JPH0646891A - Culture medium for fluorescent detection of colibacillus group - Google Patents

Abstract

PURPOSE:To provide a culture medium for fluorescent detection which can detect groups of Escherichia coli in the specimen. CONSTITUTION:The culture medium for fluorescent detection of colibacilli comprises 1 to 2.5wt.% of peptone, 0.25 to 0.5wt.% of sodium chloride, 0.4 to 0.6wt.% of yeast extract, 0.2 to 0.5wt.% of potassium hydrogen phosphate, 0.05 to 5wt.% of sodium pyruvate, 0.05 to 1wt.% of potassium nitrate, 0.01 to 1wt.% of sodium dodecylsulfate, and 4-methyl-umbellifer-beta-D-galactoside or 4-methyl-umbellifer-beta-D-glucuronide as a fluorescent substrate. The fluorescent substrate is preferably added to the culture medium in an amount of 10<-14> to 10<-2>M. When cell groups of Escherichia coli are detected using the medium, the specimen is added to the medium, cultured at 30 to 44.5 deg.C for several to teens hours, irradiated with a light of 365nm wavelength and measure the fluorescent light of 450nm wavelength.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a coliform fluorescence detection medium for rapidly, simply and highly sensitively determining whether or not microorganisms are present in a sample such as food.

[0002]

2. Description of the Related Art The safety of foods ingested by humans, such as foods and drugs, is strictly controlled.
It is especially important to determine the presence or absence of microorganisms. Of these, the determination of the presence or absence of coliform bacteria in food is particularly important as a daily inspection.

As a method for detecting coliform bacteria in foods, conventionally, a suspension obtained by suspending a food sample in sterilized physiological saline or the like and serially diluting the suspension is cultured for 24 hours using a dezoxycholate medium. After that, the number of grown colonies is calculated, or by using brilliant green lactose broth (BGLB), lactose broth, etc.
A method is known in which after culturing for 4 to 48 hours, the number of test tubes in which gas production is recognized is counted to calculate the number of bacteria.
However, in these methods, the culture time is 24 to 4
Since it takes about 8 hours, it takes more than one day to make a determination, the measurement of colonies is complicated, and the operation requires skill, and it is necessary to serially dilute the sample or prepare the culture medium for operation. There was a problem that it was complicated.

On the other hand, recently, a fluorescence method has been reported which enables rapid detection of microorganisms using a fluorescent substrate (Japanese Patent Publication No. 58-58).
-17598 and JP-A-57-144995). However, in the detection method using a fluorescent substrate, the number of coliform bacteria is required to be 10 ⁴ to 10 ⁵ or more per 1 ml of the sample stock solution, and therefore a culture operation is required to detect coliform bacteria having a relatively small number of bacteria such as food, There is a need for a medium that allows the coliforms to grow faster. Therefore, the present inventors have a fast generation time as a medium for growing coliform bacteria,
A growth medium for coliforms having a good growth rate and no lag phase was developed and reported (JP-A-4-51890).

[0005]

However, although this medium is suitable for the growth of coliforms, it has a high turbidity and a high turbidity when used as a medium for detecting fluorescence by incorporating a fluorescent substrate into this medium. However, it was not yet sufficiently satisfactory as a medium for fluorescence detection. Therefore, it has been desired to develop a medium having higher transparency and suitable for detecting the fluorescence of coliform bacteria.

[0006]

Under such circumstances, the present inventors have made various studies on the medium described in the above-mentioned Japanese Patent Laid-Open No. 4-51890, and found that the bovine bile powder contained in this medium was Escherichia coli. It has the effect of suppressing the growth of microorganisms other than Gram-negative bacteria such as colonies and promoting the growth of coliform bacteria, but on the other hand, it increases the turbidity of the medium and causes fluorescence quenching. I found it. Therefore, when we searched for ingredients that could replace the beef bile powder,
Surprisingly, the medium containing sodium dodecylsulfate instead of bovine bile powder was found to have not only an increase in turbidity, but also the use of this to shorten the fluorescence expression time and enable more rapid detection of coliform bacteria. Completed the invention.

That is, according to the present invention, by weight, peptone is 1.0 to 2.5% and sodium chloride is 0.25 to 0.5.
%, Yeast extract 0.4 to 0.6%, dipotassium monohydrogen phosphate 0.2 to 0.5%, sodium pyruvate 0.05
-5.0%, potassium nitrate 0.05-1.0%, sodium dodecyl sulfate 0.01-1.0% and 4-methyl-
It is intended to provide a coliform detection medium containing umbelliferyl-β-D-galactoside or 4-methyl-umbelliferyl-β-D-glucuronide.

The medium of the present invention is characterized by containing sodium dodecyl sulfate as described above, and the content thereof is 0.01 to 1% by weight. The control of bacterial species other than Gram-negative bacteria is not sufficient, and when it exceeds 1% by weight, the fluorescence intensity is decreased, which is not preferable.

Further, 4-methyl-umbelliferyl-β-D-galactoside (4-MU) which is a component of the medium of the present invention.
Gal) and 4-methyl-umbelliferyl-β-D-
Glucuronide (4-MUGul) is a fluorescent substrate. 4-MUGal and 4-MUGul are usually colorless, of which 4-MUGal has the presence of coliforms,
4 by the action of β-galactosidase produced by this
-Methyl-umbelliferone (4-MU) was generated and 4
-MUGul is Escherichia coli (Es) in E. coli.
chelicia coli), 4-MU is produced by the action of the glucuronidase produced by C. coli. The 4-MU thus produced is a fluorescent substance and is excited at a wavelength of 365 nm to emit fluorescence of 450 nm. Therefore, the presence of coliforms can be confirmed by measuring this.

4-MUGal or 4-MUGul is preferably added to the medium of the present invention in an amount of about 10 ^-4 to 10 ^-2 M. The Escherichia coli group to be detected by the medium of the present invention refers to a group of microorganisms having the ability to produce a lactose-degrading enzyme, which belong to the genus Escherichia, the genus Cytobacter, the genus Klebsiella, the genus Enterobacter, and the like.

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

In order to detect the presence of coliform bacteria in a subject using the culture medium for detecting coliform bacteria of the present invention, the subject is added to the culture medium at 30 to 44.5 ° C. for several hours to several tens of hours. After culturing, irradiation with light of 365 nm may be performed, and fluorescence at 450 nm may be measured.

[0013]

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 Modified mediums 1 to 4 were prepared using the coliform detection medium described in JP-A-4-51890 as a basal medium, and a test was conducted to determine which medium is suitable for fluorescence detection. Table 1
0.1 ml of medium having the composition shown in 96-well microplate
After inoculating each plate with the bacteria listed in Table 2, 3
Culture was performed at 5 ° C., and each plate was sequentially irradiated with light of 365 nm using a fluorescence reader (MTP-32 manufactured by Corona Co., Ltd.), and the time until the fluorescence of 450 nm was expressed was measured. The results are shown in Table 2.

[0015]

[Table 1]

[0016]

[Table 2]

From this result, the modified 4 which is the medium of the present invention is obtained.
It can be seen that in any of the culture mediums, the fluorescence detection time is shortened by 1 to 2 hours or more as compared with the other culture media.

Example 2 A commercial food was tested for coliforms using the medium of the present invention.
1 ml each of the medium of the present invention was dispensed into a 24-well transparent plate, and 90 ml of sterilized physiological saline was added to 10 g of the food shown in Table 3 to inoculate 1 ml of a sample that was stomached to homogeneity. Cultured at ° C. 6, after the start of culture
Fluorescence reader (Corona, M
TP-32) was used to irradiate light of 365 nm, and fluorescence at 450 nm was measured (fluorescence plate method). On the other hand, the stomached sample is serially diluted 10-fold, 1 ml thereof is put into a sterilized petri dish, and dezoxycholate medium which has been dissolved by heating in advance and cooled to about 45 ° C. is poured therein, and the medium and the sample are well mixed, and after cooling and overlaying. After culturing at 35 ° C. for 20 hours, the number of bacteria was calculated (Deso pour method). The results are shown in Table 3. From Table 3, it can be seen that the fluorescent plate method using the medium of the present invention is in good agreement with the conventional Deso pour method, and the Deso pour method requires a culture time of at least 18 hours, whereas the fluorescent plate method According to the method, the coliforms could be confirmed in a culture time of 6 to 8 hours.

[0019]

[Table 3]

[0020]

EFFECTS OF THE INVENTION According to the present invention, it becomes possible to perform a test capable of quickly and highly sensitively determining the presence or absence of coliform bacteria in a sample such as food.

Claims (1)

[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%, sodium dodecyl sulfate 0.0
1-1.0% and 4-methyl-umbelliferyl-β-
D-galactoside or 4-methyl-umbelliferyl-
A coliform fluorescence detection medium containing β-D-glucuronide.