KR20100057112A - Method for analyzing e. coli and coliforms automatically by using defined substrate technology - Google Patents

Abstract

PURPOSE: A method for measuring E.coli using chromogenic assay is provided to quickly analyze E.coli and to enhance availability of analysis result. CONSTITUTION: A method for automatically measuring E.coli is performed by adding a substrate of chromogenic assay in a water sample and measuring sample color. The sample is concentrated and culture medium dissolved oxygen is controlled by injecting air in order for shortening analysis time. A method for concentrating the sample comprises a step of inputting sample and filtering E.coli and E.coli group; a step of removing E.coli and E.coli group from a filter by blowing filter; a step of mounting overflow pipe in a filter tank and analyzing the amount of the sample.

Description

E. coli and coliforms automatically by using Defined Substrate Technology}

The present invention relates to a method for shortening the analysis time by reducing the incubation time in the automatic analysis of E. coli and E. coli group using the enzyme chromatographic substrate as a biological measurement field in the environmental field.

The present invention relates to a measuring method for automatically and quickly measuring the E. coli and E. coli group in water.

There are many methods for analyzing E. coli and E. coli group, but recently, the enzyme method has been highlighted because of the relatively simple measurement method. Enzymatic colorimetric analysis is a method that detects color or fluorescence changes that occur during the hydrolysis of a substrate using a substrate that reacts with an enzyme specifically present in E. coli and E. coli. Herein, E. coli is defined as a bacterium expressing the β-D-galactosidase enzyme, and E. coli is defined as a bacterium expressing the β-D-glucuronidase enzyme. Therefore, substrates that specifically react with these enzymes are selected, and in particular, substrates for which the product causes color or fluorescence change after the reaction are used for easy reading of the reaction results. Enzyme coloration method is also listed in Korea's drinking water quality testing method. Many patents have been filed for the composition of such substrates.

Most of the enzymatic color development method uses a substance called MUG and ONPG as a coloring reagent. When this substance is included in the substrate and analyzed, the culture medium is identified as positive for E. coli group if the culture medium is yellow, and negative for E. coli group if the color is colorless. In addition, the culture medium is irradiated with 365 nm ultraviolet rays and fluorescence is identified as E. coli positive.

E. coli and E. coli group analysis technology using the enzyme coloration method is commercialized by batch analysis and automatic analysis in the form of a kit. Kit form is sold by lyophilizing the composition of the substrate to powder and packaged in a single measurement. The kit is placed in a plastic bottle, dissolved in 100 mL of sample, and incubated for 24 hours for analysis. The automated analysis technique consists of sample introduction, measurement, drug addition, culture, and optical analysis, which were commercialized by IDEXX in the US and Colifast in Norway.

Analysis time is very important for analyzing E. coli and E. coli group in drinking water. However, in addition to the actual enzymatic coloration method, using the other methods, it takes about 24 hours to analyze the E. coli and E. coli group. Colilert and Colifast may take 18 hours or more for drinking water. In the case of drinking water, if the analysis takes a long time, it is likely that humans have already eaten even if the E. coli and E. coli groups are positive. Therefore, it is necessary to reduce the analysis time of E. coli and E. coli group.

The inventors have developed and secured a substrate capable of analyzing E. coli and E. coli populations by enzymatic coloration through the Ministry of Environment Next Generation Core Environmental Technology Development Project for two years from 2004 to 2006.

Literature Information of the Prior Art

[Patent 1] Japanese Patent Publication No. 2004-187588

[Patent 2] US Patent US005650290A

[3] Moira D. Johnston. A simple and rapid test for quality control of liquid media using the bioscreen microbiological growth analyser, J. microbiol. methods. 32. 37-43

[Reference 4] Colilert Catalog

[Reference 5] Colifast company catalog

The present invention has the biggest object of ultimately shortening the analysis time by shortening the incubation time in order to increase the utility of the analytical results in the automatic determination of the E. coli and E. coli group of water using the enzyme coloration method.

The present invention is characterized by shortening the analysis time by introducing a process for concentrating the sample and supplying and adjusting the dissolved oxygen during the cultivation in order to shorten the time required for the culture in culturing E. coli and E. coli group.

E. coli and E. coli group automatic measurement method of the present invention can analyze the E. coli and E. coli group in 3 hours can greatly increase the utility of the analysis results. When E. coli or E. coli group is detected in a water purification plant, it is possible to immediately stop supplying water or stop operations at a food and beverage plant.

In the present invention, in order to shorten the incubation time of the E. coli and E. coli group, which took 18 hours or more in the prior art, it was intended to shorten the analysis time by introducing a sample concentration process and a process for supplying dissolved oxygen during the culture.

E. coli and E. coli group 1CFU / 100mL of the sample to cultivate in the state of about 10 ¹⁴ CFU / 100mL that can be detected by the photometer E. coli is doubled in 20 minutes, so incubate for at least about 14 hours at 35 ℃ shall. In the present invention, the concentration of the sample was intended to shorten the incubation time by increasing the number of cells in the sample before incubation. Arithmetic calculations can shorten the sample 2 to 2.5 hours for 100-fold concentrations and for 3.5 to 4 hours for 1000-fold concentrations. E. coli cells were cultured using the test strains, and the number of E. coli bacteria was analyzed by MPN method, which is one of the drinking water quality test methods. This culture solution was diluted to prepare a sample of 1 CFU / 100 mL. 1 is a schematic view showing a sample concentrating device of the present invention. The sample concentrating device of the present invention includes a sample introduction pipe (1), a metering and filtration tank (3), a solenoid valve (1), a filter housing (4), and a filter. (5), solenoid valve 2 (6), solenoid valve 3 (7). Into this sample concentrator, 1 CFU / 100 mL of sample is introduced through the sample introduction pipe (1). If E. coli is present while passing through the filter through the filtration tank, it is filtered by the filter (5) and the water is discharged through the solenoid valve 2 (6). do. The amount of sample required for the cultivation is 100 mL, so if the desired concentration ratio is 100 times, 10 liters should be passed through if the desired concentration is 100 times. When all of the sample to be concentrated passes, the sample is left until it is full. Open the solenoid valve 1 (2) on the top and drain the sample on the overflow valve to quantify the amount of sample. The solenoid valve 2 (6) at the bottom is then opened to blow compressed air into the filtration tank (3). At this time, E. coli attached to the filter (5) will come off. Close the solenoid valve 2 (6) at the bottom and open the solenoid valve 3 (7) to send the sample to the culture tank. After the sample was concentrated and incubated at 35 ° C., the change in color was observed as absorbance over time using a spectrophotometer. As a result, the unabsorbed sample had a low absorbance of 0.070 at 410 nm even after 14 hours of incubation, but the absorbance of 0.124 and 1000-fold diluted at 11 hours was very high at 0.205 in 9 hours. The spectrophotometer was able to detect the color change.

In addition, the present invention added a process for increasing the dissolved oxygen to further shorten the culture time of E. coli and E. coli group. E. coli and E. coli are anaerobic and require no oxygen supply. However, the inventors noticed that the E. coli and E. coli group is aerobic anaerobic, and increased the amount of dissolved oxygen in the culture solution in the culture process. As a result, when the initial dissolved oxygen in the culture tank was adjusted to 0.2ppm or more, it was confirmed that the growth of E. coli and E. coli group was rapidly accelerated. When adjusting the dissolved oxygen of the culture medium of the culture tank, the absorbance was enough to be 0.05 or more of the absorbance of the color can be distinguished by the spectrophotometer 3 hours was enough. When the initial dissolved oxygen in the culture tank was adjusted to 0.2ppm or less, the growth did not accelerate, and even when adjusted to 0.5ppm or more, the results were not significantly different from the results of 0.2ppm to 0.5ppmm. Supplying dissolved oxygen to Escherichia coli culture increases the growth potential of Gram-positive bacteria and fungi. However, gram-positive bacteria inhibitors and mold growth inhibitors have already been added to the substrates developed and secured by the present inventors, and their growth was not observed. Dissolved oxygen may be supplied using an aerator or a stirrer.

Figure 2 is a schematic diagram showing the automatic measurement method of E. coli and E. coli group including a sample concentration step and dissolved oxygen injection step. The sample is continuously introduced and discharged through the sample introduction pipe (1). To collect the sample, open the 3-way solenoid valve 1 (2) to the metering and filtration tank (6) to introduce the sample into the metering and filtration tank (6). At this time, the sample flows through the 3way solenoid valve 3 (7), the tube pump (10), and the 3way solenoid valve 4 (11). The sample is passed through a filter mounted in the lower part of the filtration tank, while E. coli is filtered by the filter. After that time, the direction of the 3-way solenoid valve 1 (3) is changed to bypass the incoming sample so that no more sample is introduced. There is no sample in the pipe between the 3-way solenoid valve 1 (3) and the metering and filtration tank (6) and the tube pump (10) is stopped while the sample is filled in the metering and filtration tank (6). Open the 2 way solenoid valve 1 (5) and drain all the samples on the valve to quantify the sample volume. Then, the direction of the 3way solenoid valve 3 (7) is changed to allow the compressed air to flow into the metering and filtration tank (6). At this time, the E. coli filtered out of the filter and falls into the water of the metering and filtration tank (6). Then open the 2-way solenoid valve 2 (9) to transfer the sample to the culture tank (12). When the concentrated sample is transferred to the culture tank, the substrate for enzymatic color analysis is injected by using reagent injection pump 1 (16) and reagent injection pump 2 (17). The stirrer paddle 20 is then rotated to supply dissolved oxygen. Dissolved oxygen can also be controlled by injecting air. The temperature of the culture tank 12 is adjusted by circulating the water of the thermostat 14 to the outside of the culture tank 12. The incubated sample is cooled to room temperature and then transferred to the lower optical system 22. Optical system can measure the emission spectroscopy and fluorescence in the sample of 365nm, light in a general configuration. After the measurement, all the samples are discharged and chlorine is injected into the sample introduction tube (1) to flow the sample in the same manner as in the measurement process to be sterilized.

1 is a schematic view of a sample concentrator

Figure 2 is a schematic diagram of the automatic measurement method of E. coli and E. coli group

DESCRIPTION OF THE REFERENCE NUMERALS

1: 1: sample introduction pipe, 2: 2 way solenoid valve, 3: metering and filtration tank, 4: filter module, 5: filter, 6: 3 way solenoid valve, 7: 2 way solenoid valve

2: 1: sample introduction pipe, 2: sample bypass pipe, 3: 3way solenoid valve 1, 4: 3way solenoid valve 2, 5: 2 way solenoid valve 1, 6. metering and filtration tank, 7: 3way solenoid 3 , 8. 2 way solenoid valve 2, 9: air reservoir, 10: tube pump, 11: 3 way solenoid valve 4, 12: culture tank, 13: circulation pump, 14: thermostat, 15: stirrer motor, 16: reagent injection pump 1 , 17: reagent injection pump 2, 18: reagent reservoir 1, 19: reagent reservoir 2, 20: stirring paddle, 21: 2say solenoid valve 3, 22: optical system, 23: 2way solenoid valve 4

Claims (2)

In the automatic measurement method of measuring E. coli and E. coli group by adding the substrate of enzyme coloration to the water sample and incubating at 35 ° C by measuring the color of the sample with a photometer, the sample is concentrated and used to reduce the analysis time. E. coli and E. coli group automatic measurement method characterized in that by adjusting the dissolved oxygen of the culture medium by injecting air into the culture medium of the culture tank The E. coli and E. coli group adhered to the filter according to claim 1, wherein the sample is introduced into the metering and filtration tank equipped with the filter to filter the E. coli and E. coli groups through the filter. The sample concentration method, characterized in that the separation from the filter, the metering and filtration tank has an over-flow pipe so that the sample on the pipe is discharged to quantify the amount

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