KR20000030381A - A test kit for in situ detecting microorganisms and a method for manufacturing the same - Google Patents

Abstract

PURPOSE: Microorganism test in water, foods, and aqueous chemical compositions such as paint, dye, or resin, needs immediate sample collection and detection method performed on the scene. A portable detection equipment showing speedy and accurate results is provided which uses liquid phase detection medium to collect sample from viscous materials or thin membrane forming goods. CONSTITUTION: The liquid medium for the detection of microorganism consists of 91.22weight % of water, 0.1 weight % of soytone, calcium carbonate, sodium phosphate (I), sodium phosphate (II), boric acid, and antifoam (KM73); 0.5 weight % of corn starch, and potato extract; 1.0 weight % of dextrin, porcine meat extract, lactose, NaCl, sucrose, fructose, peptone, and sugar; 0.01 weight % of zinc sulfate, copper sulfate, iron sulfate, manganese chloride, and calcium chloride; 0.1 weight % of phenol red, 0.01 weight % of triphenyltetrazolium chloride, 0.02 weight % of bromoxylenolblue. Each 5ml of liquid medium is dispensed in a bottle having a capacity of 10 ml and a built in inoculation tool and then autoclaved. The inoculation tools are Pasteur pipette, needle, or cotton stick. The color of medium is changed from purple into yellow as microorganism is growing.

Description

Microbiological testing apparatus that can be measured in the field and a manufacturing method thereof {A TEST KIT FOR IN SITU DETECTING MICROORGANISMS AND A METHOD FOR MANUFACTURING THE SAME}

The present invention relates to a method for producing a microorganism testing instrument that can be measured in the field. More specifically, the present invention relates to a method for manufacturing an inspection apparatus capable of measuring the contamination state by microorganisms in manufacturing sites such as food, drinking water or aqueous industrial products, food service establishments, and food service groups. The invention also relates to a microbiological testing instrument made by such a method.

In the case of drinking water, food service establishments, and catering establishments, the drinking water management law and the food hygiene law define the pollution standards and inspection cycles for microorganisms. However, the microbial analysis process prescribed in such a law is dualized by taking samples in a sterile state and analyzing them in a sterile laboratory using a sterilized sampling container and a collecting tool in the field. Therefore, there is a problem of contamination according to the sampling process or the delay time until the analysis, and the microbial analysis process of the collected sample requires a lot of skilled experts and a lot of equipment, which is economical and controversial about accuracy. .

In addition, various cases such as food poisoning and Vibrio sepsis have been reported through the mass media as a result of the contamination of microorganisms. There is an urgent need for an inspection apparatus that can.

In addition, in the case of aqueous industrial products, namely paints, pigments, resins, detergents, cosmetics and lubricants, contamination of microorganisms causes deterioration of the product quality. It is required.

However, the prior art requires facilities and experts for the analysis of microorganisms, there is no suitable alternative to this, and in the case of a product having high viscosity or a film forming a film, it is conventionally used for microbial analysis by a laboratory method. The conventional solid media test method used is not an appropriate assay.

Accordingly, the present invention is to provide a test apparatus that can be used for microbial analysis quickly by maintaining a state capable of growing microorganisms by diluting in a liquid medium even in the case of a high viscosity material or a film-forming product using a liquid test solution. It is for.

In another aspect, the present invention is to provide a rapid and accurate measurement means that the sample collection process and the microbial analysis process is carried out in the field so that the tester can test the contamination state by the microorganism in a short time.

Hereinafter, the present invention will be described in detail.

The present invention is to prepare a liquid medium composition for optimizing the growth of microorganisms by combining water with a carbon source, nitrogen source, salts and other additives, it is possible to detect the metabolite of the liquid medium composition and microorganisms in a container containing an inoculation device A method of making a microbial testing instrument capable of measuring microbial contamination at a site, comprising the step of formulating an indicator present, and sterilizing the container.

The invention also relates to a microbiological testing instrument made by such a method.

In the invention of the test apparatus of the present invention, the design of a liquid medium for optimizing the growth of microorganisms, the development of indicators for visually confirming the growth of microorganisms, and a test container capable of sample collection directly in the field and maintain a sterile state Development was carried out.

As the composition of the liquid medium, the composition of the carbon source, nitrogen source and salts for optimizing the growth of general microorganisms was examined.

As the carbon source, monosaccharides, disaccharides, and polysaccharides, which are generally used for microbial analysis, can be used, but are not limited thereto. The concentration of application is 1 to 20 parts by weight, and the microorganisms are most easily used for rapid assay. It is preferable to use known monosaccharides and disaccharides.

Examples of nitrogen sources include pork extract, yeast powder, peptone, tryptone, and soytone, but are not limited to these, and in some cases, it is preferable to add amino acids, and the application concentration is 1 to 20 parts by weight. .

The salts are preferably boric acid, phosphates, iron salts, manganese salts, molybdenum salts, salts, copper salts, potassium salts, and magnesium salts, but are not limited to these, and the application concentration is suitably 0 to 10 parts by weight.

The addition of vitamins, antifoaming agents, buffers, chelating agents, etc. is preferable as the other compounding components, but is not limited thereto. The application concentration is preferably 0 to 5 parts by weight, and as a solvent for dissolving the above compounding components. Water is suitable and the application concentration is preferably 45 to 98 parts by weight.

It is not preferable to use the compounding components of the above liquid medium alone, and it is preferable to use them in combination for optimization of microbial growth.

Indicators that can visually detect the growth of microorganisms are chemical reactions that can detect metabolites generated by metabolism of the components of the composition medium as described above, redox indicators, organic acid detection indicators, ammonia detection indicators, Amino acid detection indicator, hydrogen ion concentration detection indicator, etc. are preferable.

Triphenyltetrazolium chloride, bromothymol blue, crystal violet, eosin, phenol red, bromo xylenol blue, neutral red, methylene blue, etc. may be used as an indicator for detecting microorganisms used in the present invention. It is not limited to, the application concentration is preferably 1 part by weight or less.

In preparing the test apparatus of the present invention, a test container capable of aseptically collecting a sample and manufacturing a test tool was used as an inoculation device, and an inoculation device was used as an inoculation dropper, a solid surface and a powder sample. Cotton swabs, needles for collecting solid samples, and the like were used.

In manufacturing the test apparatus of the present invention, a test solution containing the above-mentioned medium component and indicator was dispensed into a test container and sterilized. Sterilization conditions are preferably treated at 121 ° C. and 1.5 atm for 20 minutes. The process of sterilization treatment is essential for the manufacture of the test apparatus, and if the sterilization process is not performed, the test liquid may be deteriorated due to the growth of the microorganisms contained in the test apparatus, thereby failing to achieve the purpose of the test.

Therefore, the present inventors prepared a test solution containing an indicator for detection of metabolites due to the metabolic activity of the microorganisms in the liquid medium composition for optimizing the growth of microorganisms, the collection of samples and the preparation of test spheres in the field The test apparatus of the present invention was prepared by dispensing into a test apparatus having a built-in inoculation mechanism, and sterilizing.

The inspection apparatus of the present invention is capable of hygiene and quality inspection at the production site of food, drinking water or aqueous industrial products, or at any site where environmental hazards or deterioration of quality may occur due to microorganisms such as food service restaurants and community food service stations.

Therefore, the present invention can significantly reduce the time, cost, and manpower of the microbial analysis process that is generally employed, it is believed that it can contribute to the public health and hygiene due to microorganisms.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

Preparation Example 1

In order to prepare a microorganism test apparatus for field inspection of the present invention, 99.87 parts by weight of a liquid medium having the composition shown in Table 1 was prepared, and 0.1 parts by weight of phenol red, 0.01 parts by weight of triphenyltetrazolium chloride, and bromoza were used as indicators for detecting microorganism growth. A total amount of 100 parts by weight of the test solution was prepared by adding 0.02 parts by weight of elenol blue.

The test solution prepared above was dispensed 5 ml each into a 10 ml bottle containing an inoculation device, and sterilized for 20 minutes at 121 ° C and 1.5 atm to prepare a microorganism testing device of the present invention.

Preparation Example 2

11.5 grams of nutritious agar (Midifco Co.), which is mainly used as a solid medium for general bacterial analysis, was prepared to dissolve in water to 500 grams and sterilized under the same sterilization conditions as in Example 1 A solid medium was prepared by dispensing 25 grams aseptically into Petri dishes (87x15 mL) before the medium solidified at 60 ° C.

Example 1

Aqueous paints, aqueous resins, aqueous adhesives, lubricating oils were inoculated by one drop (10 μl) at the site using the microbiological testing apparatus of the present invention prepared by Preparation Example 1 and incubated at a constant temperature of 30 ° C. The measurement of the results recorded whether the test solution discolored and the time required for discoloration.

The test results are shown in Table 2 below, and the following test results were obtained based on 100 colonies per 1 ml of the sample to determine whether the microorganisms were grown on the high-viscosity material or the coating-forming sample. It can be judged that all contain the microorganism density | concentration of 100 colony or more per ml of samples.

Comparative Example 1

Samples were taken using a sampling container prepared aseptically at each manufacturing site for the same sample as in Example 1, and plated on a conventional solid medium of Preparation Example 2 by sequential dilution in a sterile phase. As a result of the assay, the culture conditions were the same as in Example 1. The notation of the results recorded the time taken to form colonies with the bacteria. The test results are shown in Table 3 below.

The test result of Comparative Example 1 is a test result by a conventional microbiological test method, and the sampling process is complicated, and in the test of bacteria, there is a problem in the test of the number of bacteria due to the characteristics of the sample (film formation or high viscosity sample). It was shown that the time required was also two to three times as compared to the case of Example 1.

In contrast, since the microorganism testing apparatus of the present invention detected the microorganisms in all of the tested samples, it can be seen that it is much superior in inspection time and detection range than the conventional solid medium test method.

Example 2

The cooking utensils used in the kitchen are directly related to hygiene, and were assayed using the microorganism test apparatus of Preparation Example 1.

As a kitchen utensil, use a cotton swab, dish cloth, dish dryer, and sink to contain moisture, and then rub the surface of the sample by 1 cm using a cotton swab of the microorganism testing apparatus of the present invention of Preparation Example 1, and immerse it in the testing instrument liquid. And cultured under the same conditions as in Example 1. The test results are shown in Table 4 below.

Comparative Example 2

The same kitchen utensils were taken at the same place as in Example 2, and 2 ml of sterile water was dropped on the surface aseptically, 1 ml of the sample was collected using a sterile pipette, and the samples were prepared by the same serial dilution as in Comparative Example 1. Then, the conventional solid medium of Preparation Example 2 was inoculated by the smear method to measure the number of bacteria. The test results are as follows.

From the results of Example 2, it can be seen that according to the microorganism testing apparatus of the present invention, it is possible to collect and inspect a sample in the field by a simple method. From the results of Comparative Example 2, according to the conventional solid medium testing method, It can be seen that the sampling and analysis process of the sample is dualized, and that the test results are also difficult to use for the bacterial count test of the sample.

Example 3

The microorganism test was carried out in the same manner as in Example 1 and Example 2 for the super-cleaning spring water and tap water. Using the dropper of the microorganism testing apparatus of the present invention of Preparation Example 1 was inoculated with a drop of the tap water and the tap water of the super-cleaning weak water field and incubated in the same manner as in Example 1, the results in Table 6 the same method as in Example 1 Indicated as.

Comparative Example 3

Tap water in the same place as in Example 3 was collected using a thermos filled with ice in a sterilized container, and the sample of the water bath was collected in the water container and aseptically sampled by the continuous dilution method of Comparative Example 1 after 4 hours of moving time. Was prepared and smeared on the medium using the solid medium of Preparation Example 2 to determine the number of bacteria. The test results are shown in Table 7 below.

Example 4

A test piece was prepared by the method of Example 1 using the tester sample of Comparative Example 3 using the test apparatus of the present invention of Preparation Example 1, and the test results are shown in Table 8 below.

Compared with the results of Example 3 and Comparative Example 3, in the case of Example 3, the bacterial content in the field of the spring water can be determined to be 100 or less per 1 ml within the water quality standard, but the results of Comparative Example 3 and Example 4 The results showed that more than 100 colonies were grown per ml of the sample.

From the results of Example 3, Comparative Example 3, and Example 4, it can be determined that the microorganisms grow inside the collection container if the sample collection process does not proceed aseptically or if there is an elapsed time in the sample collection and analysis. Therefore, it can be seen that the sampling and analysis of the sample are most preferably performed at the same time.

Example 5

Test apparatus of the present invention prepared by Example 1 microbial solution of 10,000,000 colonies (hereinafter referred to as test 1), 100,000 colonies (hereinafter referred to as test 2), 1,000 (hereinafter referred to as test 3) colonies per 1 ml of E. coli density Each drop was inserted into each of them and tested in the same manner as in Example 1 while incubating in a thermostat at 37 ° C. The results are shown in Table 9 below.

What can be judged from the above results is that the density is inversely related to the time required for discoloration. These results suggest that the higher the bacterial density, the shorter the time required for the test, so that the test can be promptly checked for contamination by microorganisms.

Comparative Example 4

Samples of test strips 1, 2, and 3 of Example 5 were respectively inoculated and cultured in the conventional solid medium of Preparation Example 2 using a smear method. Culture conditions were the same as in Example 5, the results are shown in Table 10 below.

The test results described above only record the incubation time after inoculation, and it takes 36 hours to visually confirm the results regardless of the sample density. However, the general microbial solid medium analysis process requires sampling, transport, pretreatment, medium preparation, smearing, cultivation, and confirmation of results, and the total time required for microbial analysis in consideration of such working time is considerably more. It can be judged to be long. In the case of food poisoning, etc., most of them are caused by microorganisms, so it is considered that the promptness of microbial testing is absolutely important.

The microbiological testing apparatus of the present invention is for the purpose of hygiene and quality inspection of drinking water, food service establishments, catering establishments, and industrial products, and can drastically reduce the time, cost, and manpower of the microbial analysis process that is generally employed. In addition, it is possible to contribute to the health and hygiene of the people due to microorganisms by enabling rapid and accurate measurement by enabling inspection in the field.

Claims (9)

Preparing a liquid medium composition optimized for the growth of microorganisms by combining water with a carbon source, nitrogen source, salts and other additives, and an indicator for detecting the metabolite of the liquid medium composition and microorganisms in a container containing an inoculation device. A method of making a microbial testing instrument capable of measuring microbial contamination at a site, comprising the step of compounding and sterilizing the container. The method according to claim 1, wherein the carbon source of the liquid medium is selected from the group consisting of monosaccharides, disaccharides, and polysaccharides. The microorganism testing apparatus according to claim 1, wherein the nitrogen source of the liquid medium is selected from the group consisting of pork extract, yeast powder, peptone, tryptone, soytone and amino acid. How to prepare. According to claim 1, wherein the salt of the liquid medium is selected from the group consisting of boric acid, phosphates, iron salts, manganese salts, molybdenum salts, salts, copper salts, potassium salts, magnesium salts, microbial contamination in the field A method of making a measurable microbial testing instrument. The method according to claim 1, wherein the other additives in the liquid medium are selected from the group consisting of vitamins, antifoams, buffers and chelating agents. The microorganism in situ of claim 1, wherein the indicator is selected from the group consisting of triphenyltetrazolium chloride, phenol red, neutral red, eosin, methylene blue, bromothymol blue and bromo xylenol blue. A method of making a microbiological testing instrument capable of measuring contamination. The method according to claim 1, wherein the inoculation device is selected from the group consisting of an eye dropper, a cotton swab, and a needle. The method of claim 1, wherein the sterilization step is performed at 121 ° C. and 1.5 atm for 20 minutes. A microbiological testing instrument capable of measuring microbial contamination in situ, prepared according to the method according to any one of claims 1 to 8.