JPH08173190A - Method for examining presence of microorganism and examination kid used therefor - Google Patents

Abstract

PURPOSE: To simply examine the existence of a microorganism and its amount in a food, a medicine, a cosmetic, etc., by judging the consumption amount of oxygen due to the growth of the microorganism in a microorganism culture solution by the change in the color tone of an oxidation-reduction indicator. CONSTITUTION: An oxidation-reduction indicator such as methylene blue or resazurin is added to a culture solution for a microorganism, and the microorganism existing in a specimen to be examined is raised in a closed container. The color tone of the oxidation-reduction indicator is changed by the consumption of oxygen with the growth of the microorganism. Thereby, the presence of the microorganism existing in the specimen to be examined, and the amount of the microorganism can be measured or examined by the simple operation in a short time.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention adds a test substance into a closed container containing a culture solution and allows microorganisms existing in the test substance to grow in the closed container to reduce oxygen consumption accompanying growth of the microorganism. The present invention relates to a method for detecting the presence of microorganisms simply and in a short time by detecting, and a microorganism test kit used in the method.

[0002]

2. Description of the Related Art Generally, there are two methods for detecting contamination of foods, pharmaceuticals, chemical products and the like with microorganisms, that is, a method of culturing microorganisms and a method of not culturing microorganisms. As a method of culturing a microorganism, for example, a method of observing the turbidity of the medium accompanied by growth of the microorganism by culturing the microorganism and observing the turbidity of the medium is added to the test medium such as the sterility test method described in the Japanese Pharmacopoeia General Test Method A general method is to form colonies on a plate solid medium and count the number. In addition to the above, a method for measuring the amount of metabolites such as ethanol produced along with the growth of microorganisms (JP-A-4-11899) and a sensor composition containing a fluorescent substance whose fluorescence intensity decreases at the same time as exposure to oxygen Method to be used (JP-A-5-137596)
Is proposed. As a method of not culturing microorganisms,
A method of directly observing the number of microorganisms under a microscope or a method of observing the microorganisms under a fluorescence microscope after staining the microorganisms with a chemical such as acridine orange (edited by Ryuichi Sudo, Environmental Microbial Experiments, P89-9).
1, Kodansha Scientific, 1991) is commonly practiced. In addition, a method of extracting and quantifying adenosine triphosphate existing in the body of a microorganism has been put into practical use.

[0003]

In many cases, there is no special device for checking the presence or abundance of microorganisms at the production or storage site, and the sample is sent to an inspection center or the like for inspection. Much time is spent on. Therefore, a simple microorganism detection method that does not require a special device is desired. The method for measuring the turbidity of the medium associated with the growth of microorganisms, such as the sterility test method described in the General Test Method of the Japanese Pharmacopoeia, is an excellent general-purpose method in that no special device is required. However, if turbidity occurs when the test substance is added to the medium due to the turbidity of the test substance, the determination is difficult. A method of forming a colony of a microorganism on a flat plate solid medium and measuring the number thereof is also a simple method, and many base materials have been commercialized. However, there are many microorganisms that have no or weak colonization ability in the environment, and such microorganisms cannot be detected by this method. In the method for quantifying the metabolites produced with the growth of microorganisms, special equipment such as gas chromatography and high performance liquid chromatography is required for measurement. Although the method of directly observing the number of microorganisms under a microscope is a simple method, it is necessary to measure not only live cells but also dead cells which are not considered to be the direct cause of contamination. As a method of measuring only viable bacteria, there is a method of staining a microorganism with a chemical such as acridine orange and observing it under a fluorescence microscope.
Since the fluorescence microscope is extremely expensive, it cannot be said to be a general-purpose method. A special instrument is also required for measurement in the method of extracting and quantifying adenosine triphosphate existing in the body of the microorganism. A method of measuring the oxygen consumption accompanying the growth of microorganisms is also an effective means. In this respect, the method using a sensor composition containing a fluorescent substance whose fluorescence intensity decreases simultaneously with exposure to oxygen (Japanese Patent Laid-Open No. 137596/1993) is an excellent method, but unfortunately this method also measures fluorescence. Equipment is needed. Similarly, other physical and electrochemical methods for measuring oxygen consumption also require special equipment such as manometers and coulometers.

[0004]

An object of the present invention is to provide a method for detecting the presence and abundance of microorganisms such as foods, pharmaceuticals and chemical products by a very simple operation without using a special inspection device, and a method for the inspection. The point is to provide a test kit to be used.

[0005]

[Structure] The present inventors grow microorganisms present in the substance to be inspected in a closed container containing a culture solution, and change the color tone of the redox indicator due to the consumption of oxygen accompanying the growth of the microorganism. It has been found that the presence of microorganisms present in the substance can be determined. Furthermore, the experiments based on the above findings
Many experiments were conducted because the amount of oxygen consumption can be performed with extremely simple operation without using a special device.
As a result, the microorganism is cultured in a closed container containing the culture solution,
The present invention has been completed by finding that it is possible to determine the abundance of microorganisms of a substance to be inspected, for example, it can be determined on an order-by-order basis by observing a decrease in oxygen accompanying growth using a redox indicator. Hereinafter, the present invention will be described in detail. A first aspect of the present invention is to add a test substance into a closed container containing a culture solution, to grow microorganisms existing in the test substance in the closed container, and to reduce oxygen by consumption of oxygen accompanying growth of the microorganism. The present invention relates to an inspection method for inspecting the presence and abundance of microorganisms according to the presence or absence of change in color tone. The redox indicator used in the present invention is not particularly limited in its type as long as it changes the color tone by the consumption of oxygen accompanying the growth of microorganisms, but for detecting the change in the color tone without using a special device. In addition, as the above-mentioned redox indicator, a substance whose color tone change can be visually confirmed is desirable. Such redox indicators include methylene blue, leuco methylene blue, 2,6-dichlorophenol indophenol,
Methylphenazonium metasulfate, cresol violet, pyocyanin, resazurin and the like can be mentioned. Particularly, methylene blue and resazurin are preferable because there is little risk of thermal decomposition even if these reagents are stored in a closed container and then sterilized or heat-treated. The addition amount or concentration of the redox indicator is not particularly limited as long as it does not inhibit the growth of microorganisms. The closed container containing the culture solution is not particularly limited in shape as long as the test substance can be added and the container can be sealed, and any kind such as an ampoule or a screw cap bottle can be used. . The material of the closed container is not particularly limited, but is preferably transparent or has a color tone similar to this so that the change in the color of the culture solution can be observed. Further, it is preferable to use a pretreatment such as sterilization or sterilization by heat treatment, radiation irradiation or the like before or after containing the culture solution in the closed container. The culture solution contained in the closed container can be selected optimally for each substance to be inspected or microorganism to be detected, but for example, the special test method described in the Japanese Pharmacopoeia General Test Method Sterility Test Method Medium and institution for Fermenta
List of Culture of Tion Osaka
It is also possible to use the various media described under e. The color of the culture solution is not particularly limited as long as the change in the color tone can be observed, but it is desirable that the color of the culture solution be transparent or have a color tone similar to this so that the change in the color tone can be easily observed. This culture solution also needs to be sterilized or sterilized by heat treatment, radiation or the like as in the case of the filter sterilization or the above-mentioned closed container. This sterilization or sterilization treatment is preferably performed after the culture solution and the redox indicator are stored in a closed container from the viewpoint of easy operation.

The substance to be inspected is not particularly limited as long as it may be contaminated with microorganisms. For example, foods such as fresh foods and processed foods, pharmaceuticals for drinking and washing, and lubrication. Examples thereof include petroleum products such as oil and fuel oil, and chemical products such as chemical raw materials. The target microorganism to be judged or inspected is not particularly limited as long as it is a microorganism that consumes oxygen during growth, and is facultative anaerobic regardless of archaea, eubacteria, or eukaryotes. And microorganisms with aerobic properties can be detected. From the viewpoint of simplifying the operation, the above-mentioned redox indicator is preferably contained in the medium, but when the redox indicator inhibits the growth of the microorganism, it may be added after the microorganism is grown to some extent. .

A second aspect of the present invention relates to a microorganism test kit used in the first testing method. The above-mentioned microorganism test kit is basically composed of the above-mentioned closed container containing a culture solution for growing microorganisms and an instrument for collecting an object to be inspected. From the viewpoint of simplifying the operation, the redox indicator is preferably contained in the medium, but if the redox indicator inhibits the growth of the microorganism, it is necessary to grow the microorganism to some extent before adding it. In some cases, a device containing a redox indicator is attached. It should be noted that the device for collecting the inspection object and the device containing the redox indicator are also preferably those which have been sterilized or heat-treated by heat treatment, radiation or the like as in the closed container.

[0008]

EXAMPLES Examples of the present invention will be specifically described below. It should be noted that these examples are for illustrating the present invention and do not limit the scope of the present invention. Example 1: Detection of microorganism using medium containing methylene blue Glycerol 1.0 g / l, succinic acid 1.0 g / l, ammonium nitrate 0.5 g / l, magnesium sulfate heptahydrate 0.1 g / l, yeast extract A culture solution containing 0.1 g / l, peptone 0.5 g / l, phosphate buffer 0.01 M, and methylene blue 0.001 g / l was prepared, and the pH was adjusted to 7.
Adjusted to 0. 1.0 ml of this culture solution is used for about 1.5
It was put in a ml screw cap bottle and sterilized by autoclaving at 120 ° C. for 15 minutes. Next, the cutting oil contaminated with microorganisms is appropriately diluted with sterilized water, and 0.1 ml is added to the screw cap bottle containing the above-mentioned culture solution as a sample to be inspected.
Was added. The screw cap bottle containing the culture solution to which the test sample was added was incubated at 30 ° C. for 20 hours and the change in the color tone of methylene blue was observed. At the same time, the same test sample was smeared on a broth medium for general bacteria, and the viable cell count (CFU) in the test sample was measured. The results are shown in Table 1.
(-) Indicates that there is no change in the color tone of methylene blue,
(+) Indicates that the tone of methylene blue changed from blue to colorless, and (±) indicates that the tone of methylene blue changed from blue to pale blue. Approximately 1.
When 0.1 ml of the test sample containing 0 × 10 ⁶ CFU / ml or more was added, the color tone of methylene blue changed from blue to colorless. From this result, it was clarified that this method can detect the presence of about 1.0 × 10 ⁶ CFU / ml of the microorganism in the sample to be inspected.

Example 2: Detection of microorganisms using medium containing resazurin Glycerol 1.0 g / l, succinic acid 1.0 g / l, ammonium nitrate 0.5 g / l, magnesium sulfate heptahydrate 0.1 g / l , Yeast extract 0.1 g / l, peptone 0.5 g / l, phosphate buffer 0.01 M, resazurin 0.001 g / l was prepared and the pH was 7.0.
Adjusted to. 1.0 ml of this culture solution is about 1.5 m
It was put in a 1-liter screw cap bottle and sterilized by autoclaving at 120 ° C. for 15 minutes. Next, cutting oil contaminated with microorganisms was appropriately diluted with sterilized water, and 0.1 ml was added to the screw cap bottle containing the above-mentioned culture solution as a sample to be inspected. The screw cap bottle containing the culture solution added with the sample to be inspected was incubated at 30 ° C. for 20 hours, and the color tone change of resazurin was observed. At the same time, the same test sample was smeared on a broth medium for general bacteria, and the viable cell count (CFU) in the test sample was measured. The results are shown in Table 2. (−) Indicates that the color tone of resazurin did not change, (+) indicates that the color tone of resazurin changed from purple to colorless, and (±) indicates that the color tone of resazurin changed from purple to pale pink. . About 1.0 × 10 ⁵ CFU / m as viable bacteria
When 0.1 ml of the test sample containing 1 or more was added, the color tone of resazurin changed from purple to light pink, and the test sample containing about 1.0 × 10 ⁶ CFU / ml or more as the viable cell count was added. When 1 ml was added, the color tone of resazurin changed from purple to colorless. From this result, it was clarified that this method can detect the presence of a microorganism of about 1.0 × 10 ⁵ CFU / ml in the sample to be inspected.

Example 3: Detection of Bacillus natto using a medium containing resazurin 1.0 g / l glycerol, 1.0 g / l succinic acid, 0.5 g / l ammonium nitrate, 0.1 g / magnesium sulfate heptahydrate 0.1 g / l 1, a yeast extract 0.1 g / l, peptone 0.5 g / l, a phosphate buffer 0.01 M, resazurin 0.001 g / l was prepared and the pH was 7.0.
Adjusted to. 1.0 ml of this culture solution is about 1.5 m
It was put in a 1-liter screw cap bottle and sterilized by autoclaving at 120 ° C. for 15 minutes. Next, the natto-derived mucus was appropriately diluted with sterilized water, and 0.1 ml was added to the screw cap bottle containing the above-mentioned culture solution as a sample to be inspected. The screw cap bottle containing the culture solution added with the sample to be inspected was incubated at 30 ° C. for 20 hours, and the color tone change of resazurin was observed. At the same time, the same test sample was smeared on a broth medium for general bacteria, and the viable cell count (CFU) in the test sample was measured. The results are shown in Table 3. (−) Indicates that the color tone of resazurin did not change, (+) indicates that the color tone of resazurin changed from purple to colorless, and (±) indicates that the color tone of resazurin changed from purple to pale pink. . When 0.1 ml of the test sample containing about 3.0 × 10 ⁴ CFU / ml or more as the viable cell count was added, the color tone of resazurin changed from purple to pale pink. From this result, it was clarified that the present method can detect the presence of a microorganism of about 3.0 × 10 ⁴ CFU / ml in the sample to be inspected.

[0011]

[Table 1]

[0012]

[Table 2]

[0013]

[Table 3]

[0014]

[Effect] According to the present invention, there is provided a method capable of measuring or inspecting the presence of a microorganism and its abundance in a simple and short time and an inspection kit used in the method.

Claims (6)

[Claims] 1. A redox indicator by adding oxygen to a test substance into a closed container containing a culture solution of microorganisms and growing the microorganisms present in the test substance in the closed container to consume oxygen accompanying the growth of the microorganisms. An inspection method that determines the presence of microorganisms by changing the color tone of. 2. The test method according to claim 1, wherein the abundance of microorganisms is determined by the change in color tone of the redox indicator. 3. The test method according to claim 1, wherein the redox indicator is a substance whose change in color tone can be visually confirmed. 4. Methylene blue, leuco methylene blue, 2,6-
The method for testing a microorganism according to claim 3, wherein the method is at least one selected from the group consisting of dichlorophenol indophenol, methylphenazonium metasulfate, cresol violet, pyocyanin and resazurin. 5. A microorganism test kit comprising a closed container containing at least a microorganism culture solution, a device for collecting an object to be inspected, and the redox indicator according to claim 3 or 4. 6. The microorganism test kit according to claim 5, wherein the closed container containing the microorganism culture solution has a redox indicator stored therein in advance.