JP2004057054A - Method for simply detecting salmonella - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simply dry medium prepared by carrying at least a water-soluble gelling agent, nutritive ingredients for the salmonella, a pH indicator, and a lysine decarboxylase substrate on a fibrous water-absorbing sheet and used for detecting the salmonella. <P>SOLUTION: A method for detecting the salmonella, using a simply dry culture medium prepared by carrying at least the water-soluble gelling agent, the nutritive ingredients for the salmonella, the pH indicator, and the lysine decarboxylase substrate on the fibrous water-absorbing sheet is characterized by inoculating a test sample in the culture medium in an amount penetrating only a part of the culture medium, inoculating water or an aqueous solution in the culture medium in an amount penetrating all parts of the culture medium, and then culturing the penetrated culture medium. Thereby, the salmonella can simply and accurately be detected from the test sample containing many various bacteria, such as a food, in a short time. <P>COPYRIGHT: (C)2004,JPO

Description

【００３１】
表１のように、接種方法（Ａ）〜（Ｅ）のいずれにおいても培地の黄変が認められ、サルモネラの存在は確認できた。しかしながら、（Ｄ）法では滅菌水注加後菌が流れやすい傾向が認められた。
【００３２】
【表２】

【００３３】
表２より、サルモネラが比較的他の夾雑菌を排除した、純培養に近い状態で分離されたのは、試料接種後と滅菌水を注加した（Ａ）〜（Ｃ）の方法、特に（Ａ）と（Ｂ）の方法が良好であった。サルモネラが運動性により移動した接種部から離れたところを釣菌した場合、分離性が極めて良好であった。ただし、試料の接種量が多い場合（１ｍＬ）は、試料の希釈操作を行なわなければ分離不可能であった。
【００３４】
【発明の効果】
本発明によれば、食品等の多くの夾雑菌が共存する検体から、サルモネラを簡便かつ短時間で正確に検出できる。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for detecting Salmonella in various samples such as foods in a short time and easily without requiring special skills and techniques, and a medium used in the method.
[0002]
[Prior art]
A conventional method for detecting Salmonella is that a specimen is cultured at 35 ° C. for 20 to 24 hours in a pre-enrichment culture medium (EEM broth or buffered peptone water BPW), and a part of the pre-enrichment culture is selected. The medium is inoculated into an enrichment medium (Hana tetrathionate medium, Rapaport basiliadis medium, etc.) and cultured at 42 ° C. for 20 to 24 hours. Then, a part of the selective enrichment culture was spread on a plate medium such as a MLCB agar medium, a DHL agar medium, an XLD agar medium, a Rambach agar medium, or an SS agar medium, which is a Salmonella selective separation medium. After culturing for a time, colonies suspected of Salmonella are confirmed on a TSI agar medium, a LIM agar medium or the like. Furthermore, it is a complicated, time-consuming and time-consuming method, such as performing serotyping and DNA probe analysis in immunological tests as necessary. Therefore, the presence of Salmonella in the sample will be screened on day 3 or later after pre-enrichment culture, selective enrichment culture, and smearing with a selective separation medium. Therefore, as an attempt to detect Salmonella quickly or simply, (1) a method in which the pre-enrichment culture solution is brought into contact with a through-hole of a container filled with a semi-fluid selective medium and the motility of the target bacterium is used to detect the Salmonella (Japanese Patent Application Laid-Open No. 2001-136998), (2) A method of inoculating a loopful (or 0.1 mL) of a loop in a semi-fluid selective medium and detecting the target bacteria by motility (Herman Goossens et al., 1984. J. Clin. Microbiol. 19 (6), DIASALM), (3) Sheet-shaped medium for detecting Salmonella (JP-A-2001-321196) and the like have been reported. However, in (1), the target bacterium can be detected by a simple operation just by putting the container in the pre-enrichment culture solution, but there is a problem in storage stability due to the live culture medium. It is difficult to separate the target bacterium because it must be germ-free. (2) is a semi-fluid raw medium, which has a problem in storage stability, and requires careful handling since it is easy to flow after inoculating a sample. The method (3) requires a diluting operation of the culture solution, and is used in place of the selective separation medium, and thus has a problem in rapidity.
[0003]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a method for detecting Salmonella more simply and more quickly.
[0004]
[Means for Solving the Problems]
Accordingly, the present inventors have conducted various studies on the detection of Salmonella using a simple dry medium, and as a result, first, using a dry medium carrying a pH indicator and a lysine decarboxylase substrate in addition to a water-soluble gelling agent and a cell nutrient, If a small amount of the test sample is inoculated to support only a part of the medium, then water is inoculated so as to penetrate the entire medium, and then cultured, if Salmonella is motile, it will be supported on a part of the medium because of its motility. Salmonella moves in the culture medium, and in particular in parts other than the inoculated part of the test sample, an enzymatic reaction and a coloring reaction occur due to the action of lysine decarboxylase secreted by Salmonella, and simple and quick Salmonella by visual observation. Was found to be able to be detected, and the present invention was completed.
[0005]
That is, the present invention relates to a method for detecting Salmonella using a simple dry medium comprising a fibrous water-absorbent sheet carrying at least a water-soluble gelling agent, a cell nutrient, a pH indicator, and a lysine decarboxylase substrate. It is intended to provide a method for detecting Salmonella, comprising inoculating a test sample solution in an amount permeating only a part of the medium, inoculating water or an aqueous solution in an amount permeating the entire medium, and then culturing the salmonella.
[0006]
The present invention also provides a simple dry culture medium for detecting Salmonella, comprising a fibrous water-absorbent sheet carrying at least a water-soluble gelling agent, a cell nutrient, a pH indicator and a lysine decarboxylase substrate.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
Salmonella to be detected in the present invention mainly includes motility and lysine decarboxylation-causing bacteria causing food poisoning, such as Salmonella Enteritidis, Salmonella Typhimurium and the like.
[0008]
The simple dry medium used in the present invention is a dry medium in which a fibrous absorbent sheet carries at least a water-soluble gelling agent, a cell nutrient, a pH indicator and a lysine decarboxylase substrate. It is preferable that the culture medium further supports a water-soluble adhesive.
[0009]
Specific examples of these simple media include, for example, the following simple media (1) to (6).
(1) A culture medium in which an adhesive layer, a cold water-soluble gelling agent powder layer containing a nutrient component, and a cover sheet are sequentially laminated on the upper surface of a waterproof substrate (Japanese Patent Laid-Open No. 57-502200).
(2) A culture medium in which an air-permeable membrane, a cold water-soluble gelling agent powder layer containing nutrients, and a cover sheet are sequentially laminated on the upper surface of a waterproof substrate (Japanese Patent Laid-Open No. 15379/1991).
(3) Detection paper obtained by impregnating a filter paper or the like with a bacterial nutrient and covering the surface with a cover sheet (Japanese Patent Application Laid-Open No. 2-65798).
(4) A medium in which a cold water-soluble gelling agent, a microorganism culture medium, and a fibrous water-absorbing sheet are sequentially laminated on the surface of a waterproof flat plate (Japanese Patent Laid-Open No. 6-181741).
(5) a medium composition containing (a) an adhesive soluble in water and alcohol, (b) a gelling agent soluble in water and insoluble in alcohol, and (c) a medium composition containing cell nutrients. A simple medium supported on a fibrous water-absorbent sheet having a mesh larger than the agent (JP-A-9-19282).
(6) (a) 0.01 to 0.4% by weight of an adhesive soluble in water and alcohol, (b) a gelling agent soluble in water and insoluble in alcohol, and (c) a nutrient component of bacterial cells. The contained alcohol suspension is impregnated into a fibrous absorbent sheet having a mesh larger than the particle size of the gelling agent placed on a waterproof flat plate, and dried while suppressing rapid evaporation of alcohol. Then, a simple medium in which a water-absorbent sheet is fixed to a waterproof flat plate (Japanese Patent Laid-Open No. 2000-325072).
Of these simple dry media, (5) and (6) are preferred, and (6) is particularly preferred.
[0010]
The gelling agent may be any water-soluble one, but is particularly preferably water-soluble and insoluble in alcohol, for example, natural gelling agents such as xanthan gum, locust bean gum, guar gum and carrageenan, and hydroxyethyl cellulose. And the like, and among them, xanthan gum is particularly preferable. As such a gelling agent, it is preferable to use a powder having an average particle size of 500 μm or less, particularly 0.5 to 50 μm.
[0011]
The adhesive used as needed in the medium composition is preferably soluble in water, particularly preferably soluble in water and alcohol, such as hydroxypropylcellulose, polyvinylpyrrolidone, and polyethylene oxide. Among them, hydroxypropyl cellulose is particularly preferred.
[0012]
Further, as the cell nutrients, those suitable for the growth of Salmonella are selected. For example, a nutrient component such as meat extract, peptone, and yeast extract, a saccharide, and a medium component containing a selective agent or an antibiotic for suppressing bacteria other than Salmonella are used. As saccharides, lactose and saccharose, which are particularly assimilated by the coliform bacteria likely to be mixed in the test sample, are preferably used in order to improve the selectivity to Salmonella. As the selective agent, malachite green, alkyl sulfates such as magnesium chloride and sodium lauryl sulfate, bile salts and the like are preferable. Further, addition of novobiocin, which is effective against Proteus bacteria, is effective as an antibiotic.
[0013]
The pH indicator may be any indicator capable of detecting a pH change (alkalization) caused by decarboxylation of lysine as a substrate by lysine decarboxylase secreted from Salmonella, such as bromcresol purple and bromthymol blue. Phenol red, neutral red, litmus and the like.
[0014]
Examples of the lysine decarboxylase substrate include lysine, lysine-containing oligopeptides and salts thereof. The lysine-containing oligopeptide has a carboxyl group that can be decarboxylated by lysine decarboxylase, and any substance produced by decarboxylation may be alkaline, and examples thereof include dipeptide and tripeptide having a lysine residue. . Examples of these salts include hydrochloride. Of these, lysine hydrochloride is particularly preferred.
[0015]
Further, the medium of the present invention can contain components for detection utilizing the property that Salmonella is a bacterium that produces hydrogen sulfide, for example, thiosulfate and ferric salt. Examples of the thiosulfate include sodium thiosulfate. Examples of the ferric salt include a ferric salt of a polybasic organic acid, such as iron ammonium citrate. When these components are contained, the hydrogen sulfide generated by Salmonella reacts with iron ammonium citrate to form black iron sulfide, and the medium becomes black.
[0016]
Further, in the medium of the present invention, in order to facilitate color observation, a suitable coloring agent other than the pH indicator, for example, triphenyltetrazolium chloride, 3- (p-iodophenyl) -2- (p-nitro Dyes such as phenyl) -5-phenyl-2H-tetrazolium chloride and 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide; 5-bromo-3-indolyl-β- D-galactopyranoside, 5-bromo-3-indolyl-α-D-galactopyranoside, 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside, 5-bromo-6 It may contain an enzyme substrate such as -chloro-3-indolyl-α-D-galactopyranoside.
[0017]
The amount of the gelling agent in the medium of the present invention is preferably an amount that causes gelation to such an extent as not to inhibit the motility of Salmonella, and is preferably smaller than that of a normal simple medium. Specifically, it is preferable to adjust the amount of the test sample solution and the medium after inoculation with water or an aqueous solution to a state in which the medium becomes a semi-fluid medium.
[0018]
Alcohol is preferable as the medium for suspending the medium composition during the preparation of the medium, and for example, ethanol, 2-propanol and the like are particularly preferable.
[0019]
The fibrous water-absorbing sheet supporting the medium composition is such that the inoculated test liquid easily diffuses by capillary action, and the gelling agent in the medium composition can be supported in the network structure. Is preferred. The fibrous absorbent sheet has a mesh larger than the particle size of the gelling agent, and preferably has a thickness larger than the particle size. For example, the mesh size is 15 to 100 mesh, particularly 20 to 50 mesh. The thickness is preferably 10 to 1000 μm, particularly preferably 50 to 600 μm.
[0020]
Examples of such a fibrous water-absorbent sheet include a synthetic fiber nonwoven fabric represented by a rayon nonwoven fabric and a natural fiber nonwoven fabric represented by a cotton nonwoven fabric. The shape of these sheets is not particularly limited, and may be any of a square, a rectangle, a circle, and the like. Although the size is not particularly limited, those having a major axis of 1 to 15 cm are preferable.
[0021]
The waterproof flat plate to which the fibrous water-absorbing sheet is fixed may be any one of a waterproof material such as plastic and glass, but a transparent one is preferable for external observation.
[0022]
In the Salmonella detection method of the present invention, first, a test sample solution is inoculated in an amount that permeates only a part of the medium. If the test sample solution is inoculated in an amount that penetrates the entire medium, selectivity due to the motility of Salmonella cannot be obtained, and accurate detection cannot be performed. The term "part of the culture medium" means that a part to which the test sample liquid does not permeate remains, but it is preferable that at least 1/2 of the surface area of the culture medium remains without permeation for Salmonella detection. Specifically, the inoculation amount of the test sample solution is sufficient to be 1/100 to 1/2 of the amount that permeates the whole medium. In addition, the inoculation site is preferably a site closer to the end than the center of the culture medium in order to detect Salmonella motility well.
[0023]
The test sample liquid includes an aqueous suspension of food or a pre-culture liquid. In the present invention, a pre-culture solution (pre-enrichment culture solution) may be used as necessary. Here, as the preculture, a method in which a test sample such as a food sample is mixed with a pre-enrichment medium (EEM broth, buffered peptone water BPW, etc.) in an amount that is 10 times and cultured at 35 ° C for 20 to 24 hours. No.
[0024]
Next, water or an aqueous solution is inoculated so as to penetrate the entire medium. Here, purified water is preferable as the water, and an aqueous solution containing a component that does not affect color development due to a pH change is preferable as the aqueous solution. The amount that permeates the entire medium can be determined by the size, porosity, etc. of the medium. The inoculation site may be the same site as the test sample solution inoculation site, but a site different from the test sample solution inoculation site is preferable in terms of Salmonella separability. For example, when the test sample solution is inoculated below the center, it is preferable to inoculate water or an aqueous solution on the opposite side.
[0025]
Then, after culturing and observing the color of the medium, Salmonella can be accurately detected. The culture is preferably carried out at 41 to 43 ° C. for 20 to 24 hours, or aerobically by further inverting.
[0026]
When the test sample solution is inoculated into a part of the simple medium, the microorganisms in the test sample are carried between the meshes. Thereafter, when a large amount of water is inoculated, the entire medium swells and gels. Among the supported microorganisms, motile Salmonella moves in the gelled medium and moves to a site different from the test sample inoculation site. At the site, lysine decarboxylase secreted by Salmonella decarboxylates the substrate, and the liquidity at the site changes to the alkaline side, so that the pH indicator causes a color change. Thus, Salmonella can be detected simply and in a short time.
[0027]
As described above, since only Salmonella proliferates at the site distant from the test sample inoculation site, only Salmonella can be picked up. Can be subjected to chemical differentiation tests.
[0028]
【Example】
Next, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.
[0029]
Example 1
(1) Preparation of simple medium 21 g of peptone, 1 g of yeast extract, 10 g of lactose, 10 g of sucrose, 0.03 g of pH indicator (neutral red), 0.036 g of malachite green, 13.58 g of magnesium chloride, 1 g of sodium citrate, iron citrate 1 g of ammonium, 2 g of sodium thiosulfate, 0.3 g of chromogenic enzyme substrate (5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside), 5 g of L-lysine hydrochloride, 0.02 g of novobiocin, xanthan gum 15 g was added to 1000 mL of an ethanol solution containing 1 g of hydroxypropyl cellulose to form a suspension. The ethanol suspension was dispensed 1 mL at a time into a container (50 mm) containing a fibrous water-absorbent sheet (50 mm), dried, and then capped to prepare a simple medium. After this medium was hermetically sealed in an aluminum packaging material together with a desiccant, it was sterilized by γ-irradiation at a surface dose of 10 to 20 kGy.
(2) Test sample chicken minced meat (3) Inoculation into simple medium 25 g of chicken minced meat was pre-enriched with 225 mL of EEM broth at 35 ° C for 24 hours, and then inoculated into the medium of (1). The methods were the following five types. The sample inoculation volume was 6 steps of 1 mL, 0.1 mL, 0.05 mL, 0.025 mL, 0.01 mL, and 0.005 mL, and 1 mL of sterilized water was injected.
(A) Inoculate the sample in the center → Inject sterilized purified water slowly avoiding the inoculation point.
(B) Inoculate the sample at the end → Pour sterilized purified water from the opposite side.
(C) Inoculate the sample at the center → Inject sterilized purified water from the same place.
(D) Inject 1 mL of sterile purified water → Inoculate the sample in the center.
(E) 1 mL inoculation of a 10-fold serial dilution from stock solution to 8-fold.
(4) Observation After inoculation, the cells were cultured at 42 ° C. for 18 hours, and the simple medium was observed.
(5) Confirmation One platinum loop was collected from the simple medium and streaked with MLCB (Nissui) and Rambach Agar (Merck).
(6) Results The results are shown in Tables 1 and 2.
[0030]
[Table 1]

[0031]
As shown in Table 1, in all of the inoculation methods (A) to (E), yellowing of the medium was observed, and the presence of Salmonella was confirmed. However, in the method (D), it was observed that the bacteria tended to flow after pouring sterilized water.
[0032]
[Table 2]

[0033]
From Table 2, it is found that Salmonella was isolated in a state close to a pure culture, in which other contaminants were relatively excluded. The methods A) and B) were good. When Salmonella was isolated from the inoculated area where it had moved due to motility, the separability was extremely good. However, when the inoculation amount of the sample was large (1 mL), separation was not possible without diluting the sample.
[0034]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, Salmonella can be simply and accurately detected in a short time from a sample in which many contaminating bacteria such as foods coexist.

Claims (9)

A method for detecting Salmonella using a simple dry medium comprising a fibrous water-absorbent sheet carrying at least a water-soluble gelling agent, a cell nutrient, a pH indicator, and a lysine decarboxylase substrate, wherein the test sample solution is used in the medium. A method for detecting Salmonella, comprising inoculating an amount inoculating only part of the medium, inoculating the medium with water or an aqueous solution in an amount permeating the entire medium, and then culturing. The method for detecting Salmonella according to claim 1, wherein the test sample solution is a test sample suspension or a test sample pre-culture solution. 3. The method for detecting Salmonella according to claim 1, wherein the lysine decarboxylase substrate is lysine, a lysine-containing oligopeptide or a salt thereof. The method for detecting Salmonella according to any one of claims 1 to 3, wherein the simple dry medium further supports lactose and sucrose. The method for detecting Salmonella according to any one of claims 1 to 4, wherein the simple drying medium further supports an adhesive soluble in water. A simple dry medium for detecting Salmonella comprising a fibrous absorbent sheet carrying at least a water-soluble gelling agent, a cell nutrient, a pH indicator and a lysine decarboxylase substrate. The simple dry medium according to claim 6, wherein the lysine decarboxylase substrate is lysine, a lysine-containing oligopeptide or a salt thereof. 8. The simple dry medium according to claim 6, which further supports lactose and sucrose. The simple dry culture medium according to any one of claims 6 to 8, which further supports an adhesive soluble in water.