JP2002045197A - Colorimetric solution for identifying microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a colorimetric solution capable of clearly identifying the positive limit of microorganism by naked eyes in a specific enzyme-substrate medium method and capable of being supplied together with a culture medium as a kit for identifying microorganism since the solution shows no color change even in storing at room temperature and has good storage stability. SOLUTION: The colorimetric solution is used for judging the color of a medium which has been developed by an indigo-related substance formed by the decomposition from a coloring enzyme substrate in specific enzyme-substrate medium method, and the solution contains indigo carmine and o-nitrophenol.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a colorimetric liquid for judging microorganisms, which is used for judging the color of a medium formed by an indigo-based substance.

[0002]

2. Description of the Related Art The determination of the presence of Escherichia coli and coliforms in food and drinking water is important as a daily test for confirming the presence of fecal contamination of food and water. In particular, for the detection of coliform bacteria in drinking water, a method which can be determined easily and quickly is desired. In the water test method, the lactose bouillon-brilliant green lactose bile bouillon medium method (LB-BGLB) which has been conventionally used has been desired. Law), and in 1993,
A specific enzyme substrate medium method was added as a rapid method.

[0003] Such a specific enzyme substrate medium method utilizes β-glutaronidase or galactosidase which is specifically present in a microorganism, for example, Escherichia coli or Escherichia coli, and comprises o-nitrophenyl-β-added to the medium. D-galactopyranoside (ONPG), 5-bromo-3-indolyl-β-D-galactoside (Blugal), 5-
Chromogenic enzyme substrates such as bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and 4-methylumbelliferyl-β-D-glucuronide (MU
Microorganisms are detected by the color or fluorescence of a coloring substance or a fluorescent substance generated by decomposing the fluorescent enzyme substrate such as G) by the enzyme. Above all, when 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) is used as the chromogenic enzyme substrate, the resulting blue reaction product (bromochloroindigo) is visually apparent. Therefore, the medium supplemented with X-Gal is a suitable medium for rapidly and specifically detecting coliform bacteria.

[0004] However, when the concentration of bacteria is low, it is difficult to determine with the naked eye, and it is necessary to measure the absorbance value using a spectrophotometer, which is problematic in terms of simplicity and speed. On the other hand, when the medium contains a plurality of organic and inorganic substances such as nutrients and the medium itself is colored, the color tone of the indigo blue in the medium changes, and a positive judgment is made with the naked eye. Need to show a control color, but the indigo-based dye generated from the chromogenic enzyme substrate has low water solubility and could not be used as a water-soluble dye solution.

[0005]

DISCLOSURE OF THE INVENTION The present invention provides a colorimetric liquid which is used in the specific enzyme substrate medium method for reliably determining, with the naked eye, the color of the medium that has developed indigo blue due to the presence of microorganisms. With the goal.

[0006]

Means for Solving the Problems In view of such circumstances, the present inventors have conducted various studies on a water-soluble dye which can serve as a positive control of the medium color in which indigo blue color develops and develops in the medium. The present inventors have found that a solution in which o-nitrophenol and o-nitrophenol are mixed is extremely close to the color of the medium in which indigo blue has developed, is stable, and can be a colorimetric liquid showing a positive limit for judging microorganisms, and completed the present invention.

That is, the present invention provides a colorimetric liquid for determining the color of a medium formed by an indigo-based substance produced by decomposition from a chromogenic enzyme substrate in a specific enzyme substrate medium method, comprising indigo carmine and o- An object of the present invention is to provide a colorimetric liquid for determining microorganisms, which comprises nitrophenol.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The colorimetric liquid for judging microorganisms of the present invention comprises:
In the specific enzyme substrate medium method, it is used to determine the color of the medium that has been colored by an indigo-based substance generated by decomposition of a color-forming enzyme substrate due to the presence of a microorganism.

Here, the specific enzyme substrate medium method refers to an enzyme in which a chromogenic enzyme substrate or a fluorescent enzyme substrate added to the medium is specifically present in a microorganism, for example, Escherichia coli or an Escherichia coli group (such as β-glutaronidase or galactosidase). Is a method of detecting microorganisms by the color or fluorescence of a color-forming substance or a fluorescent substance generated by the method, which is also employed in a water supply test method. In the present invention, in particular, among these, indigo-based Using a chromogenic enzyme substrate that produces a chromogenic substance.

Such a chromogenic enzyme substrate includes, for example, 5-
Bromo-3-indolyl-β-D-galactoside (Bl
ugal), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal), 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside (X-Gluc) ), 5-bromo-4-chloro-
3-indolyl-α-D-galactopyranoside, 5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside, 5-bromo-4-chloro-3-indolyl-
β-D-glucuronic acid cyclohexylammonium salt, 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid sodium salt, etc. Gal is preferred.

The colorimetric solution of the present invention is used when judging the presence of a microorganism by using a specific enzyme substrate medium method. If it is a kind, it is not particularly limited,
For example, Escherichia coli (E. coli) and coliforms are exemplified.

The Escherichia coli group refers to a group of microorganisms having the ability to produce a lactose-degrading enzyme, and belongs to the genus Escherichia, the genus Cytobacter, the genus Klebsiella, the genus Enterobacter, and the like.

The colorimetric liquid for judging microorganisms of the present invention contains indigo carmine and o-nitrophenol. By mixing o-nitrophenol as a yellow component together with indigo carmine which is a blue component, The color tone can be very close to the medium color in which indigo blue color has developed.

The color tone of the color changes depending on the number of bacteria in the medium, and the colorimetric liquid must be the same as the color showing the positive limit. From such a viewpoint, the colorimetric liquid of the present invention has an absorbance at a wavelength of 610 nm of 0.07 to 0.07.
It is preferably 0.12, and more preferably the absorbance at a wavelength of 420 nm is 0.1 to 0.3 (see Reference Example 1).

In order to obtain such an absorption value, for example, the concentration of indigo carmine is 0.2 to 0.8.
mg / 100 ml, the concentration of o-nitrophenol is 0.
4 to 2 mg / 100 ml, and the mixing ratio is 1: 9.
~ 4: 6 is preferable. X as a chromogenic enzyme substrate
For a medium using -Gal, it is most preferable to use a colorimetric liquid in which the concentration of indigo carmine is 0.2 mg / 100 ml and the concentration of o-nitrophenol is 0.48 mg / 100 ml.

The colorimetric liquid of the present invention may be prepared by dissolving a specified amount of indigo carmine and o-nitrophenol in a transparent solvent such as water, ethanol and methanol.
Other components, for example, components contained in the medium to be used, buffers, preservatives, preservatives, and the like can be appropriately added to the extent that the coloring stability is not affected.

For example, in order to detect E. coli, a fluorescent enzyme substrate, 4-methyl-umbelliferyl-
When β-D-glucuronide (MUG) is added, by adding 4-methyl-umbelliferone to the colorimetric solution of the present invention, it can also be used as a positive control solution for detecting E. coli at the same time. . In this case, the wavelength 36
It can be confirmed by the presence or absence of fluorescence due to 6 nm ultraviolet irradiation, and the concentration of 4-methyl-umbelliferone added to the colorimetric liquid is preferably 0.1 to 1 mg / 100 ml.

[0018]

The present invention will be described below in more detail with reference to examples and reference examples.

Reference Example 1 Examination of marginal color of positive reaction E. coli ATCC 11775, K. ascorbata ATCC 33433, A. hydro
philia JCM 3976, C. freundii ATCC 8090, C. diversus
ATCC 25407, S. marcescens ATCC 8100, E. aerogenes AT
CC 13048, E.cloacae ATCC 23355, E.intermedium ATCC
33421, E.cloaceae ATCC 13047, K.oxytoca ATCC 1318
2. Bacteria (Escherichia coli, coliform group) total of K. pneumoniae ATCC 13883, K. ozaenae ATCC 11296 and H. alvei ATCC 13337 1
The strains obtained by culturing the four strains with tryptosome bouillon at 35 ° C. for 24 hours are tested, and the strains other than E. coli are diluted to 10 ^{−9 with} sterile physiological saline, and E. coli is diluted to 10 ^−10. And dilute 10 ml of each dilution to a medium containing X-Gal ("E
C Blue 10 "manufactured by Nissui Pharmaceutical Co., Ltd.)
After culturing for 24 hours, the positive reaction of each bacterial species was determined by naked eyes and measured at 610 nm (using a photoelectric photometer (ANA-7S) and a round sample cell). As shown in Table 1,
When the absorbance at a wavelength of 610 nm was 0.07 or more, it was determined to be positive in the naked eye determination.

[0020]

[Table 1]

Reference Example 2 Indigo carmine solution and o-
Examination of Formulation of Nitrophenol Solution A phosphate buffer was prepared by dissolving 1 g of potassium dihydrogen phosphate and 4 g of dipotassium hydrogen phosphate per 1000 ml of purified water. Indigo carmine solution is 0.2 mg or 0.5 m of indigo carmine in 100 ml of phosphate buffer.
g-solution, and the o-nitrophenol solution was similarly prepared by dissolving 0.2 mg, 0.5 mg, 0.8 mg or 1 mg of o-nitrophenol in 100 ml of phosphate buffer. The two solutions of each concentration were mixed, and the absorbance was measured at a wavelength of 610 nm (photometer (ANA-7S), round sample cell). Further, an indigo carmine solution of each concentration was added to an X-Gal-containing medium (“EC Blue 10” manufactured by Nissui Pharmaceutical Co., Ltd.) to obtain a positive color index.

As shown in Table 2, an indigo carmine solution (0.2 mg / 100 ml) was added to EC Blue 10
By adding ml, the color became close to the positive limit color. Using this as an index, the combination of the indigo carmine solution and the o-nitrophenol solution gave the closest color to the indigo carmine solution (0.5 mg / 100 ml) 4 m
l and o-nitrophenol solution (0.8 mg / 100 m
l) It was a 6 ml combination. The wavelength of the solution at this time is 61
The absorbance at 0 nm was 0.07, and the absorbance at 420 nm was 0.11. On the other hand, an indigo carmine solution (0.2 mg / 100 ml) was added to EC Blue 10 for 10 times.
The absorbance at a wavelength of 610 nm of the solution added with 0.1 ml was 0.10,
Since the absorbance at a wavelength of 420 nm was 0.15, substantially the same colorimetric liquid could be prepared.

[0023]

[Table 2]

Reference Example 3 Examination of Fluorescence Confirmation Solution 10 mg of 4-methylumbelliferone (7-hydroxy-4-methylcoumarin) sodium salt (SIGM) was dissolved in 100 ml of purified water and serially diluted 10 times with purified water. The solution was 366 nm in wavelength (MODEL UVBL-2).
5, UVP, INC) with ultraviolet light irradiation and measurement with an MTP-32 fluorescence reader (365 nm).

As shown in Table 3, the concentration of the sodium 4-methylumbelliferone solution was 0.1 mg / 100 ml.
At that time, it was recognized that it was a limit that could be confirmed as fluorescence.
Therefore, 1 mg of 4-methylumbelliferone sodium was added per 1000 ml of purified water to make a fluorescence confirmation solution.

[0026]

[Table 3]

Example 1 Preparation of colorimetric liquid Indigo carmine 2 mg, o-nitrophenol
4.8 mg, sodium 4-methylumbelliferone
1 mg, 1 g of potassium dihydrogen phosphate and 4 g of dipotassium hydrogen phosphate were dissolved in purified water to make a total volume of 1 L.
Note that the container used was the same as the medium used.

Example 2 Performance of colorimetric liquid E. coli ATCC 11775 (2.1 × 10 ^{9 /} ml), C. diversus ATCC
25407 (4.0 × 10 ^{8 /} ml), C. freundii ATCC 8090 (2.0 × 1
0 ⁷ / ml), E. aerogences ATCC 13048 (9.0 × 10 ⁸ / ml),
E.cloacae ATCC 23355 (8.0 × 10 ^{8 /} ml), K.oxytoca ATC
C 13182 (1.5 × 10 ^{9 /} ml), K. ozaenae ATCC 11296 (2.7
× 10 ⁸ / ml), K.pneumoniae ATCC 13883 (9.0 × 10 ⁸ / ml)
And a test strain of K. ascorbata ATCC 33433 (6.3 × 10 ⁸ / ml) (the number in parentheses indicates the number of bacteria in trifoil soy broth after cultivation at 35 ° C. for 24 hours) so as to be several per 1 ml. (10 ^-7 to 10 ^-10 ), and the medium containing X-Gal containing 9 ml of purified water ("EC Blue 10").
Nissui Pharmaceutical Co., Ltd.), cultured at 35 ° C., and observed a positive reaction for 18 to 48 hours. At this time, the colorimetric liquid prepared in Example 1 was set as a control, and the limit of positivity was determined. Then, it was confirmed whether or not the colorimetric liquid was actually positive. In the culture medium of the positive limit showing the same color development as that of, all the cultures became positive when the culture was continued, and it was confirmed that the performance of the colorimetric liquid was sufficient. E.coli ATCC l1775 and C.diversus ATCC 2
The results in the case of 5407 are shown in Tables 4 and 5 as examples.

[0029]

[Table 4]

[0030]

[Table 5]

[0031]

By using the colorimetric liquid of the present invention, the positive limit of microorganism determination in the specific enzyme substrate medium method can be clearly judged with the naked eye. Further, since the colorimetric liquid does not change its color tone even when stored at room temperature and has good storage stability, it can be provided together with a culture medium as a kit for determining microorganisms.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Taya Teramura 1075-2 Kita Minamiro, Yuki City, Ibaraki Prefecture Nissui Pharmaceutical Co., Ltd. Diagnostic Drug Research Department (72) Inventor Shingo Mizuochi 1075-2 Kita Minamiro, Yuki City, Ibaraki Prefecture F-term (reference) in Diagnostics Research Dept., Nissui Pharmaceutical Co., Ltd.

Claims (5)

[Claims] Claims: 1. A colorimetric liquid for judging a medium color developed by an indigo-based substance produced by decomposing a chromogenic enzyme substrate in a specific enzyme substrate medium method, wherein indigo carmine and o-nitrophenol are used. A colorimetric liquid for determining microorganisms, comprising: 2. The chromogenic enzyme substrate is 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X
-Gal), the colorimetric liquid for judging microorganisms according to claim 1. 3. An absorption value at a wavelength of 610 nm of 0.1.
The colorimetric liquid for judging microorganisms according to claim 1 or 2, wherein the colorimetric liquid is from 07 to 0.12. 4. 0.01 to 1 mg of indigo carmine
/ 100 ml, 0.1 to 10 m with o-nitrophenol
The colorimetric liquid for determining microorganisms according to any one of claims 1 to 3, which contains g / 100 ml. 5. The colorimetric liquid for determining microorganisms according to claim 1, wherein the microorganism to be determined is Escherichia coli and / or a group of coliforms.