JP2002010799A - Cell examination medium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a medium enabling the simple determination of E.coli group in high reliability, the simultaneous determination of E.coli group and the number of viable bacteria and the quick discrimination of E.coli group. SOLUTION: The medium for the examination of viable bacteria contains a β-galactosidase color-developing substrate, an alanine aminopeptidase color- developing substrate and their enzyme-deriving substrates.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a 1) simple and highly reliable test for coliform bacteria, 2) a simultaneous test for coliform bacteria and common bacteria, and 3) a medium capable of rapidly distinguishing coliform bacteria. About. The Escherichia coli group referred to in the present invention is a group of microorganisms having the ability to degrade lactose (lactose), and includes Escherichia, Cytobacter, Klebsiella, Enterobacter, and Serratia. It means the microorganism to which it belongs.

[0002]

2. Description of the Related Art In the food manufacturing industry, the quantity of microorganisms in a product is routinely used as an index for asking whether or not the product has been subjected to safety, preservability, and hygienic handling. ing. Among various microorganisms existing in nature, aerobic or facultatively anaerobic gram-negative non-spore bacterium having lactose fermentation called "coliforms" is detected from various foods. This is also very often used as an indicator of hygiene and safety standards for food, such as determining that the food is a dirty food that may be subject to fecal contamination and cause food poisoning. I have.

[0003] Among the microorganisms, methods for testing general bacteria include ordinary agar medium, standard agar medium, Trypto-
A method using an agar medium (a general bacterial test medium) such as Soy Agar (trypto soy agar medium) is known. However, these methods are not suitable for specimens
Can be accurately measured, but other specific bacteria (for example, coliforms) cannot be measured in the same medium. On the other hand, as a method for testing coliform bacteria, Des
oxycholate Agar (Desoxycholate agar medium), Brilliant Green Lac
tose Bile Broth (BGLB medium), E
osin Methylene Blue Agar
There is known a method of selectively cultivating and detecting only the coliform bacteria using an agar medium or a liquid medium such as (EMB agar medium). These media are used to inhibit the growth of bacteria other than the coliforms (hereinafter sometimes referred to as "microbes"), so that a bacterial selection agent (for example, sodium desoxycholate,
Beef liver powder, brilliant green, etc.). Therefore, any bacteria cannot be detected.
In other words, this method can test coliform bacteria,
General bacteria cannot be tested. In addition, the presence of the bacterial selection agent has a risk of slightly affecting not only the germs but also the growth of Escherichia coli itself, and there is a problem that the test results of the Escherichia coli group lack reliability. In addition, when the number of coliforms contained in the sample is relatively small compared to various bacteria, the coliforms may not be detected. Furthermore,
In a modern food manufacturing plant, the inspection of products and semi-finished products, semi-finished products, and products and semi-finished products, especially immediately after heat sterilization, processed by sterilization equipment with improved sterilization capacity, in a sanitary environment. In such a case, the coliforms are examined in a state where the number of coliforms is very small or the growth activity of the coliforms themselves is weak, and there is a problem that the coliforms cannot be detected accurately.

On the other hand, β-galactosidase has conventionally been used as a convenient medium for testing Escherichia coli group, as one medium for growing Escherichia coli group selected from the group consisting of brain-hard infusion agar medium, tryptosia agar medium and K3 medium. A medium for detecting Escherichia coli containing a bacterium belonging to the genus Escherichia, which is characterized by adding a chromogenic substrate, and a method for examining Escherichia coli using this medium are known (Patent No. No. 2879698). The feature of this method is that when a medium is inoculated with a specimen and cultured, if the specimen contains an Escherichia coli group containing a bacterium belonging to the genus Escherichia, an enzyme (β-galactosidase) is specifically produced at the stage of development of the microorganism.
Is produced, and the chromogenic substrate contained in the medium reacts with the chromogenic substrate to cause coloration, so that the presence of Escherichia coli can be easily confirmed.

On the other hand, a chromogenic substrate for β-galactosidase and E. coli E. coli and E. coli containing a chromogenic substrate of β-glucuronidase as a moiety metabolizable by E. coli. A medium for simultaneously detecting the presence or absence of E. coli is known (Japanese Patent Publication No. 7-4272 and
000-100). A medium for detecting the presence or absence of a gram-negative bacterium in a specimen, which contains a chromogenic substrate for L-alanine aminopeptidase, is also known (Japanese Patent Publication No. H07-27139).
-4272).

[0006] However, these methods all involve various bacteria,
Particularly, in order to eliminate gram-positive bacteria, a bacterial selection agent such as sodium lauryl sulfate or vancomycin (antibiotic) is added to the medium. Therefore, general bacteria cannot be used as a bacterial test medium because their growth is inhibited. In addition, it is not possible to quickly distinguish between coliforms and various bacteria. On the other hand, in preparing the above-mentioned known E. coli group test medium, it is conceivable that a bacterial selection agent is not added, or even if it is added, the amount is extremely small. However, in this case, Gram-positive bacteria (ie, non-Escherichia coli) such as B. licheniforms and B. megaterium also proliferate vigorously, and since these microorganisms have β-galactosidase producing ability, they are erroneously identified as E. coli. It has the disadvantage of making a decision. As a result, the accuracy of the test medium for the coliform group decreases, and the reliability of the result decreases.

[0007]

DISCLOSURE OF THE INVENTION The present invention is not only capable of 1) testing of coliform bacteria which is simple and highly reliable in test results, and 2) simultaneous testing of coliform bacteria and the number of common bacteria is possible, 3) An object of the present invention is to provide a medium capable of rapidly distinguishing coliform bacteria.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that a commercially available standard agar medium, ordinary agar, which is usually used for inspection (detection measurement) of the number of general bacteria. In a method for preparing a general bacterial test medium such as a medium, a β-galactosidase chromogenic substrate (for example, 5-bromo-4-chloro-3-indolyl-
β-D-galactopyranoside, its enzyme-derived substrates (eg, L-lactoses, isopropyl-β-D-thiogalactoside, etc.), alanine aminopeptidase chromogenic substrates (eg, L-alanine-p- Nitroanilide)
And its enzyme-derived substrate (for example, bovine serum albumin, reduced glutathione, etc.), which is used to specifically discolor the Escherichia coli group having β-galactosidase activity and having alanine aminopeptidase for detection. At the same time, they discovered that general bacteria including these coliforms could be detected at the same time. In addition, desoxyco-
By discontinuing the use of conventionally used bacterial selection agents (particularly, gram-positive bacteria inhibitors) such as sodium lactate, bovine liver powder, brilliant green, etc., the coliform group becomes better in the culture process. It showed growth, and it was discovered that the coliform group could be detected with high sensitivity and high precision, and that it could be quickly identified. Furthermore, the present inventors have also discovered that since the composition of the medium is similar to the composition of a general general bacterial test medium, the test for coliform bacteria and the measurement of general bacteria can be simultaneously performed on a single agar medium.

The present invention has been completed on the basis of these findings, that is, the present invention relates to a general bacterial test medium, in which a β-galactosidase chromogenic substrate, alanine aminopeptidase is contained. -A bacterial test medium characterized by comprising a zeogenic substrate and an enzyme-derived substrate thereof. The present invention also relates to a method for testing coliform bacteria, which comprises inoculating and culturing a sample into the bacterial test medium obtained above and testing a colony colored yellow-green. The present invention also provides an Escherichia coli characterized in that a specimen is inoculated and cultured in the bacterial test medium obtained above, and the total number of colonies formed on the medium and the number of colonies colored yellow-green are examined. This is a simultaneous test method for group and general bacteria. The present invention also relates to a method for identifying coliform bacteria, which comprises inoculating and culturing a sample into the bacterial test medium obtained above and testing a colony colored yellow-green.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION As a medium used in the present invention, an ordinary general bacteria test medium (the growth of the target general bacteria is active, but at the same time, the growth and growth of microorganisms other than bacteria such as yeasts and molds). A culture medium is prepared according to the method for preparing a medium to be inhibited). At this time, the following chromogenic substrate and enzyme-derived substrate are added without adding a bacterial selection agent (for example, an antibiotic) or a pigment that adversely affects the growth of all bacteria including the coliform group. . As an example of such a medium, almost a colorless and transparent medium obtained by adding the following chromogenic substrate and enzyme-inducing substrate to an agar medium containing peptone, yeast extract and inorganic salts as appropriate and adjusted to pH 5.7 to 7.0 is provided. Bacterial test media.

The chromogenic substrate used in the present invention includes β-galactosidase chromogenic substrate and alanine aminopeptidase.
Ze color-developing substrates.

The β-galactosidase chromogenic substrate is 5
-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside or a salt thereof (eg, hydrochloride). The chromogenic substrate is preferably 0.01 to 0.1% (hereinafter,% means weight% unless otherwise specified) in the medium, and more preferably 0.02 to 0.03%. Since this substrate has little solubility in water, it is a fine white turbid state in water at the above concentration, which is barely noticeable to the naked eye, but becomes dark blue when hydrolyzed by the β-galactosidase reaction. Discolors to. Therefore, when bacteria having β-galactosidase enzyme grow on the medium, dark blue colonies are observed.

The alanine aminopeptidase chromogenic substrate includes L-alanyl-p-nitroanilide or a salt thereof (for example, hydrochloride). The chromogenic substrate is preferably contained in a medium in an amount of preferably 0.01 to 0.1%, and more preferably 0.07 to 0.
09% is more preferable. This substrate is colorless in aqueous solution, but turns yellow when hydrolyzed by the alanine aminopeptidase reaction. Therefore, when gram-negative bacteria having an enzyme of alanine aminopeptidase grow on the medium, yellow colonies are observed.

The present invention is characterized in that a β-galactosidase chromogenic substrate and an alanine aminopeptidase chromogenic substrate are simultaneously contained in a medium. When the substrate is contained, the object of the present invention cannot be achieved. That is, β
-Galactosidase has an ability to produce not only Escherichia coli but also other bacteria, so that accurate examination of Escherichia coli is not possible. In addition, since alanine aminopeptidase has the ability to produce not only coliforms but also some gram-positive bacteria and gram-negative bacteria other than Escherichia coli, accurate testing of the coliforms cannot be performed. On the other hand, the property of expressing two kinds of enzymes, β-galactosidase and alanine aminopeptidase, on the agar medium of the present invention is specific to the Escherichia coli group. Upon growth, a distinctive yellow-green or yellowish-blue colony is formed due to a dark blue discoloration and a yellow discoloration due to two types of enzymatic reactions.

One of the two enzyme-color-forming substrates of the present invention is L-alanyl-p-nitroanilide or a salt thereof, which changes its color to a deep blue color by a hydrolysis reaction with β-galactosidase. Is L-alanyl-p-nitroanilide or a salt thereof, which turns yellow by hydrolysis with alanine aminopeptidase. In the Ostwald color system of the Ostwald color system, dark blue (Blue) and yellow (Yellow) are in a relationship between opposing colors, and dark blue and yellow are very distinct from each other. Not only is it easy to color, but when two enzymatic reactions take place at the same time and the deep blue and the yellow fuse, they differ from these colors and are very easy to distinguish (discriminate) and beautiful yellow-green (or yellowish blue) ). Therefore, the present invention has a feature that inspection of coliform bacteria is very easy with the naked eye (visually).

The enzyme-derived substrate used in the present invention includes β
-Galactosidase derived substrates and alanine aminopeptidase derived substrates.

The β-galactosidase-derived substrate includes
Lactose and isopropyl-β-D-thiogalactoside. In the case of lactose, the enzyme-derived substrate is preferably 0.5 to 2.0% in the case of lactose.
8-1.0 is more preferable. Also, isopropyl-β-D
In the case of -thiogalactoside, 0.01 to 0.1% is preferable, and 0.03 to 0.07% is more preferable.

The alanine aminopeptidase-derived substrate includes bovine serum albumin, reduced glutathione, and gelatin peptone. In each case, the enzyme-derived substrate is preferably contained in the medium in an amount of 0.5 to 2.0%, more preferably 0.8 to 1.2%.

In the present invention, it is also very important to add an enzyme-inducing substrate to the medium, and in the absence of an inducing substrate, β-galactosidase and alanine aminopeptidase are produced in the medium. Since it is weak (slow), it has a disadvantage that the enzyme color reaction is slow and the test requires a long time. However, when the enzyme-derived substrate is added to the medium, β-galactosidase and alanine aminopeptidase are induced and generated in the medium in a short time, and as a result, the enzyme color reaction is also rapidly performed, and Differentiation between groups and other general bacteria can be performed in a very short time (for example, 90 minutes).

In the present invention, it is preferable to obtain a colorless and transparent or almost colorless and transparent medium. A large amount of insoluble components are contained in the medium, and it becomes difficult to detect bacterial colonies (colonies) in an opaque to translucent medium. When the medium is not colorless, it becomes difficult to distinguish the colored colonies from the uncolored colonies.

[0021]

The present invention will be described more specifically with reference to the following examples.

Example 1 (Preparation of bacterial test medium)

[Medium composition, a] Polypeptone 0.05% Casein peptone 0.05% Yeast extract 0.05% Sodium chloride 0.5% Magnesium sulfate 0.05% Calcium chloride 0.01% Isopropyl-β-D- Thiogalactoside (β-galactosidase-derived substrate) 0.05% L-alanyl-p-nitroanilide hydrochloride (alanine aminopeptidase coloring substrate) 0.08% Bovine serum albumin (alanine aminopeptidase-induced) Substrate) 1.0%

[Medium composition, b] 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside hydrochloride) (β-galactosidase chromogenic substrate) 0.02% Agar 1.6%

(Preparation of bacterial test medium)
Solution A is prepared by dissolving each of the components in "a" in distilled water so as to have a concentration twice as high as that described above. Next, the “medium composition, b” is suspended in a 0.05 M potassium phosphate buffer solution previously adjusted to pH 7.2 to a concentration twice as high as that described above to prepare a suspension B. Then, the solution A is sterilized by filtration using a sterilizing membrane filter having pores having a diameter of 0.2 to 0.5 μm and the like, and the suspension B is autoclaved (autoclave). ) At 121 ° C, 5
Sterilize for ~ 15 minutes. While heating the solution A and the suspension B after the sterilization and sterilization in a warm bath kept at 45 to 50 ° C. (while keeping the agar of the suspension B from hardening),
The bacterial test medium of the present invention, which was mixed aseptically and was almost colorless and transparent, was prepared. This medium may be aseptically dispensed into test tubes and cooled and solidified, or may be poured into a dish and cooled and solidified before use.

Example 2 (Method for testing the number of general bacteria and coliform bacteria) A sample (sample solution containing bacteria) was taken in a dish, and heated at 45 to 50 ° C. after heat dissolution prepared in Example 1 above. A suitable amount of the liquefied bacterial test medium, which has been kept warm, is poured in, and while shaking the dish, the sample and the medium are mixed so as to be substantially uniform, and quickly cooled and solidified, and the dish is cooled in a thermostat at 30 ° C. After culturing for one day, all colonies formed on the dishes were counted and used as the general bacterial count. Also those colonies
Among them, colonies having a yellow-green (or yellowish blue) color were measured and used as the number of coliforms.

Example 3 (Testing Method for the Number of General Bacteria and Escherichia coli Group) An appropriate amount of the liquefied bacterium test medium prepared in Example 1 was poured in advance in a dish and solidified to obtain the plate bacterium test medium of the present invention. Prepared. Using a spatula on this medium, a specimen (sample containing solution)
Smear evenly. This dish was cultured in a thermostat at 30 ° C. for 1 day, and the number of all colonies formed on the dish was counted, which was regarded as the number of general bacteria. Of those colonies, the yellow-green (or yellowish blue) colonies were measured and used as the number of coliforms.

Example 4 (Determination of Escherichia coli group) An appropriate amount of the liquefied bacterial test medium prepared in Example 1 was poured in advance into a dish and solidified to prepare a plate bacterial test medium of the present invention. On the other hand, a strain (bacteria) identified by a predetermined organization was inoculated and cultured on a common agar medium in advance to obtain colonies. A colony of an identified strain obtained by culturing the plate bacterial test medium of the present invention on a normal agar medium
The platinum wire to which was adhered was spotted (inoculated) and cultured in a thermostat at 37 ° C., and after 90 minutes, the colony spotted on the dish and changes in color tone around the colony were observed. Table 1 shows the results.

From the results shown in Table 1, it can be seen that the bacterial test medium of the present invention does not contain a bacterial selection agent, so that not only many gram-negative strains but also gram-positive strains grow.

Further, it can be seen that there are many gram-positive bacteria as well as Escherichia coli and Escherichia coli group which form blue spots. This is because the enzyme chromogenic substrate 5 is produced by a β-galactosidase reaction produced by a bacterium in a medium.
-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside hydrochloride is hydrolyzed and changes its color to dark blue. Therefore, β
-Even if a chromogenic substrate of galactosidase is added, E. coli,
Not only the coliforms but also many gram-positive bacteria are detected as positive, indicating that the coliforms cannot be tested and differentiated.

The yellow spots are formed not only by Escherichia coli and Escherichia coli but also to a small extent by gram-positive bacteria (Micrococcus flavus, Micrococcus luteu).
s), indicating that there are many types of Gram-negative bacteria. The yellow spot is based on the fact that the enzyme chromogenic substrate L-alanyl-p-nitroanilide hydrochloride is hydrolyzed by the alanine aminopeptidase reaction in the medium and changes its color to yellow. Therefore, when a chromogenic substrate of alanine aminopeptidase is added to the medium, not only Escherichia coli and Escherichia coli but also gram-positive bacteria (Mi
Crococcus flavus, Micrococcus luteus) and various types of Gram-negative bacteria were detected as positive, indicating that the coliforms could not be tested and differentiated.

Further, it can be seen that Escherichia coli and Escherichia coli group are present as those which form yellow-green spots. This is because, on the medium, the chromogenic substrate of β-galactosidase and the chromogenic substrate of alanine aminopeptidase are each hydrolyzed, and the resulting dark blue and yellow are fused to form a yellow-green spot. It is based on that. Therefore, it can be seen that by adding the two enzyme coloring substrates to the medium, only Escherichia coli and Escherichia coli group can be distinguished from other bacteria. From these results, it can be seen that the present invention can quickly test and differentiate Escherichia coli from any group of bacteria. That is, a part of the colony of the previously identified strain was scraped off with a platinum loop, attached to a platinum wire, and spotted on the bacterial test medium of the present invention, and cultured in an incubator. Proliferates almost without a lag period and begins to react with the chromogenic substrate. Then, in a very short time of 90 minutes, it can be seen that it is possible to determine whether the inoculated bacteria are coliforms or not by the discoloration reaction of the medium.
That is, it can be seen that rapid identification of coliform bacteria is possible.

The inventor of the present invention used a conventionally known medium for testing coliform bacteria and a desoxycholate agar medium to isolate a strain (bacteria) that had been identified by a predetermined organization in the same manner as in Example 4.
Inoculation culture was performed. As a result, due to the presence of a bacterial selection agent (desoxycholate), the culture medium may be a bacterium other than the coliforms, for example, Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomona
s convexa, Pseudomonas fluorescens, Pseudomonas ov
fluorescen fluorescen
t pseudomonads) was inhibited from growing, but the bacterial test medium of the present invention was found to proliferate vigorously and form a yellow colony without being inhibited from growing. That is, it can be seen that these fluorescent Pseudomonas genus bacteria groups are recognized as Gram-negative bacteria in a daylight room on the bacterial test medium of the present invention. However, these fluorescent Pseudomonas spp. Cultivated on the bacterial test medium of the present invention produce a fluorescent dye (fluorescin).
It was found that when these colonies were irradiated with ultraviolet light in the dark, they emitted fluorescence. Therefore, it can be seen that the bacterial test medium of the present invention can be used for testing and differentiating a group of fluorescent Pseudomonas bacteria that simultaneously produce a fluorescent dye, in addition to the above characteristics. From this fact, the bacterial test medium of the present invention can not only be capable of testing coliform bacteria with high reliability of test results, simultaneous testing of the total bacterial counts of coliform bacteria and general bacteria, rapid differentiation of coliform bacteria, but also the same. It turns out that the fluorescent Pseudomonas genus bacterial group can also be tested on the medium.

[0034]

[Table 1]

Example 5 (Inspection of Number of General Bacteria by Pour Method) After appropriately diluting domestic wastewater (specimen) discharged from a general household, a part thereof is taken in a petri dish, and the above-mentioned example is taken from there. Pour an appropriate amount of the bacterial test medium (liquefied matter) prepared in Step 1, mix the specimen and the medium so as to be almost uniform while shaking the dish, and quickly cool and solidify the dish. After culturing at 30 ° C. for 18 hours in the inside, all colonies formed on the dish were counted and determined as the number of general bacteria. Also, of those colonies,
The yellowish-green (or yellowish blue) colonies were counted and used as the number of coliforms. Table 2 shows the results.

Comparative Example 1 For the purpose of comparison, in the above "Inspection of the number of common bacteria by the pour method", "Bacteria test medium (liquefied matter) prepared in Example 1"
In place of the above, a colony was measured in exactly the same manner except that a “normal agar medium (liquefied product)” having the following composition was used, and this was used as the general bacterial count. Table 2 shows the results.

[Common Agar Medium Composition] Meat extract 0.5% Peptone 1.0% Sodium chloride 0.5% Agar 1.5% pH 7.0

COMPARATIVE EXAMPLE 2 For comparison, in the above "Inspection of Number of General Bacteria by Pour Method", "Desoxycholate Agar Medium (Liquid) prepared in Example 1 was replaced with" Desoxycholate Agar Medium " (Manufactured by Eiken Chemical Co., Ltd.) (liquefied product) "
The colony was measured in exactly the same manner as in the above, except that “24 hours at 37 ° C.” was used instead of “18 hours”. Table 2 shows the results.

[0039]

[Table 2]

Example 6 (Inspection of the number of common bacteria in a sample by the pour method) A certain amount (sample) of domestic wastewater discharged from a general household is appropriately diluted, and the diluted sample is taken in a petri dish. A suitable amount of the culture medium (liquefied matter) for bacterial test prepared in Example 1 was poured, and while shaking the dish, the specimen and the medium were mixed so as to be substantially uniform, and quickly cooled and solidified. Was cultured in a thermostat at a culture condition of 30 ° C. for 18 hours, and the number of all colonies formed on the dish was counted.
This was defined as the general bacterial count. The results are shown in FIG. 1 (evaluation of the analysis accuracy of the number of general bacteria in the bacterial test medium of the present invention by comparison with a known test method). Of those colonies, the yellow-green (or yellowish blue) colonies were measured and used as the number of coliforms. The results are shown in FIG. 2 (Evaluation of the analysis accuracy of the number of coliform bacteria in the bacterial test medium of the present invention by comparison with a known test method).

Comparative Example 3 (Inspection of the Number of General Bacteria in a Sample by the Pour Method) For comparison, the “Example of the number of general bacteria in the sample by the pour method” in Example 6 was used for comparison. The colony was measured in exactly the same manner as in Example 1 except that the “normal agar medium (liquefied substance)” prepared in Comparative Example 1 was used instead of the “bacterial test medium (liquefied substance) prepared in Step 1”. The bacterial count was used.
The results are shown in FIG. 1 (same as above).

From the results shown in FIG. 1, when the correlation between the bacterial test medium of the present invention and the conventional ordinary agar medium was investigated, almost the same results were obtained, indicating that the bacterial test medium can be effectively used as a bacterial test medium by the pour method. I understand.

COMPARATIVE EXAMPLE 4 For comparison, in the above “Examination of the number of general bacteria in a sample by the pour method” in Example 6, instead of using the bacterial test medium (liquefied matter) prepared in Example 1, , Except that "Desoxycholate agar medium (manufactured by Eiken Chemical Co., Ltd.) (liquefied material)" is used, and "24 hours at 37 ° C" is used instead of "18 hours at 37 ° C" during culture. Similarly, the colonies were measured and used as the number of coliforms. The results are shown in FIG.

From the results shown in FIG. 2, when the correlation between the bacterial test medium of the present invention and the conventional coliform group test medium and desoxycholate agar medium was examined, almost identical results were obtained. It turns out that it can be used effectively as a culture medium.

In the desoxycollate agar medium,
Due to the presence of the bacterial selection agent, gram-positive bacteria other than the coliform group are inhibited from growing and thus cannot be used as a bacterial test medium. However, the bacterial test medium of the present invention is, as apparent from the results of FIGS. Bacteria other than the coliforms also vigorously grow and proliferate to form colonies, indicating that they can be suitably used as a bacterial test medium.

[0046]

EFFECTS OF THE INVENTION Escherichia coli groups are normally present in the intestinal tract of humans and animals, and when they are present in water or foods, it is contaminated with water and foods and excrement of humans and animals. May have been contaminated by digestive system pathogens. For this reason, instead of detecting pathogenic bacteria from water, food, and the like, it has been widely practiced to test coliforms that are easily detected as faecal contamination indicator bacteria. The bacterial test medium of the present invention is useful for testing coliforms in water and foods and has the following features. That is, since the medium of the present invention does not contain a bacterium selection agent, not only a large number of gram-negative strains but also gram-positive strains proliferate, and thus can be used as a general bacterial test medium. In addition, by extremely simple means, it is possible to perform a test of coliform bacteria with a high reliability of test results, a simultaneous test of the total bacterial count of coliform bacteria and general bacteria, and a rapid differentiation of coliform bacteria, and further, on the same medium, Fluorescent shoe
It can also test for Domonas bacteria. In addition, by using an additional coliform group confirmation test (confirmation test) such as an oxidase test or a catalase test on the medium, a more accurate coliform group or bacteria identification test can be performed.

[Brief description of the drawings]

FIG. 1 shows the results of a test for general bacteria in a sample using the bacterial test medium of the present invention (the method of the present invention) and the conventional ordinary agar medium (the conventional method), respectively, and the correlation between the present method and the conventional method. FIG.

FIG. 2 shows the results of testing the coliform bacteria of a sample using the bacteria test medium of the present invention (the method of the present invention) and the conventional test medium for coliform bacteria (the conventional method), respectively, and the correlation between the present method and the conventional method. FIG.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) C12Q 1/37 C12Q 1/37 (C12Q 1/06 (C12Q 1/06 C12R 1:19) C12R 1:19)

Claims (11)

[Claims] 1. A general bacterial test medium, comprising β medium
-A bacterial test medium comprising a galactosidase chromogenic substrate, an alanine aminopeptidase chromogenic substrate and their enzyme-derived substrates. 2. The bacteria test medium according to claim 1, wherein the β-galactosidase chromogenic substrate is 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside or a salt thereof. 3. An alanine aminopeptidase chromogenic substrate,
The culture medium for bacterial tests according to claim 1, which is L-alanyl-p-nitroanilide or a salt thereof. 4. The bacterial test medium according to claim 1, wherein the β-galactosidase-derived substrate is lactose or isopropyl-β-D-thiogalactoside. 5. The alanine aminopeptidase-derived substrate,
The bacterial test medium according to any one of claims 1 to 4, which is bovine serum albumin, gelatin peptone, or reduced glutathione. 6. The bacterial test medium according to claim 1, wherein the bacterial test medium is a test medium for coliform bacteria. 7. The bacterial test medium according to claim 1, wherein the bacterial test medium is a simultaneous test medium for coliform bacteria and general bacteria. 8. The bacterial test medium according to any one of claims 1 to 7, wherein the bacterial test medium is a differentiation medium for coliform bacteria. 9. A method for testing coliform bacteria, which comprises inoculating and culturing a sample on the culture medium for bacterial test according to claim 1, and testing colonies colored yellow-green. 10. A group of Escherichia coli, wherein a specimen is inoculated and cultured on the culture medium for bacterial test according to claim 1, and the total number of colonies formed on the culture medium and the number of colonies colored yellow-green are inspected. And simultaneous testing of common bacteria. 11. A method for identifying coliform bacteria, comprising inoculating and culturing a sample on the bacterial test medium according to claim 1 and testing a colony colored yellow-green.