JP3142962B2 - Medium for fluorescence detection of coliform bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a medium for detecting Escherichia coli group fluorescence 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 humans, such as foods and medicines, which are ingested by humans, is strictly controlled.
It is particularly important to determine the presence or absence of microorganisms. Of these, the determination of the presence or absence of coliforms in food is particularly important as a daily test.

[0003] As a method for detecting coliforms in foods, a suspension obtained by suspending a food sample in sterile physiological saline or the like and serially diluting the suspension is serially diluted and cultured for 24 hours using a desoxycholate medium. After that, a method of calculating the number of grown colonies, or brilliant green lactose broth (BGLB), lactose broth, etc.
It is known to calculate the number of bacteria by counting the number of test tubes in which gas production is observed after culturing for 4 to 48 hours.
However, in these methods, the culture time is 24 to 4 hours.
Since it takes about 8 hours, it takes more than one day for the determination, and the counting of the colonies is complicated, the operation requires skill, and the operation is necessary because the sample is serially diluted and the medium is prepared. Is complicated.

On the other hand, recently, a fluorescence method using a fluorescent substrate and capable of detecting microorganisms more rapidly has been reported (Japanese Patent Publication No. 58-58).
-17598 and JP-A-57-144995). However, in the detection method using a fluorescent substrate, the number of coliforms is required to be 10 ⁴ to 10 ⁵ or more per 1 ml of the sample stock solution. There is a need for a medium that allows the coliforms to grow faster. Therefore, the present inventors have a rapid generation time as a medium for growing Escherichia coli,
A growth medium for Escherichia coli with a good growth rate and no lag phase was developed and reported (Japanese Patent Application Laid-Open No. 4-51890).

[0005]

However, although this medium is a medium suitable for the growth of Escherichia coli, it has a high turbidity when a fluorescent substrate is incorporated into this medium and used as a medium for fluorescence detection. Quenching was not yet fully satisfactory as a medium for fluorescence detection. Therefore, development of a medium having higher transparency and suitable for detecting the fluorescence of coliform bacteria has been desired.

[0006]

Under such circumstances, the present inventors have conducted various studies on the medium described in Japanese Patent Application Laid-Open No. 4-51890, and found that the bovine bile powder contained in this medium is Although it has the effect of suppressing the growth of microorganisms other than Gram-negative bacteria such as groups and promoting the growth of Escherichia coli, it increases the turbidity of the culture medium and causes quenching of fluorescence. I found it. Therefore, when searching for a component that can replace the bovine bile powder,
Surprisingly, the medium to which sodium dodecyl sulfate was added instead of bovine bile powder not only did not increase the turbidity, but also found that the use of this shortened the fluorescence expression time and enabled a more rapid detection of coliform bacteria. Completed the invention.

That is, the present invention relates to a peptone of 1.0 to 2.5% by weight and a sodium chloride of 0.25 to 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 0.01-1.0% and 4-methyl-
It is intended to provide a medium for detecting coliform bacteria containing umbelliferyl-β-D-galactoside or 4-methyl-umbelliferyl-β-D-glucuronide.

[0008] The medium of the present invention is characterized by containing sodium dodecyl sulfate as described above. The amount of the medium is 0.01 to 1% by weight. Of the bacteria other than Gram-negative bacteria is not sufficient, and if it exceeds 1% by weight, the fluorescence intensity decreases, which is not preferable.

Further, 4-methyl-umbelliferyl-β-D-galactoside (4-MU) which is a component of the medium of the present invention is used.
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 is
By the action of β-galactosidase produced by this, 4
-Methyl-umbelliferone (4-MU) produces 4
-MUGul is Escherichia coli (Es
In the presence of C. cherichia coli, 4-MU is produced by the action of the glucuronidase it produces. The thus generated 4-MU is a fluorescent substance and is excited at a wavelength of 365 nm to emit fluorescence at 450 nm. By measuring this, the presence of the coliform group can be confirmed.

4-MUGal or 4-MUGul is preferably added to the culture medium of the present invention in an amount of about 10 ^-4 to 10 ^-2 M. The Escherichia coli group to be detected in the medium of the present invention refers to a group of microorganisms having the ability to produce a lactose-degrading enzyme, belonging to the genus Escherichia, the genus Cytoobacter, 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.
1.5% by weight may be used as an agar medium.

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

[0013]

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

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

[0015]

[Table 1]

[0016]

[Table 2]

From these results, it is found that the medium of the present invention, modified 4
It can be seen that the fluorescence detection time is shortened by 1 to 2 hours or more in any of the strains in any of the strains.

Example 2 A commercially available food was tested for coliform bacteria using the medium of the present invention.
1 ml of the medium of the present invention was dispensed into a 24-well transparent plate, and 90 ml of sterile physiological saline was added to 10 g of the food shown in Table 3 to inoculate 1 ml of a uniformly stomached sample. Incubated at ℃. After the start of culture 6,
Fluorescence reader (Corona, M
Using TP-32), light of 365 nm was irradiated and fluorescence of 450 nm was measured (fluorescence plate method). On the other hand, the stomached sample is serially diluted 10-fold, and 1 ml of the diluted sample is placed in a sterile petri dish. A desoxycholate medium, which has been heated and dissolved and cooled to about 45 ° C., is poured therein, and the medium and the sample are mixed well. After culturing at 35 ° C. for 20 hours, the number of bacteria was calculated (Deso pour method). Table 3 shows the results. 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 at least 18 hours of culture time, whereas the fluorescent plate method requires at least 18 hours. The method was able to confirm the coliform bacteria in a culture time of 6 to 8 hours.

[0019]

[Table 3]

[0020]

According to the present invention, it has become possible to carry out a test capable of quickly and highly sensitively determining whether or not coliforms are present in a sample such as food.

──────────────────────────────────────────────────の Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C12Q 1/10 C12N 1/20 BIOSIS (DIALOG)

Claims (1)

(57) [Claims] 1. Peptone 1.0-2.5%, sodium chloride 0.25-0.5%, yeast extract 0.4-2.5% by weight
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-
An Escherichia coli group fluorescence detection medium containing β-D-glucuronide.