JP2000000100A - Test sheet for detecting coliform bacteria - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject sheet for detection of Escherichia coli, of food, etc., by impregnating a detected liquid to which 5-bromo-4-chloro-3-indolyl-β- galactopyranoside is added to culturing reagent-containing bouillon medium into a substrate sheet. SOLUTION: Culturing reagents of L-asparagine, Casamino acid and yeast extract, etc., are added to bouillon culture media and further, 5-bromo-4-chloro-3- indolyl-β-galactopyranoside and 4-methylumbelliferyl-β-D-glucuronide is added as a reaction substrate thereto to prepare a detection solution. Then, sheet-like substrate 2 comprising a filter paper, etc., cut in paper strip-like shape is immersed into the detection solution up to the cut line 3 to impregnate the detection solution in a test part 5 on the sheet-like substrate 2 and the impregnated sheet is dried to provide the objective test sheet 1 for detection of coliform bacteria. Further, the test sheet can surely detect coliform bacteria which becomes index of contamination of food and beverage due to feces and cause of pollution of Escherichia coli in a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an inexpensive test sheet for detecting coliform bacteria, which can easily and reliably detect an indicator of fecal contamination of food and drink and a coliform group causing contamination of coliform bacteria in a short time.

[0002]

2. Description of the Related Art Since coliform organisms are resident bacteria in the intestinal tract of a warm-blooded animal, if these bacteria are detected from water or food, the coliforms are usually found in water and food. It is suspected that water or food is directly or indirectly contaminated by human or animal feces, and there is a possibility that various digestive system infectious bacteria and food poisoning bacteria may be present. Is considered less favorable.

[0003] Therefore, the coliform group has heretofore been referred to as a stool pollution indicator bacterium or simply as a pollution indicator bacterium, and a test of the coliform group is generally performed as an indicator of fecal contamination of food and drink.

[0004] Recently, Escherichia coli (Escherichia coli) has been used for some fresh foods, milk and processed meat products, which are relatively contaminated with coliform bacteria, rather than the coliform bacteria test. Direct testing is more specific and more efficient, and is used to detect Escherichia coli instead of the coliforms.

[0005] Therefore, a method for simply detecting the coliform group and a method for specifically detecting Escherichia coli (E. coli) from the coliform group are desired.

Accordingly, it is a first object of the present invention to provide an inexpensive coliform bacteria detection device capable of easily and reliably detecting coliform bacteria in a short time. A second object of the present invention is to provide an Escherichia coli group detector capable of reliably detecting Escherichia coli from the Escherichia coli group thus detected.

[0007]

In order to achieve the above object, the present invention provides 5-bromo-4-chloro-3-indolyl-β as a reaction substrate in a broth medium containing a culture reagent. -To provide a test sheet for detecting coliform bacteria, wherein a sheet-like substrate is impregnated with a detection solution to which galactopyranoside is added.
The present invention also provides a test sheet obtained by further adding 4-methylumbelliferyl-β-D-glucuronide as a reaction substrate to the test sheet.

That is, when detecting coliform bacteria using the test sheet of the present invention, a sample is attached to the test sheet,
The fungi in the sample are cultured in the detection solution contained in the test sheet. At this time, if the coliform group is present in the sample, β-galactosidase, a lactose-degrading enzyme possessed by the coliform group,
The above-mentioned 5-bromo-4-chloro-3-indolyl-β-galactopyranoside is hydrolyzed to form a blue colored spot, and Escherichia coli (E. coli) and bacteria belonging to the coliform group other than E. coli, for example, Citrobacta (Citro
bacter), Klebsiell
a) Genus, Enterobacter
r) The genus Proteus, Salmonella (Sal) belonging to other intestinal bacterial groups while the genus is detectable
monella) is not detected.

In this case, 5-bromo-4-chloro-
3-indolyl-β-galactopyranoside plus 4-
When methylumbelliferyl-β-D-glucuronide was added, E. coli The E. coli β-D-glucuronidase enzyme hydrolyzes 4-methylumbelliferyl-β-D-glucuronide to produce a fluorescent substance, e.g.
m, the 5-bromo-4-chloro-
Among blue spots due to hydrolysis of 3-indolyl-β-galactopyranoside, E. coli was used. only blue spots based on E. coli fluoresce light blue, c
Since blue spots based on coliforms other than oli do not emit such fluorescence, E. coli. E. coli can be distinguished from other coliforms.

Therefore, the test sheet of the present invention can specifically detect Escherichia coli and also specifically detect Escherichia coli (E. coli) from the Escherichia coli group. Moreover, as described above, the specimen was attached to the test sheet, cultivation was performed, E. coli was easily detected in a short time only by irradiating ultraviolet rays, and the sheet-like substrate was impregnated with the detection liquid. Therefore, the test can be carried out at a low cost with a very small amount of medium, culture reagent and reaction substrate, and after the test, it can be easily and safely discarded by incineration.

Hereinafter, the present invention will be described in more detail. The test sheet for detecting coliform bacteria according to the present invention comprises a culture reagent and 5-bromo-4-chloro-3-indolyl-β-galactopyranoside as described above. The detection solution added to the bouillon medium is impregnated into a sheet-like substrate.In this case, as the bouillon medium, a bouillon having a basic composition conventionally used generally for culturing bacteria can be used. Specifically, a bouillon having the following composition can be exemplified.

Peptone 20 g / L Lactose 5 g / L Bile salt 1.5 g / L Monopotassium hydrogen phosphate 4.0 g / L Dipotassium hydrogen phosphate 1.5 g / L Sodium chloride 5.0 g / L

Also, 5-bromo-4-chloro-3-indolyl-β- added to the broth as a reaction substrate.
As described above, galactopyranoside is the lactose-degrading enzyme β-galactosidase (β-galac) possessed by the Escherichia coli group.
is hydrolyzed to form a blue colored spot. Although the amount of addition is not particularly limited, it is usually 0.1 ml per 1000 ml of the bouillon medium.
It can be about 01 to 0.5 g, particularly about 0.03 to 0.2 g.

On the other hand, 4-methylumbelliferyl-β-
D-glucuronide is a substance in which the glucuronide bond is hydrolyzed by an enzyme β-D-glucuronidase produced by E. coli to produce a fluorescent substance (4-methylumbelliferyl). Is to detect Escherichia coli from the fluorescence reaction of The amount of addition is not particularly limited, but is usually about 0.05 to 0.1 g per 1000 ml of the above-mentioned broth medium.

Further, the above-mentioned culture reagent is added for the purpose of culturing Escherichia coli effectively in a short time, and may be any one which does not affect the reaction of the above-mentioned reaction substrate. Can be suitably used. Specific examples include L-asparagine, casamino acid, yeast extract, glucose, sodium citrate, etc. Among them, it is preferable to use L-asparagine, casamino acid and yeast extract in combination. The amount of the culture reagent to be added can be a usual amount, specifically, about 0.5 to 3 g per 1000 ml of the above bouillon. Particularly, the above L-asparagine, casamino acid and yeast extract can be used. When used in combination, it is preferable to use about 1 g of L-asparagine, about 0.5 g of casamino acid, and about 1 g of yeast extract.

The test sheet of the present invention is obtained by impregnating the above-mentioned broth medium with a detection solution obtained by adding the above-mentioned reaction substrate and culture reagent, and it is preferable that the detection solution has a pH of about 6.5. In addition, as long as the detection solution does not inhibit the growth of Escherichia coli, additives other than those described above may be added, and a culture reagent or a bacterium selection agent other than the above may be appropriately added.

The sheet-like substrate impregnated with the detection liquid is not particularly limited in its material and shape, and may be paper or cloth having water absorption. Those formed in a strip shape using paper having good properties are preferably used. Specifically, the sheet-like substrate shown in FIG. 1 is exemplified. That is, FIG. 1 shows an example of the test sheet of the present invention. This test sheet 1 is a sheet-like substrate 2 formed in a strip shape using a paper having good water absorption such as filter paper.
Is impregnated with the above-mentioned detection solution. In this case, 3
Is a score line formed on one end side of the sheet-like substrate 2, one side is configured as a knob portion 4 and the other side is defined as a test portion 5 with the score line 3 as a boundary, whereby the test sheet 1 is inoculated with a specimen. Other than picking up the knob 4 with a finger and lifting it up, the other parts of the test sheet 1, especially the test section 5, are not directly touched by a finger, so that the test section 5 is not contaminated and the test section 5 is not contaminated. Can be determined to be derived from the sample, and the test results can be determined without error.

The means for impregnating the sheet-like substrate 2 with the detection liquid is not particularly limited, but a method of immersing the sheet-like substrate 2 in the detection liquid and impregnating the sheet-like substrate 2 with the detection liquid is preferably employed. You. In this case, there is no particular limitation on the amount of the detection solution impregnated in the sheet-like substrate, and the test sheet for detecting coliform bacteria of the present invention can be obtained by drying the sheet-like substrate impregnated with the detection solution.

The detection test sheet of the present invention can be used for various foods and drinks, for example, fresh foods (vegetables, fruits, tofu, etc.), processed livestock products (meat, ham, cheese, etc.), furthermore, pool water, tap water, It can be widely used for well water, etc. and is not limited to the indicator of fecal contamination of food and drink, but also a simple public health inspection test such as water quality survey, contamination of sandboxes in parks, and hygiene management in restaurants and homes. It can be used effectively as a sheet.

In the method of using the test sheet for detecting coliform bacteria of the present invention, first, a specimen is attached to the test sheet of the present invention. In this case, when the test substance is water-soluble, the test substance can be used as it is, and when the test substance is in a viscous or solid state, the test substance can be appropriately diluted and mixed with physiological saline to prepare the test substance.
The amount of the sample attached to the test sheet is not particularly limited, but is preferably about 1 ml. When the test substance is a solid such as various foods, the test sheet can be brought into direct contact with the test substance to adhere the sample.

Next, the test sheet to which the sample is attached is subjected to culture. The culture conditions are preferably at a temperature of 36 to 44.5 ° C, and particularly preferably at a temperature of 35 to 37 ° C, which is a general culture condition. The culturing time is not particularly limited, but is generally 18 to
It is about 24 hours. As a specific culturing method, for example, a test sheet to which a sample is attached is placed in a petri dish or a transparent bag made of plastic film or the like in order to avoid germs and drying, and stored in an incubator at 35 to 37 ° C. for about 24 hours. Can be performed.

After culturing, if the test sheet is in a transparent container such as a transparent bag, the test sheet is taken out if necessary, the number of blue spots is counted, and the degree is determined. In addition, this blue spot indicates that β-galactosidase possessed by Escherichia coli is 5-bromo-4-chloro-3-.
Indolyl-β-D-galactopyranoside is hydrolyzed to give 5-bromo-4-chloro-3-indoline and β-
It is based on the reaction which produces D-galactose and oxidizes the former to bromochloroindigo.

When the test sheet further contains 4-methylumbelliferyl-β-D-glucuronide, the test sheet is further irradiated with ultraviolet rays using a UV lamp, etc. E. coli can be counted by counting blue fluorescence.
Can be determined. In this reaction, the glucuronide bond of the reaction substrate 4-methylumbelliferyl-β-D-glucuronide contained in the detection solution was hydrolyzed by the enzyme β-D-glucuronidase of Escherichia coli, and the fluorescent substance 4-methyluncuronide was hydrolyzed. It is based on the release of veriferyl.

Here, when the test sheet having the spots is stored, refrigerated storage is suitable, and in the frozen storage, the generated spots attenuate in about 10 days.

The method for storing the test sheet for detecting coliform bacteria of the present invention is carried out in a cool and dark place (such as a refrigerator) in view of the stability of the reagent.
Preservation is desirable. However, spots are observed even after 7 months of storage at room temperature, and storage for a certain period of time is possible even at room temperature.

The used test sheet may be contaminated with other bacteria even if the coliform bacteria are not detected. Therefore, the used test sheet must be disposed of by incineration in order to prevent infection and the like. In this case, the test paper sheet of the present invention is
Since it is a sheet made of paper or cloth, it can be easily incinerated and disposed, and disposal is simpler and more economical than the conventional detection kit method using a test tube or the like.

This test paper does not observe the formation of spots due to the coloring reaction of the dye in the culture medium, and therefore does not contain a dye or a reducing agent, so that the test paper does not discolor during storage. The color development on the test paper is based on an enzyme reaction, and the color of the reaction is proportional to the amount of bacteria, and the color can be developed even with a small amount of bacteria. Further, by irradiating ultraviolet rays, Thus, only E. coli is fluorescently colored in a specific light blue to easily distinguish it from other coliforms, and the method of use is extremely simple.

[0028]

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

Example 1 5-Bromo-4-chloro-3-indolyl-β was added to 1000 ml of broth medium.
-Galactopyranoside (X-GAL) 0.05 g, 4-
Methylumbelliferyl-β-D-glucuronide (MU
G) A detection solution was prepared by adding 0.05 g, 1.0 g of L-asparagine, 0.5 g of casamino acid and 1.0 g of yeast extract, and the filter paper cut into strips was immersed in the detection solution to prepare the detection solution. After the filter paper (sheet-like substrate) was impregnated with the above-mentioned detection solution, it was dried to obtain a test sheet for detecting coliform bacteria.

On the other hand, the following E. coli, coliforms and other bacterial species other culture was diluted with sterile 0.1% peptone pressurized saline, number 1 to 10 to 10 ³ or more live bacteria A diluent was prepared. E. coli: Escherichia col
i) Type I coliform bacteria: Citrobacter
r fresh) Enterobacter aerogenes (Enterobac)
ter aerogenes Klebsiella pneumoniae
pneumonia) Other fungi: Salmonella typhimurium
typhimurium Proteus Pseudomonas

Next, the test sheet was immersed in the bacterial solution and cultured at 35 ° C. for 24 hours, and the number of blue spots was determined. In addition, UV light (wavelength 365n)
m), and the number of spots emitting light blue fluorescence was examined. Table 1 shows the results. The results were evaluated according to the following criteria. The amount of inoculated bacteria was calculated according to the official method.

The number of spots +1 to 10 ++ 10 to 100 ++ 100 or more or the entire test paper is blue

[0033]

[Table 1]

As shown in Table 1, E. coli In E. coli and E. coli, the number of spots corresponding to each dilution was observed, but no spot was observed in other bacterial species. When this was irradiated with ultraviolet light, Only the blue spot on the test paper inoculated with E. coli fluoresced light blue, and the test papers of coliforms and other bacterial species did not fluoresce at all.

[Embodiment 2] Bacterial liquids of various amounts of E. coli and the other bacterial species described above were prepared, and an experiment was carried out in the same manner as in Example 1. Table 2 shows the results.

[0036]

[Table 2]

From the above results, E.I. E. coli bacteria quantity 1-1
0 microbial and 1-10 microbial of other species
Even in the case of a combination of ⁴ or more large bacteria, the number of blue spots generated is Only the number of spots corresponding to the bacterial mass of E. coli is formed, and the reverse mixed bacterial mass is not affected by the bacterial content of other bacterial species. The number of spots corresponding to the amount of E. coli was formed.

When this was irradiated with ultraviolet rays, all the blue spots emitted fluorescence, and were not affected by the presence of other bacterial species. It was confirmed that only E. coli grew and formed spots.

[Example 3] Bacterial liquids of various amounts of the Escherichia coli group and other bacterial species were prepared, and an experiment was carried out in the same manner as in Example 1. Table 3 shows the results.

[0040]

[Table 3]

From the results in Table 3, it was found that the blue spots developed for the coliform group were formed without being affected by the content of other bacterial species, and corresponding to the respective bacterial content of the coliform group.
The test paper having the blue spot was irradiated with ultraviolet light, but no fluorescence was observed.

From the above, the blue spots generated on the test paper were E. coli. E. coli and Escherichia coli group only, are not affected by other bacterial species, and by further irradiating ultraviolet rays,
E. FIG. It was confirmed that only E. coli specifically developed fluorescence.

Example 4 Using the test sheet of Example 1, water (30 samples) such as well water, side dishes (135 samples), raw meat and raw fish (17 samples) such as pig, cow, chicken, tuna sashimi, etc.
Samples), tofu, fresh confectionery, cut vegetables (10 samples), cookware (15 samples) such as manita, etc., were tested for the presence of coliform bacteria, and the results were compared with the results of the LB-BGLB fermentation tube method and desoxycholate agar medium. Was. In addition, the LB-BGLB fermentation tube method and the test method using desoxycholate agar medium as controls followed the Food Sanitation Law. For prepared foods, raw meat and raw fish, and tofu, raw confectionery and cut vegetables, a test was performed on a mixture of 10 g of a sample and 90 ml of sterile 1% peptone-added physiological saline, and a wipe test was performed on cookware. Table 4 shows the results. In this case, LB-BGL for well water and used water
Compare the number of positive acids and gases produced by the B fermentation tube method with the number of blue spots formed on this test paper.For various foods, those with red colonies formed on a desoxycholate agar medium are regarded as positive. On the test paper, the one that formed a blue spot was regarded as positive, and the agreement rates of both test methods were compared.

[0044]

[Table 4]

[0045]

According to the present invention, coliforms such as foods can be detected reliably and easily.

[Brief description of the drawings]

FIG. 1 is a plan view showing an example of a test paper used in the present invention.

[Explanation of Signs] 1 Test paper 2 Sheet 3 Cut line 4 Knob 5 Test section

Claims (2)

[Claims] 1. A broth medium containing a culture reagent is prepared as a reaction substrate with 5-bromo-4-chloro-3-indolyl-
A test sheet for detecting Escherichia coli, wherein a sheet-like substrate is impregnated with a detection solution to which β-galactopyranoside is added. 2. The test sheet according to claim 1, wherein 4-methylumbelliferyl-β-D-glucuronide is further added as a reaction substrate.