JP3442865B2 - Film-like medium and method for detecting microorganisms using the same - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to the detection of various microorganisms,
The present invention relates to a film medium useful for identification and transportation, and a method for detecting microorganisms using the same.

[0002]

2. Description of the Related Art In detecting and identifying various microorganisms, a plate smear method, a pour method, a liquid medium method, etc. are generally used as a conventional method for culturing microorganisms. These methods require preparation of sterilization of the medium and instruments before culturing, and require operation requiring skill such as smearing and pour after inoculating the test sample into the medium.
Further, transportation of the medium after inoculation of the test sample and sterilization of the medium after measuring the colonies that have grown after culturing require a great deal of labor due to the bulky equipment and medium.

Therefore, a simple inspection method using a film-shaped medium that can be easily cultured has been reported. Examples thereof include (1) a medium in which an adhesive layer, a cold water-soluble gelling agent powder layer containing a nutrient component, and a cover sheet are sequentially laminated on the upper surface of a waterproof substrate (JP-A-57-502200).
(2) A medium in which an air-permeable membrane, a cold water-soluble gelling agent powder layer containing a nutrient component, and a cover sheet are sequentially laminated on the upper surface of a waterproof substrate (JP-A-3-15379),
(3) Detection paper obtained by impregnating a filter paper or the like with a nutrient component of bacteria and covering the surface with a cover sheet (Japanese Patent Laid-Open No. 2-65798).
Issue).

[0004]

However, in the film-shaped medium of the above (1) and (2), it is necessary to apply a pressure with a spreader after the sample inoculation in order to diffuse the sample into a certain area. There was a problem. Further, in the film-like medium of (3) above, since colonies are formed inside the filter paper, it is difficult to observe the colonies, so that there is a problem that the accuracy is lowered and further the bacilli cannot be formed. Therefore, it is an object of the present invention to provide a film medium which is excellent in operability such as inoculation of a sample, which allows easy observation of colonies and allows accurate detection, and a method for detecting microorganisms using the same.

[0005]

Therefore, as a result of various investigations on the film-shaped medium from both aspects of the operability and the detectability of colonies, the present inventors have used a sheet capable of diffusing water by capillary action as a substrate, If a thin film containing a medium composition containing two specific polymer compounds and nutrients for cell culture is formed on one side of the sheet, the sample solution can be easily diffused simply by dropping it on the film-shaped medium. Further, they have found that clear colonies are formed on the formed thin film, and that a film-shaped medium having good detectability can be obtained, and thus the present invention has been completed.

That is, according to the present invention, (a) a polymer compound soluble in water and alcohol, (b) a water-soluble but alcohol-insoluble high-molecular compound on one surface of a sheet capable of diffusing water by a capillary phenomenon. It is intended to provide a film-shaped medium characterized by forming a thin film containing a medium composition containing a molecular compound and a nutrient for cell culture (c).

Further, the present invention comprises detecting a colony formed on a thin film forming surface by inoculating a non-thin film forming surface of the above film-like medium with a sample liquid and culturing, and detecting a colony formed on the thin film forming surface. It provides a method.

The sheet used as the substrate of the film-like medium of the present invention diffuses water by a capillary phenomenon because the sample solution inoculated on the sheet diffuses and allows microorganisms in the sample solution to reach the medium composition. It must be able to do. Examples of the sheet having such properties include filter paper, rayon nonwoven fabric, polyester nonwoven fabric,
Examples thereof include synthetic fiber nonwoven fabrics typified by polypropylene nonwoven fabrics and natural fiber nonwoven fabrics typified by cotton nonwoven fabrics. Among them, filter paper, rayon nonwoven fabric and long fiber cellulose nonwoven fabric are preferable, and filter paper and rayon nonwoven fabric are particularly preferable.

These sheets are not particularly limited as long as they have the above-mentioned properties, but have a density of 5 to 200 g / m ² in consideration of both water diffusivity and thin film forming property, and have a density of 0. Sheets having a thickness of 05 to 0.5 mm, particularly filter paper and rayon non-woven fabric, are preferred.

The shapes of these sheets are square,
It is not particularly limited to a rectangle, a circle and the like. The size is also not particularly limited, but in the case of simple detection of microorganisms, the major axis is preferably 1 to 15 cm.

In the present invention, a thin film containing a medium composition containing the above-mentioned components (a), (b) and (c) is formed on one side of the sheet, and the medium composition is a sheet. It is desirable that it hardly penetrates into the structure. When the medium composition penetrates into the structure of the sheet, colonies of microorganisms are formed in the structure of the sheet, so that it is not easy to observe the colonies with the naked eye, and the bacilli cannot be formed. On the other hand, in the present invention, since the thin film containing the medium composition is formed on one side of the sheet, colonies are formed in the thin film, so that the observation is easy with the naked eye and the bacillus is also easy.

The thin film is formed, for example, in the above (a) and (b).
And a method in which an alcohol suspension containing the component (c) is applied to one side of a sheet and then dried; and a method in which a thin film obtained by drying the alcohol suspension is laminated on one side of the sheet. In alcohol, the component (a) is dissolved, but the component (b) is not dissolved, so that even if the coating method is adopted, it hardly penetrates into the structure of the sheet and is uniformly coated on the surface. . When the laminating method is adopted, it is preferable that the thin film is formed by, for example, applying a sheet to a thin film obtained by applying the alcohol suspension on the surface of glass or plastic such as a petri dish and then drying, and fixing the sheet. The film medium obtained by the method of laminating on the glass or plastic surface inside the petri dish may be used as it is in a state where the film medium is fixed in the petri dish. In addition, since the film-shaped medium prepared by the laminating method has high transparency, it is easy to observe with the naked eye and is particularly preferable.

Examples of the (a) water- and alcohol-soluble polymer compound used in the medium composition include hydroxypropyl cellulose, polyvinylpyrrolidone and polyethylene oxide, with hydroxypropyl cellulose being particularly preferred. The component (a) is preferably added to the medium composition in an amount of 0.1 to 10% by weight, particularly 0.5 to 3% by weight.

Examples of the polymer compound (b) soluble in water and insoluble in alcohol include polymer compounds usually used as gelling agents, such as xanthan gum, locust bean gum, guar gum, carrageenan and pectin. A natural gelling agent; a synthetic gelling agent such as polyacrylamide is preferable. Of these, xanthan gum is particularly preferable. The component (b) is preferably added to the medium composition in an amount of 1 to 30% by weight, particularly 5 to 15% by weight.

As the nutrient for cell culture (c), a water-soluble nutrient is selected, which is suitable for the growth of the microorganism to be detected. For example, when multiplying various types of microorganisms,
A general nutrient medium component is used, and a selective medium component is used when selectively growing a specific microorganism. The component (c) is usually added to the medium composition in an amount of 0.5 to 20% by weight, preferably 1 to 5% by weight.

Further, in order to facilitate the observation of colonies, it is preferable to add various coloring agents to the medium composition. Such coloring agents include dyes that color colonies, such as triphenyltetrazolium chloride, 3- (p-
Iodophenyl) -2- (p-nitrophenyl) -5-
Phenyl-2H-tetrazolium chloride, 3-
Dyes such as (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide; 5
Enzyme substrates such as -bromo-3-indolyl-β-D-galactoside: pH indicators such as bromthymol blue and neutral red are included. Further, in order to facilitate observation of colonies, the sheet itself may be colored without adding a color former.

The concentration of the medium composition in the alcohol suspension in forming a thin film containing the medium composition containing these components does not allow the medium composition to easily penetrate into the substrate sheet during coating. The concentration is preferably about 1 to 70% by weight, particularly 6 to 30% by weight.
Is preferred. Here, the alcohol has 1 to 5 carbon atoms.
Are preferred, and ethanol is particularly preferred.

The alcohol suspension may be applied to a sheet or a glass surface by coating with a bar coater, a doctor blade, a baker applicator, or spraying (spraying). The baker applicator is preferable. Then, the drying is natural drying,
Vacuum drying and hot air drying are preferred. Furthermore, it is preferable to sterilize with gas, γ ray, electron beam or the like.

The thin film layer on the sheet in the obtained film medium is preferably 0.01 to 2 mm, particularly preferably 0.1 to 1 mm.

In the film-shaped medium of the present invention, a thin film containing one type of medium composition may be uniformly formed on one surface of the sheet (for example, FIG. 1), and the nutrient for cell culture and the coloring agent may be changed. A thin film containing a plurality of types of culture medium compositions may be formed (for example, FIGS. 2 and 3).

The obtained film medium of the present invention is covered with a bag of a water-repellent film, a plastic petri dish, a glass petri dish or the like for the purpose of preventing contamination by various bacteria until it is subjected to the test. preferable.

Cultivation and detection of microorganisms using the film-form medium of the present invention is carried out by inoculating a non-thin film forming surface with a sample solution and culturing, and detecting the colonies formed on the thin film forming surface. . The microorganisms in the sample liquid inoculated on the non-thin film forming surface diffuse with the liquid in the sheet by the capillary phenomenon and reach the thin film forming surface. Since the components (a), (b) and (c) are present in a solid state on the thin film forming surface, the components (a) and (b) grow among the particles by utilizing the component (c) to form colonies. To form. Therefore, the formed colonies can be easily observed by observing the thin film formation surface. If the sample is quantitatively inoculated into the film-shaped medium, the number of bacteria can be easily calculated by measuring the colonies appearing after the culture. A method of inoculating a fixed amount with a pipette or the like is usually adopted for inoculation of the bacterial cell liquid, but a method of stamping a solid with a large amount of water on the non-thin film forming surface may be used. Further, the culture after inoculation of the sample liquid may be allowed to stand still, and may also serve as the culture during the transportation period.

[0023]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 Peptone 1.7 g, soybean peptone 0.3 g, sodium chloride 0.5 g, glucose 0.2 as the components of the medium composition.
5 g, potassium monohydrogen phosphate 0.25 g, xanthan gum (San-eigen F.F.A.I.) 10 g, hydroxypropyl cellulose (HPC, Wako Pure Chemical Industries, Ltd.) 1 g, triphenyltetrazolium chloride 0.002 g, and suspended in ethanol 100 ml, It was prepared by stirring well. Next, this ethanol suspension was filtered with a filter paper (Advantech No. 1
55 .mu.mm), 1.5 ml of each solution was uniformly coated with a baker applicator, dried and then inserted into a polyethylene bag (thickness 40 .mu.m, 60.times.85 mm), and sterilized with ethylene oxide gas for testing.

35 various trypsin broths with various strains
After culturing at ℃ for 24 hours, 10-fold serial dilution was performed.
Inoculate each non-thin film surface of the film medium with ml
Alternatively, the number of bacteria was measured after inoculating a trypto soya agar medium (TSA, Nissui Pharmaceutical Co., Ltd.) and culturing at 35 ° C. for 24 hours. The results are shown in Table 1. As a result, various strains showed almost the same number of bacteria in the film medium and TSA.
When colonies formed on the film-shaped medium were stabbed with a platinum wire, the bacteria were picked up, and the TSA plates were further inoculated and cultured at 35 ° C. for 24 hours.

[0026]

[Table 1]

Example 2 Soybean peptone 1.0 g, peptone 0.5 g, sodium chloride 0.5 g, yeast extract 0.
5 g, dipotassium hydrogen phosphate 0.25 g, sodium pyruvate 0.1 g, potassium nitrate 0.1 g, sodium lauryl sulfate 0.02 g, xanthan gum (San-eigen F.F.A.I.) 10 g and hydroxypropyl cellulose (HPC, Wako Pure) The drug was suspended in 100 ml of ethanol using 1 g of the drug and 0.03 g of 5-bromo-3-indolyl-β-D-galactoside (X-Gal), and stirred well to prepare. The method for producing the film-shaped medium was the same as in Example 1.

35 different trypsin broths
After culturing at ℃ for 24 hours, 10-fold serial dilution was performed.
ml of the film-shaped medium (X-Gal) on the non-thin film forming surface, or x-Gal agar medium (xanthan gum and HPC were excluded from the medium components, and 1.5% was used instead).
% Agar was added) and cultured at 35 ° C. for 24 hours, and the number of bacteria was measured. The results are shown in Table 2. As a result, in each strain, the number of bacteria was almost the same in both the film-like medium (X-Gal) and the X-Gal agar plate.

[0029]

[Table 2]

Example 3 The production of a film medium was carried out in the same manner as in Example 1. A commercially available food was purchased, 90 ml of sterile physiological saline was added to 10 g of the food, and the sample was serially diluted 10 times, and 1 ml of the sample was inoculated on the non-thin film forming surface of the film medium. Further, 1 ml of the same sample was placed in a sterile petri dish, and a standard agar medium (Nissui Pharmaceutical Co., Ltd.) which had been sterilized in advance and cooled to about 45 ° C. was poured and poured. The culture is
After culturing at 35 ° C. for 48 hours, emerged colonies were calculated.
The results are shown in Table 3. As a result, in various foods, the film medium and the standard agar medium showed almost the same number of bacteria.

[0031]

[Table 3]

Example 4 Ethanol suspension 1 having the same medium composition as in Example 1
ml was dropped on a 47 mm dish, spread evenly and dried, and then a semitransparent rayon nonwoven fabric having a density of 13.5 g / m ² was contact-fixed. This was tested after sterilization with ethylene oxide gas. After culturing various strains in trypto soya broth at 35 ° C for 24 hours, 10-fold serial dilution was performed, and 1 ml of each was inoculated on the non-thin film-formed surface of the above film medium, and the bacteria were observed after culturing at 35 ° C for 24 hours. went. As a result, the medium and the diffusion sheet became transparent, and the grown colonies could be easily observed from both the front and back sides.

[0033]

EFFECTS OF THE INVENTION By using the film medium of the present invention, it is easy to inoculate a sample solution, and moreover, colonies can be observed and bacilli can be accurately and easily provided, so that various microorganisms can be easily tested. it can.

[Brief description of drawings]

FIG. 1 is an explanatory view showing a film-shaped medium of the present invention in which a thin film containing a medium composition having the same composition is formed on one entire surface of a sheet.

FIG. 2 is an explanatory view showing the film medium of the present invention in which a thin film containing a medium composition having a different composition is formed on one surface of a sheet.

FIG. 3 is an explanatory view showing a film-shaped medium of the present invention in which a thin film containing a medium composition having a different composition is formed on one surface of a rectangular sheet.

─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Kazuki Horimei 1075-2 Kitanan Mororo, Yuki City, Ibaraki Prefecture Nissui Pharmaceutical Co., Ltd., Diagnostic Drug Research Department (56) References JP-A-49-20385 (JP, A) JP-A-52-154589 (JP, A) JP-A-50-125081 (JP, A) JP-B-46-28813 (JP, B1) JP-A-57-502200 (JP, A) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12Q 1/00-3/00

Claims (5)

(57) [Claims] 1. A polymer compound soluble in water and alcohol, (b) a polymer compound soluble in water and insoluble in alcohol, on one side of a sheet capable of diffusing water by a capillary phenomenon. (C) A film-shaped medium comprising a thin film containing a medium composition containing a nutrient for cell culture. 2. The thin film containing the medium composition is formed by coating an alcohol suspension of the medium composition on one side of the sheet and then drying it, or the medium composition. The film-shaped culture medium according to claim 1, which is formed by laminating a thin film obtained by drying the alcohol suspension of 1. on one side of the sheet. 3. The film-shaped medium according to claim 1, wherein the medium composition further contains a coloring agent. 4. A thin film containing a plurality of medium compositions having different compositions is formed on one sheet.
The film-shaped medium according to any one of 3 above. 5. Detection of a microorganism in a sample, which comprises inoculating a non-thin film-formed surface of the film-form medium according to claim 1 with a sample solution and culturing the mixture to detect colonies formed on the thin film-formed surface. Method.