JPH0662893A - Method for rapidly detecting e. coli - Google Patents

Abstract

PURPOSE:To surely detect E. coli in excellent sensitivity by a simple operation in a short time. CONSTITUTION:E. coli is determined by adding a specimen to a medium for detecting E. coli, comprising 1.0-2.5wt.% peptone, 0.25-0.5wt.% sodium chloride, 0.4-0.6wt.% yeast essence, 0.2-0.5wt.% dipotassium hydrogenphosphate, 0.05-5.0wt.% sodium pyruvate, 0.05-1.0wt.% potassium nitrate, 0.01-1.0wt.% 5- bromo-4-chloro-3-inodolyl-beta-D-glucuronide or its salt and 0.01-1.0wt.% sodium dodecyl sulfate and/or 1.0-2.0wt.% bovine bile powder, culturing and detecting presence of coloring of the medium.

Description

Detailed Description of the Invention

[0001]

The present invention relates to urine and other clinical materials,
For food, medicine, cosmetics, drinking water, etc. The present invention relates to a method for easily, quickly and accurately determining whether or not E. coli exists.

[0002]

2. Description of the Related Art E. coli is a representative highly pathogenic bacterium in the coliform group, and is used in foods, medicines, clinical materials, etc. determining whether E. coli exists
Important for clinical bacteriology and microbiological quality control.
Conventional E. As a method for detecting E. coli,
24 using intestinal bacteria differentiation medium such as MacConkey medium
A method of confirming colonies of bacteria that have grown after culturing for a time and further identifying various biochemical properties, a method of making a determination using a fluorescent substrate such as 4-methyl-umbelliferyl-β-D-glucuronide, and the like are available. is there.

[0003]

However, the method of identifying by biochemical properties has a problem that the operation is complicated and that it requires skill and time to carry out. Further, the method using a fluorescent substrate requires an operation of irradiating the generated fluorescent substance with ultraviolet rays to measure the excited fluorescence intensity, and is not sufficiently satisfactory in terms of simplicity. Therefore, the object of the present invention is to perform E. An object of the present invention is to provide a method for detecting E. coli.

[0004]

[Means for Solving the Problems] In view of the above situation, the present inventors have investigated various media and color substrates for simplicity and speed, and as a result, 5-bromo-4-chloro, which is a color substrate, has been investigated. If a medium having a specific composition to which 3--3-indolyl-β-D-glucuronide or a salt thereof is added is used, E. The inventors have found that E. coli can be detected and completed the present invention.

That is, the present invention is by weight, peptone 1.
0-2.5%, sodium chloride 0.25-0.5%, yeast extract 0.4-0.6%, dipotassium monohydrogen phosphate 0.2-0.5%, sodium pyruvate 0.05 ~
5.0%, potassium nitrate 0.05 to 1.0%, 5-bromo-4-chloro-3-indolyl-β-D-glucuronide or a salt thereof, and sodium dodecyl sulfate 0.01
~ 1.0% and / or bovine bile powder 1.0-2.0%, the test substance is added to the medium, and the mixture is cultured to detect the presence or absence of coloration of the medium. E. coli detection method. The present invention also provides an E. A medium for detecting E. coli is provided.

The E. 5-Bromo-4-chloro-3-indolyl-β-D added to E. coli detection medium
-Glucuronide or a salt thereof is a coloring substrate,
It is decomposed by an enzyme specifically produced at the stage of growth of E. coli, that is, glucuronidase, to produce a condensate showing blue or blue-green as shown in the following reaction formula.

[0007]

[Chemical 1]

As a result of such a reaction, E. coli
The area in which the enzyme produced by is present is colored, and the area apparently larger than the size of the actual colony formed on the medium (usually agar plate) is colored. Therefore, the colony can be visually confirmed at an earlier stage.

As the salt of 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, a cyclohexyl ammonium salt is preferable. Further, the amount of the substrate to be added to the medium is preferably 1 × 10 ⁻⁴ to 1 × 10 ⁻² M.

The medium of the present invention contains peptone, sodium chloride, yeast extract, dipotassium monohydrogen phosphate, sodium pyruvate, potassium nitrate, and sodium dodecyl sulfate and / or bovine bile powder in addition to the above-mentioned substrates. Although sodium sulfate and bovine bile powder may be mixed alone or in a mixture, it is preferable to mix either one. Further, glycerol can be further added to the medium of the present invention in an amount of 0.5 to 1.5% by weight.

The medium of the present invention can be used as a liquid medium by mixing the above components.
It is preferable to use 1.5% by weight of agar as an agar medium.

The E. E. coli in the subject using the E. coli detection medium. In order to detect the presence of E. coli, a test substance may be added to the culture medium, the mixture may be cultured at 30 to 44.5 ° C. for several hours to several tens of hours, and then the presence or absence of coloration of the culture medium may be visually observed.

Here, examples of the test substance include clinical materials such as urine and blood plasma, foods, drugs, cosmetics, beverages, etc., but it is preferable to use a culture solution obtained by culturing these test samples in a medium such as trypto soya broth in advance. You can also

[0014]

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

Example 1 The strains shown in Table 2 isolated from urine as a clinical material were cultured in trypto soya broth at 37 ° C. for 24 hours. After streaking 10 μl of this culture medium onto the medium shown in Table 1 below, the medium was allowed to stand at 37 ° C. for 24 hours.
After culturing for a period of time, the presence or absence of coloration of the medium was visually observed. The results are shown in Table 2.

[0016]

[Table 1]

[0017]

[Table 2]

As is apparent from Table 2, E. While 100% coloring was observed in E. coli, no coloring was observed in other strains, confirming that the method of the present invention is accurate.

Example 2 E. 65 clinical isolates of E. coli were cultured in trypto soya broth at 37 ° C. for 24 hours. 10 μl of this culture
Streaks were smeared on the composition α medium or MacConkey medium of Table 1 and cultured at 37 ° C. for 24 hours, and the presence or absence of coloration of the medium was visually observed. The results are shown in Table 3.

[0020]

[Table 3]

As is clear from Table 3, 100% was colored when the medium of the present invention was used, whereas when the MacConkey medium was used, 7 strains did not become red turbid and could not be judged as positive. .

Example 3 The strains shown in Tables 4 and 5 were cultured in trypto soya broth at 37 ° C. for 24 hours. 10 μl of this culture solution
The described medium, peptone-terditol medium [J. Food
Prot. 51, 402-404 (1988)], and lauryltryptose medium [Appl. E
nviron. Microbiol. , 54, 1874
~ 1875 (1988)] or mTEC medium [Appl. Environ. Microbio
l. , 54, 1874-1875 (1988)], and cultured at 37 ° C. for 24 hours to compare the growth and color development of each strain. The results are shown in Tables 4 and 5.

[0023]

[Table 4]

[0024]

[Table 5]

As is clear from Tables 4 and 5, when the medium of the present invention is used, E. Both the growth and the color development were observed only in E. coli, and when other media were used, the color development was insufficient or the growth was poor, resulting in lack of sensitivity and accuracy.

[0026]

According to the detection method of the present invention, E. col
It is possible to detect i with high sensitivity and reliability by a simple operation in a short time.

Claims (2)

[Claims] 1. By weight, peptone 1.0-2.5%, sodium chloride 0.25-0.5%, yeast extract 0.4-
0.6%, dipotassium monohydrogen phosphate 0.2-0.5%,
Sodium pyruvate 0.05-5.0%, potassium nitrate 0.05-1.0%, 5-bromo-4-chloro-3-
Indolyl-β-D-glucuronide or a salt thereof, and culturing by adding a test substance to a medium containing 0.01 to 1.0% of sodium dodecyl sulfate and / or 1.0 to 2.0% of bovine bile powder, E. coli characterized by detecting the presence or absence of coloration of the medium. E. coli detection method. 2. By weight, peptone 1.0-2.5%, sodium chloride 0.25-0.5%, yeast extract 0.4-
0.6%, dipotassium monohydrogen phosphate 0.2-0.5%,
Sodium pyruvate 0.05-5.0%, potassium nitrate 0.05-1.0%, 5-bromo-4-chloro-3-
E. coli containing indolyl-β-D-glucuronide or a salt thereof and sodium dodecyl sulfate 0.01 to 1.0% and / or bovine bile powder 1.0 to 2.0%. E. coli detection medium.