the form of simple culture equipment. Moreover, the gel solidified using the said gelling agent is not suitable also for a point with low transparency.
In addition, polyvinyl alcohol has problems that it is difficult to mix uniformly with a liquid specimen and that water is easily separated.
Since carboxymethyl cellulose cannot solidify a liquid specimen and becomes a fluid gel, it is not suitable for quantitative detection of microorganisms.

  The concentration at the time of use of guar gum and / or xanthan gum in the present invention is not particularly limited, but from the viewpoint of solidification ability, 0.01 to 0.1 g / mL is preferable as a total amount, and 0.01 to 0.05 g / mL. Is more preferable. Therefore, it is preferable to apply the medium component so that the concentration at the time of use falls within the above range according to the specimen volume targeted by the culture equipment.

(C) The nutrient component (c2) contained in the medium component is for growing the target microorganism.
Although it does not specifically limit as a nutrient component, Peptone, a beef meat extract, a yeast extract, a fish meat extract etc. are mentioned preferably.
In the water supply test method, which is one of the methods for measuring the number of microorganisms, it is recommended to use the standard agar medium and the R2A agar medium in the pharmaceutical water and dialysis water tests. Therefore, when used in such applications, it is preferable that the medium component in the present invention contains a bouillon medium excluding the agar of the agar medium or a component equivalent thereto.

It is preferable that the (c) medium component further contains (c3) a color reagent. This is to make it easier to detect and measure colonies of microorganisms generated by culture as colored ones.
Examples of the color reagent include redox indicators such as 2,3,5-triphenyltetrazolium chloride (TTC) and tetrazolium violet. This can be preferably used when it is desired to measure all types of microorganisms present in the specimen. When TTC is used, the concentration during use is preferably 1 to 100 mg / L, more preferably 10 to 50 mg / L.

Moreover, as the color reagent, a compound that is a substrate (hereinafter referred to as an enzyme substrate) for an enzyme possessed only by a specific microorganism species and can release a coloring compound by being decomposed may be used. This can be preferably used when it is desired to measure the specific microorganism.
Here, the dye compound may be either a colored compound or a fluorescent compound that develops color under visible light. Examples of the functional group that can be liberated as a colored compound under visible light include 5-bromo-4-chloro-3-indoxyl group, and the liberated 5-bromo-4-chloro-3-indole is oxidized and condensed. 5,5′-dibromo-4,4′-dichloro-indigo, which is blue. Examples of the functional group that can be liberated as a fluorescent coloring compound include a 4-methylumbelliferyl group, and the released 4-methylumbelliferone fluoresces under ultraviolet irradiation.

Examples of enzyme substrates include 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-GAL) and 5-bromo-4- if the target microorganism is a coliform group. Chloro-3-indoxyl-β-D-glucuronic acid, etc., in the case of Staphylococcus aureus, 5-bromo-4-chloro-3-indoxyl phosphate (X-phos), etc., enterococci, etc. Is 5
-Bromo-4-chloro-3-indoxyl-β-D-glucopyranoside (X-GLUC) or the like in the case of fungi, X-phos, 5-bromo-4-chloro-3-indoxyl acetate or butyric acid 5 -Bromo-4-chloro-3-indoxyl and the like can be preferably used. Furthermore, when it is desired to detect all microbial species, all of these may be used in combination.
The concentration of these enzyme substrates when used is, for example, preferably 0.01 to 1.0 g / L, more preferably 0.1 to 0.5 g / L.

The (c) medium component preferably further contains (c4) a thickener. This serves as an adhesive for stably applying the medium component to the upper member and / or the lower member.
Such a thickener is not particularly limited as long as it does not affect the growth of microorganisms. For example, hydroxypropyl cellulose, polyvinyl pyrrolidone, polyvinyl alcohol, carboxymethyl cellulose, methyl cellulose, starch and derivatives thereof, polyether, hyaluron An acid, collagen, etc. are mentioned.

(C) The medium component may further optionally contain a selective substance, an antibacterial substance, an inorganic salt, a saccharide, a thickener, a pH adjuster and the like as long as the effects of the present invention are not hindered.
Examples of the selective substance include antibiotics such as polymyxin B and vancomycin, and surfactants such as bile salts such as sodium lauryl sulfate (SDS), Tween 80, and sodium cholate.
Examples of the antibacterial substance include polylysine, protamine sulfate, glycine, sorbic acid and the like.
Examples of the inorganic salts include inorganic acid metal salts such as sodium chloride and sodium thiosulfate, and organic acid metal salts such as sodium pyruvate, ammonium iron citrate, and sodium citrate.
Examples of the saccharide include glucose, lactose, sucrose, xylose, cellobiose, and maltose.
Examples of the pH adjuster include sodium carbonate and sodium hydrogen carbonate. In addition, the composition of the present invention is such that the pH during use is preferably 6.0 to 8.0, more preferably 6.5 to 7.5, from the viewpoint of the growth of the target microorganism. .

Although the culture device of the present invention can be produced by any method, an example will be described.
The upper member and the lower member are made of an acrylic plate having an appropriate size. What is necessary is just to produce the convex part of an upper member, and the recessed part of a lower member by the shaping | molding by adhesion | attachment or a punching of an acrylic board, or the press using a metal mold | die, etc., or injection.
(C) A culture medium component dissolved or suspended in a non-aqueous solvent is applied to a part or the whole of the convex part of the upper member and / or the concave part of the lower member, and then dried to obtain the culture equipment. Can be painted.
Here, the non-aqueous solvent is preferably one that can volatilize at room temperature and normal pressure, and preferred examples include lower alcohols such as ethanol, methanol, propanol, and butanol. If these non-aqueous solvents are used, medium components can be applied without gelation of (c1) guar gum or xanthan gum at the time of production, so that the culture equipment can be easily produced.

The culture device of the present invention described above can be suitably used for a method of culturing microorganisms in a specimen and measuring the number of microorganisms.
Specifically, the measurement method includes (b) a step of adding a specimen to the concave portion of the lower member, (a) a step of covering the upper member with the concave portion of the lower member, and culturing microorganisms contained in the specimen. Preferably, the method includes a step and a step of measuring the number of colonies of the microorganism. In the step of covering the lower member with the upper member, the concave portion is more preferably pressed from outside the equipment. Thereby, the specimen added to the concave portion of the lower member is uniformly spread over the entire culture medium region. In addition, (c1) guar gum or xanthan gum in the medium component gels more rapidly due to the moisture of the specimen, and the medium is easily formed.
In addition, when the upper member uses a culture device having a concave portion of (b) the lower member and (c) a convex portion having a shape that can be fitted to each other via the medium component, (b) the lower member of the culture device A step of adding a specimen to the concave portion, (a) a step of fitting the convex portion of the upper member into a concave portion of the lower member, (b) a step of culturing microorganisms contained in the specimen, and measuring the number of colonies of the microorganisms It is preferable to include a process.
By fitting the convex portion of the upper member into the concave portion of the lower member, the specimen added to the concave portion of the lower member is uniformly spread over the entire culture medium region. In addition, (c1) guar gum or xanthan gum in the medium component gels more rapidly due to the moisture of the specimen, and a thin, flat, non-fluid medium is easily formed.

The culture conditions of the microorganism are not particularly limited, but are appropriately selected depending on the type of the target microorganism. For example, the culture condition is preferably 35 ± 2 ° C. for 24 to 48 hours.
Growing colonies of the target microorganism appear in the cultured medium, and this is measured. The number of colonies of microorganisms can be measured without disassembling the culture equipment, and it can be measured by visual confirmation from the outside or by analyzing what was captured with a camera or the like with image analysis software. . According to the measuring method of the present invention, the number of colonies can be accurately measured.

The sample to which the measurement method of the present invention can be applied is not particularly limited, but preferably includes liquid samples such as drinking water, soft drinks, industrial water, pharmaceutical water, dialysis water, and urine. Moreover, the culture solution which culture | cultivated these specimens beforehand with trypto soy broth etc. may be sufficient.
The measurement method of the present invention can also be applied to a diluted sample, and can be preferably used for the measurement method of the present invention even when the amount of microorganisms in the sample is, for example, 300 CFU / mL or less.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

(1) Production of culture equipment A transparent 0.4 mm thick PET plate was molded, and an upper member having a cylindrical convex part with a diameter of 61.433 mm and a height of 2.13 mm, a diameter of 62 mm and a depth of 2.53 mm. A lower member having a columnar recess at the center of a 107.425 mm triangle on each side was produced. Note that the upper member and the lower member are joined by a hinge (FIG. 5).
100 mL prepared dose (3 g) trypto soy bouillon medium powder (Becton Dickinson), 0.0025 g TTC, 1.5 g xanthan gum, 1.5 g guar gum, and 0.1 g hydroxypropylcellulose in 80 mL ethanol. Suspended. 1200 μL of the mixed solution was added to the concave portion of the lower agent made of the PET plate prepared above, and spread uniformly, and then dried at 65 ° C. for 5 minutes.

(2) Test of strain Using Escherichia coli NBRC102203, the test strain was pre-cultured for 24 hours on trypsoy agar medium, and then equivalent to McFarland turbidity # 1 (about 3.0 × 10 ⁸ CFU / mL). Thus, it was suspended in a sterilized physiological saline using a sterilized cotton swab to obtain a bacterial stock solution. Using each bacterial stock solution, 10-fold serial dilution was repeated up to 10 ⁻⁸ with sterile physiological saline to prepare a tens of CFU / 1 mL bacterial dilution sample. Inoculate 1 mL of the bacterial dilution sample into the concave part of the lower member of the culture equipment prepared in (1), immediately fit the convex part of the upper member, spread the bacterial diluted sample uniformly into the concave part, and penetrate the medium components To form a medium. After culturing at 35 ° C. for 24 hours, the presence or absence of growth was confirmed.

FIG. 6 shows colonies of Escherichia coli NBRC102203.
When the culture device of the present invention was used, the liquid sample could be uniformly spread over the entire culture medium region by simply fitting the upper member and the lower member, regardless of the capillary phenomenon caused by the spreader or other instrument or non-woven fabric. Moreover, the gelling agent in the medium component quickly solidified uniformly, and a thin, flat medium without fluidity was formed. After culturing, as shown in FIG. 6, red colonies could be visually confirmed in an almost transparent gel, and the number could be easily measured. Moreover, since the culture device of the present invention is not complicated, it can be easily produced.

  According to the present invention, microorganisms in a specimen can be cultured by a simple operation, and the number thereof can be easily measured. In addition, the culture device of the present invention is industrially useful because it is not complicated and can be easily manufactured.

1: Culture equipment 10: Lower member 20: Medium component 30: Upper member

Claims (9)

(A) an upper member, (b) a lower member having a recess, and (c) a medium component,
(C) The culture medium component comprises (c1) one or more selected from guar gum and xanthan gum, and (c2) a nutrient component.   The culture device according to claim 1, wherein (a) the upper member has (b) a concave portion of the lower member and (c) a convex portion having a shape that can be fitted to each other via a medium component.   In a state in which the convex portion of the upper member and the concave portion of the lower member are fitted together, the distance between the upper surface of the convex portion of the upper member and the bottom surface of the concave portion of the lower member is 0. The culture equipment according to claim 2, which is 0.01 to 1 mm.   (A) In a state where the convex portion of the upper member and the concave portion of (b) the lower member are fitted, (a) the upper surface of the convex portion of the upper member and (b) the bottom surface and side surfaces of the concave portion of the lower member are surrounded. The culture equipment according to claim 2 or 3, wherein the volume of the space is 1.0 to 1.5 mL.   The culture medium according to any one of claims 2 to 4, wherein the (c) medium component is applied to at least a part of (a) the convex portion of the upper member and / or (b) the concave portion of the lower member. Equipment.   The culture equipment according to any one of claims 1 to 5, wherein (a) the upper member and / or (b) the lower member is transparent.   A method for culturing microorganisms in a specimen using the culture device according to any one of claims 1 to 6 and measuring the number of microorganisms. (B) adding a specimen to the recess of the lower member;
(A) (b) a step of covering the concave portion of the lower member with the upper member,
The method according to claim 7, comprising a step of culturing a microorganism contained in the specimen, and a step of measuring the number of colonies of the microorganism. (A) the upper member of the culture equipment has (b) a concave portion of the lower member and (c) a convex portion having a shape that can be fitted to each other via a medium component;
(B) adding a specimen to the recess of the lower member;
(A) the step of fitting the convex portion of the upper member into the concave portion of (b) the lower member;
The method according to claim 7, comprising a step of culturing a microorganism contained in the specimen, and a step of measuring the number of colonies of the microorganism.