JP4681386B2 - Simple medium and microorganism detection method - Google Patents
Simple medium and microorganism detection method Download PDFInfo
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Description
本発明は、カテキンを含有する被検液であっても被検試料中の微生物を簡便に精度よく検出する簡易培地及びこれを用いた微生物の検出方法に関する。 The present invention relates to a simple medium for simply and accurately detecting microorganisms in a test sample even in a test solution containing catechin, and a method for detecting microorganisms using the same.
近年、カテキンが体脂肪の増加抑制効果、血中コレステロール低下効果等の生理効果があることが明らかになり、カテキンを含有する緑茶飲料は注目を集め、広く飲用されている。かような緑茶飲料は、食品衛生法の清涼飲料水に属し、含まれる微生物数は1ｍｌ中100個以下でなければならないとされていることから、緑茶飲料に含まれる微生物の正確かつ簡便な検出方法が求められている。 In recent years, it has become clear that catechin has physiological effects such as a body fat increase inhibitory effect and a blood cholesterol lowering effect, and green tea beverages containing catechin have attracted attention and are widely drunk. Such green tea beverages belong to soft drinks under the Food Sanitation Law, and the number of microorganisms contained must be 100 or less in 1 ml. Therefore, accurate and simple detection of microorganisms contained in green tea beverages There is a need for a method.
微生物を検出、同定するにあたって、従来から行われている微生物の培養方法としては、平板塗抹法や混釈法、液体培地法などが一般的に用いられている。これらの方法は、培養を行う以前に培地や器具類の滅菌の準備が必要であり、被検試料を培地に接種した後は塗抹や混釈等の熟練を要する操作が必要である。更に、被検試料接種後の培地の輸送や培養後発育したコロニーを計測したあとの培地等の滅菌ではかさばる器具や培地により多大な労力を要する。 In order to detect and identify microorganisms, a plate smearing method, a pour method, a liquid medium method, and the like are generally used as conventional microorganism culturing methods. These methods require preparation for sterilization of the medium and instruments before culturing, and after inoculating the test sample into the medium, operations requiring skill such as smearing and pour are necessary. Furthermore, the transportation of the medium after inoculation of the test sample and the sterilization of the medium after measuring the colonies grown after the culture require a lot of labor due to the bulky instruments and medium.
そのため手軽に培養できるフィルム状簡易培地を用いた簡易検査法が報告されている。その例として、（１）防水性基体の上面部に、接着剤層、栄養成分を含む冷水可溶性ゲル化剤粉末層及びカバーシートを順次積層した培地（特許文献１）、（２）防水性基体の上面部に、空気透過性膜、栄養成分を含む冷水可溶性ゲル化剤粉末層及びカバーシートを順次積層した培地（特許文献２）、（３）濾紙等に菌体栄養成分を含浸させ、その表面をカバーシートで覆ってなる検出紙（特許文献３）が報告されており、さらにこれらの培地に比して被検液が容易に拡散し、かつ明確なコロニーが形成される培地として、各種の簡易培地が報告されている（特許文献４〜７）。 Therefore, a simple inspection method using a film-like simple medium that can be easily cultured has been reported. Examples thereof include (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 the waterproof substrate (Patent Document 1), (2) the waterproof substrate A medium (Patent Document 2) in which an air-permeable membrane, a cold water-soluble gelling agent powder layer containing a nutrient component, and a cover sheet are sequentially laminated on the upper surface portion of the liquid, (3) impregnating the cell nutrient component in a filter paper, etc. Detection paper (Patent Document 3) whose surface is covered with a cover sheet has been reported. Further, various types of culture media can be used as a culture medium in which a test solution can be easily diffused and distinct colonies can be formed as compared with these culture media. Simple media have been reported (Patent Documents 4 to 7).
しかしながら、本発明者らが見出したところによれば、このような簡易培地にカテキン含有緑茶飲料を接種し培養させる方法では、カテキンの抗菌作用により微生物の生育が阻害されるため、従来から行われている平板塗抹法、混釈法、液体培地法等ほどの検出精度が得られないという問題があった。
したがって、本発明の目的は、平板塗抹法、混釈法、液体培地法等と同等の高い精度で、カテキンを含有する被検液中の微生物を簡便、正確かつ迅速に検出することができる簡易培地及びそれを用いた微生物の検出方法を提供することにある。 Therefore, the object of the present invention is to be able to detect microorganisms in a test liquid containing catechin simply, accurately and rapidly with high accuracy equivalent to the plate smear method, pour method, liquid medium method and the like. An object of the present invention is to provide a medium and a method for detecting a microorganism using the same.
本発明者らは、上記課題を解決すべく鋭意検討を重ねた結果、被検体中にチオ硫酸塩を添加して測定した場合には十分な効果が得られないにもかかわらず、乾燥状態でチオ硫酸塩を含む簡易培地を用いれば、従来の平板塗抹法、混釈法、液体培地法等と同等の高い精度で、カテキンを含有する被検液中の微生物を検出することができることを見出し、本発明を完成させた。 As a result of intensive studies to solve the above-mentioned problems, the present inventors have obtained a dry state even though a sufficient effect cannot be obtained when measurement is performed by adding thiosulfate to a specimen. It has been found that if a simple medium containing thiosulfate is used, microorganisms in a test liquid containing catechin can be detected with high accuracy equivalent to that of the conventional plate smearing method, pour method, liquid medium method, etc. The present invention has been completed.
すなわち本発明は、カテキンを含有する被検液中の微生物を検出するための簡易培地であって、（ａ）水及びアルコールに可溶な接着剤、（ｂ）水に可溶でアルコールに不溶なゲル化剤、（ｃ）菌体栄養成分及び（ｄ）チオ硫酸塩を含有する培地組成物を乾燥状態で含有する繊維状吸水性シートが、防水性平板に積層されてなり、乾燥状態の培地組成物中に（ｄ）チオ硫酸塩が８〜４７重量％含まれるものであることを特徴とする簡易培地を提供するものである。
That is, the present invention is a simple medium for detecting microorganisms in a test liquid containing catechin, comprising: (a) an adhesive soluble in water and alcohol; and (b) soluble in water and insoluble in alcohol. gelling agent, (c) a fibrous absorbent sheet the cells nutrients and (d) medium composition containing thiosulfate containing in a dry state is, Ri Na are laminated waterproof flat plate, dry (d) thiosulfate medium composition of is to provide a simple culture medium, characterized in der Rukoto those contained 8-47 wt%.
また、本発明は、上記の簡易培地にカテキンを含有する被検液を接種して培養し、そこに形成されるコロニーを検出することを特徴とする微生物の検出方法を提供するものである。
The present invention also provides a microorganism detection method characterized by inoculating and culturing a test solution containing catechin in the above-mentioned simple medium and detecting colonies formed there.
さらに、本発明は、カテキンを含有する被検液中の微生物を検出するための簡易培地の製造方法であって、（ａ）水及びアルコールに可溶な接着剤、（ｂ）水に可溶でアルコールに不溶なゲル化剤、（ｃ）菌体栄養成分及び（ｄ）乾燥状態の培地組成物中に８〜４７重量％となる量のチオ硫酸塩を含有する培地組成物のアルコール懸濁液を調製し、防水性平板上に載置された該ゲル化剤の粒径より大きいメッシュを有する繊維状吸水性シートに含浸させ、これを乾燥して、繊維状吸水性シートを防水性平板に固着させることを特徴とする簡易培地の製造方法を提供するものである。 Furthermore, the present invention is a method for producing a simple medium for detecting microorganisms in a test liquid containing catechin, comprising: (a) an adhesive soluble in water and alcohol; and (b) soluble in water. An alcohol suspension of a medium composition comprising an alcohol-insoluble gelling agent, (c) a microbial nutrient, and (d) a thiosulfate in an amount of 8 to 47% by weight in the dried medium composition liquid was prepared by impregnating a fibrous water-absorbent sheet having a larger mesh than the grain size of the waterproof flat plate onto the placed the gelling agent, which was dried, waterproof flat fibrous absorbent sheet The present invention provides a method for producing a simple culture medium characterized by adhering to a medium.
本発明の簡易培地を用いれば、被検試料がカテキンを含有する被検液であっても、被検試料中の微生物を、簡便かつ正確に精度よく検出することができる。 If the simple culture medium of the present invention is used, even if the test sample is a test solution containing catechin, microorganisms in the test sample can be detected simply and accurately with high accuracy.
培地組成物に用いられる（ａ）水及びアルコールに可溶な接着剤としては、ヒドロキシプロピルセルロース、ポリビニルピロリドン、ポリエチレンオキサイド等が挙げられるが、ヒドロキシプロピルセルロースが特に好ましい。 Examples of (a) water and alcohol-soluble adhesives used in the medium composition include hydroxypropyl cellulose, polyvinyl pyrrolidone, polyethylene oxide and the like, and hydroxypropyl cellulose is particularly preferable.
培地組成物に用いられる（ｂ）水に可溶でアルコールに不溶なゲル化剤としては、例えばキサンタンガム、ローカストビンガム、グアーガム、カラギーナン、ペクチン等の天然ゲル化剤；ポリアクリルアミド、ヒドロキシエチルセルロース等の合成ゲル化剤が好ましい。斯かるゲル化剤は、平均粒径５００μm 以下、好ましくは０．５〜５０μm の粉体が繊維状吸水性シート中に良好に担持される点で好ましい。なお、培地組成物に用いられる培地成分（ｂ）に、（１）キサンタンガム又はカラギーナン及び（２）ローカストビンガムが含まれ、（１）及び（２）の重量比が６：４〜０．５：９．５であれば、作製された簡易培地に試料液を培地上に滴下するのみで容易に拡散しやすくなる点で好ましい。 Examples of (b) water-soluble and alcohol-insoluble gelling agents used in the medium composition include natural gelling agents such as xanthan gum, locust bin gum, guar gum, carrageenan and pectin; synthesis of polyacrylamide, hydroxyethyl cellulose and the like Gelling agents are preferred. Such a gelling agent is preferable in that a powder having an average particle diameter of 500 μm or less, preferably 0.5 to 50 μm is favorably supported in the fibrous water-absorbent sheet. The medium component (b) used in the medium composition includes (1) xanthan gum or carrageenan and (2) locust bin gum, and the weight ratio of (1) and (2) is 6: 4 to 0.5: If it is 9.5, it is preferable at the point which becomes easy to spread | diffuse only by dripping a sample liquid on a culture medium to the produced simple culture medium.
培地組成物に用いられる（ｃ）菌体培養用栄養分は、検出しようとする微生物の生育に適し、水可溶性のものが選択される。例えば、多種の微生物を増殖させる場合には、一般的な栄養培地成分が用いられ、特定の微生物を選択的に増殖させる場合には選択培地成分が用いられる。 The nutrient for cell culture (c) used in the medium composition is selected to be water-soluble and suitable for the growth of the microorganism to be detected. For example, when a variety of microorganisms are grown, a general nutrient medium component is used, and when a specific microorganism is selectively grown, a selective medium component is used.
本発明において、チオ硫酸塩は簡易培地中に乾燥状態で含有していることが必要である。培地組成物に用いられる（ｄ）チオ硫酸塩としては、チオ硫酸ナトリウム、チオ硫酸アンモニウム、チオ硫酸カリウム等が挙げられる。乾燥状態の培地組成物中のチオ硫酸塩の含有量は、カテキンによる微生物の増殖抑制作用に基づく、検出感度低下防止の点から８〜４７重量％、さらに１５〜３０重量％、特に１５〜２１重量％が好ましい。 In the present invention, thiosulfate must be contained in a simple medium in a dry state. Examples of (d) thiosulfate used in the medium composition include sodium thiosulfate, ammonium thiosulfate, and potassium thiosulfate. The content of the thiosulfate in the dry medium composition is 8 to 47% by weight, more preferably 15 to 30% by weight, particularly 15 to 21% from the viewpoint of preventing the detection sensitivity from being lowered based on the microbial growth inhibitory action by catechin. % By weight is preferred.
さらに、本発明の培地組成物には、コロニーの観察を容易にするために、種々の発色剤を配合するのが好ましい。かかる発色剤には、コロニーを着色する色素、例えばトリフェニルテトラゾリウムクロライド、３−（ｐ−ヨードフェニル）−２−（ｐ−ニトロフェニル）−５−フェニル−２Ｈ−テトラゾリウムクロライド、３−（４，５−ジメチル−２−チアゾリル）−２，５−ジフェニル−２Ｈ−テトラゾリウムブロマイド等の色素；５−ブロモ−３−インドリル−β−Ｄ−ガラクトシド等の酵素基質；ブロムチモールブルーやニュートラルレッドのようなpH指示薬等が含まれる。また、コロニーの観察を容易にするため、発色剤を配合せず、シート自体を着色してもよい。 Furthermore, it is preferable to mix various color formers in the medium composition of the present invention in order to facilitate observation of colonies. Such color formers include dyes that color colonies such as triphenyltetrazolium chloride, 3- (p-iodophenyl) -2- (p-nitrophenyl) -5-phenyl-2H-tetrazolium chloride, 3- (4 Dyes such as 5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide; enzyme substrates such as 5-bromo-3-indolyl-β-D-galactoside; such as bromthymol blue and neutral red pH indicator etc. are included. Further, in order to facilitate observation of colonies, the sheet itself may be colored without adding a color former.
本発明の培地組成物を含有させる繊維状吸水性シートとしては、シート上に接種された検体液が拡散し、当該検体液中の微生物を培地組成物に到達せしめるため、毛細管現象により水を拡散することのできるものであればよい。かかる性質を有するシートとしては、濾紙、レーヨン不織布、ポリエステル不織布、ポリプロピレン不織布に代表される合成繊維不織布、コットン不織布に代表される天然繊維不織布などが挙げられる。このうち、濾紙、レーヨン不織布及び長繊維セルロース不織布が好ましく、特に濾紙及びレーヨン不織布が好ましい。 As the fibrous water-absorbent sheet containing the medium composition of the present invention, the sample liquid inoculated on the sheet diffuses, and the microorganisms in the sample liquid reach the medium composition, so that water is diffused by capillary action. Anything that can be done. Examples of the sheet having such properties include filter paper, rayon nonwoven fabric, polyester nonwoven fabric, synthetic fiber nonwoven fabric represented by polypropylene nonwoven fabric, and natural fiber nonwoven fabric represented by cotton nonwoven fabric. Among these, filter paper, rayon nonwoven fabric, and long fiber cellulose nonwoven fabric are preferable, and filter paper and rayon nonwoven fabric are particularly preferable.
これらのシートは上記の性質を有するものであれば、特に制限されないが、水の拡散性及び薄膜形成性の両者を考慮すれば５〜２００ｇ／m2の密度を有し、０．０５〜０．５mmの厚さを有するシート、特に濾紙及びレーヨン不織布が好ましい。 These sheets are not particularly limited as long as they have the above-described properties, but have a density of 5 to 200 g / m 2 in consideration of both water diffusibility and thin film formability, and 0.05 to 0 Sheets having a thickness of .5 mm, especially filter papers and rayon nonwovens are preferred.
また、これらのシートの形状は、正方形、長方形、円形等特に限定されない。またその大きさも特に制限されないが、微生物の簡易検出用の場合には長径で１〜１５cmが好ましい。 Further, the shape of these sheets is not particularly limited, such as a square, a rectangle and a circle. Further, the size is not particularly limited, but in the case of simple detection of microorganisms, the long diameter is preferably 1 to 15 cm.
また、上記の繊維状吸水性シートは、接種された被検液が毛細管現象により容易に拡散し、かつ培地組成物中のゲル化剤をその網目構造中に担持できる点から、繊維状吸水性シートはゲル化剤の粒径よりも大きいメッシュを有し、かつ厚さも当該粒径より厚いことが好ましく、例えばゲル化剤として平均粒径５００μm 以下、特に０．５〜５０μm の粉体のものを用いた場合には、網目の大きさが１５〜１００メッシュ、特に２０〜５０メッシュで、厚さが１０〜１０００μm、特に５０〜６００μmのものを用いるのが好ましい。 The fibrous water-absorbent sheet is a fibrous water-absorbent sheet because the inoculated test solution can be easily diffused by capillary action and the gelling agent in the medium composition can be supported in the network structure. The sheet preferably has a mesh larger than the particle size of the gelling agent and is preferably thicker than the particle size. For example, the gelling agent is a powder having an average particle size of 500 μm or less, particularly 0.5 to 50 μm. Is used, it is preferable to use a mesh having a mesh size of 15 to 100 mesh, particularly 20 to 50 mesh, and a thickness of 10 to 1000 μm, particularly 50 to 600 μm.
上に挙げた（ａ）〜（ｄ）成分を含有する培地組成物を乾燥状態で含有してなる繊維状吸水性シートの作製は、以下のようにして行われる。まず、（ａ）〜（ｄ）の培地成分をエタノール、2-プロパノール等のアルコールに添加してアルコール懸濁液を調製する。この際、各成分の濃度が、成分（ａ）は０．０１〜５重量％、特に０．３〜２重量％、成分（ｂ）は０．１〜２０重量％、さらに２〜１０重量％、特に５〜８重量％、成分（ｃ）は０．５〜２０重量％、特に１〜５重量％、成分（ｄ）は１〜１０重量％、さらに２〜５重量％、特に２〜３重量％になるようにするのが好ましい。 Fabrication of a fibrous water-absorbent sheet containing the above-described medium composition containing the components (a) to (d) in a dry state is performed as follows. First, the medium components (a) to (d) are added to an alcohol such as ethanol or 2-propanol to prepare an alcohol suspension. At this time, the concentration of each component is 0.01 to 5% by weight, particularly 0.3 to 2% by weight for component (a), 0.1 to 20% by weight for component (b), and further 2 to 10% by weight. 5 to 8% by weight, component (c) 0.5 to 20% by weight, especially 1 to 5% by weight, component (d) 1 to 10% by weight, further 2 to 5% by weight, especially 2 to 3%. It is preferable to make it weight%.
次いで、このアルコール懸濁液を繊維状吸水性シートに注下、噴霧又は塗布等によって含浸させた後、乾燥してアルコールを蒸発除去する。アルコール懸濁液はシート１cm3 当り上記濃度のものを０．５〜２ml含浸させるのが好ましい。乾燥は自然乾燥、減圧乾燥、加熱乾燥の何れであってもよい。 Next, the alcohol suspension is poured onto a fibrous water-absorbent sheet, impregnated by spraying or coating, and then dried to evaporate and remove the alcohol. The alcohol suspension is preferably impregnated with 0.5 to 2 ml of the above concentration per cm 3 of the sheet. Drying may be any of natural drying, vacuum drying, and heat drying.
かくして乾燥状態となった培地組成物中に占めるゲル化剤の割合が、３１〜９０重量％、特に４０〜６０重量％であれば、微生物のコロニーの広がりが抑制され、かつ微生物の生育が抑制されず、菌数の計測が容易である点で好ましい。３１重量％よりも低い場合には、コロニーの広がりの抑制が不十分である点で好ましくなく、９０重量％によりも高い場合には、微生物の生育が抑制されるおそれがある点で好ましくない。 If the ratio of the gelling agent in the medium composition thus dried is 31 to 90% by weight, particularly 40 to 60% by weight, the spread of microbial colonies is suppressed and the growth of microorganisms is also suppressed. This is preferable because the number of bacteria can be easily measured. If it is lower than 31% by weight, it is not preferable in that the suppression of colony spread is insufficient, and if it is higher than 90% by weight, it is not preferable in that the growth of microorganisms may be suppressed.
本発明簡易培地は、シートの片面全体に１種の培地組成物を含む薄膜が均一に形成されていてもよく、また菌体培養用栄養分と発色剤を変化させた複数種の培地組成物を含む薄膜を形成したものでもよい。 In the simple medium of the present invention, a thin film containing one type of medium composition may be uniformly formed on one side of the sheet, and a plurality of types of medium compositions in which nutrients for cell culture and color formers are changed are used. What formed the thin film containing may be used.
繊維状吸水性シートを積層させる防水性平板の材質としては、防水性であれば特に制限されず、例えばポリエチレン系、ポリプロピレン系、ポリスチレン系等のプラスチック、ガラスなどが挙げられ、特にポリスチレン系プラスチックが好ましく、外部からの観察のため透明のものが好ましい。また、平板の表面は、薄膜を形成し得る状態であれば特に制限されず、例えば薄膜が形成し易いように梨子地に仕上げたものであってもよい。 The material of the waterproof flat plate on which the fibrous water-absorbent sheet is laminated is not particularly limited as long as it is waterproof, and examples thereof include polyethylene-based, polypropylene-based, polystyrene-based plastics, glass, and the like. A transparent material is preferable for observation from the outside. The surface of the flat plate is not particularly limited as long as a thin film can be formed. For example, the surface of the flat plate may be a satin finish so that the thin film can be easily formed.
また、これらの平板の形状は、正方形、長方形、円形等特に限定されない。またその大きさも特に制限されないが、微生物の簡易検出用の場合には長径で１〜１５cmが好ましい。 Moreover, the shape of these flat plates is not particularly limited, such as a square, a rectangle, and a circle. Further, the size is not particularly limited, but in the case of simple detection of microorganisms, the long diameter is preferably 1 to 15 cm.
繊維状吸水性シートの防水性平板への積層は、繊維状吸水性シートに含浸させたアルコール懸濁液の乾燥後に行ってもよいが、防水性平板上に載置された繊維状吸水性シートに対して含浸及び乾燥を行うことによるのが、繊維状吸水性シートを防水性平板へ固着できるなどの点で好ましい。 Lamination of the fibrous water absorbent sheet to the waterproof flat plate may be performed after drying the alcohol suspension impregnated in the fibrous water absorbent sheet, but the fibrous water absorbent sheet placed on the waterproof flat plate It is preferable that the fibrous water-absorbent sheet can be fixed to the waterproof flat plate by impregnating and drying.
含浸及び乾燥を防水性平板上に載置された繊維状吸水性シートに対して行う場合、含浸させるアルコール懸濁液に含まれる成分（ａ）の濃度を、アルコール懸濁液中0.01〜0.4重量％、好ましくは0.01〜0.15重量％とし、かつ、乾燥を、アルコールの急速な蒸発を抑制しつつ行う乾燥とすれば、乾燥後の吸水性シートの防水性平板への固着が強固となる点、及び吸水性シートの表面付近では繊維質吸水性シートの繊維が高い吸水性を保持したまま乾燥され、当該繊維目と防水性平板との立体的空洞部の毛細管現象と相俟って、被検液の拡散速度が高くなる点で好ましい。アルコール懸濁液中の成分（ａ）がこの範囲より高濃度の場合、懸濁液中での培地成分の動きが高濃度の成分（ａ）により制限され、そのまま培地成分がシート内部でのみ乾燥固化してしまい、防水性平板への固定化が不十分となり、またシートの吸水性も劣るものとなるので好ましくない。アルコールの急速な蒸発を抑制しつつ乾燥を行うための手段としては特に限定されるものではないが、例えばアルコールの蒸気圧の高い雰囲気中で乾燥を行う、低温で乾燥を行う等の方法が挙げられる。ここで行う乾燥が急速な場合には、アルコール懸濁液中の培地成分の沈降が不十分なまま固化してしまうため、吸水性シートの防水性容器への固定が不十分となり、また被検液の拡散性に劣るものとなってしまうので好ましくない。 When impregnation and drying are performed on a fibrous water absorbent sheet placed on a waterproof flat plate, the concentration of the component (a) contained in the alcohol suspension to be impregnated is 0.01 to 0.4 weight in the alcohol suspension. %, Preferably 0.01 to 0.15% by weight, and if drying is performed while suppressing rapid evaporation of alcohol, the water-absorbent sheet after drying is firmly fixed to the waterproof flat plate, In the vicinity of the surface of the water-absorbent sheet, the fibers of the fibrous water-absorbent sheet are dried while maintaining high water absorbency, and coupled with the capillary phenomenon of the three-dimensional cavity between the fibers and the waterproof flat plate, This is preferable in that the diffusion rate of the liquid is increased. When the concentration of the component (a) in the alcohol suspension is higher than this range, the movement of the medium component in the suspension is restricted by the high concentration component (a), and the medium component is dried only inside the sheet as it is. Since it solidifies, the fixing to the waterproof flat plate becomes insufficient, and the water absorption of the sheet is also inferior, which is not preferable. The means for drying while suppressing rapid evaporation of alcohol is not particularly limited, and examples thereof include drying in an atmosphere having a high alcohol vapor pressure, drying at low temperature, and the like. It is done. If the drying performed here is rapid, the medium components in the alcohol suspension will solidify with insufficient settling, so that the water-absorbent sheet will be insufficiently fixed to the waterproof container, and Since it becomes inferior to the diffusibility of a liquid, it is unpreferable.
本発明の簡易培地は、汚染及び乾燥を防止するために、その表面をフィルムで覆うか、容器に収納するのが好ましい。特に、防水性平板として容器一体型の防水性容器を用い、これに培地成分を担持させた繊維質吸水性シートを積層固着させるのが好ましい。 The simple medium of the present invention is preferably covered with a film or stored in a container in order to prevent contamination and drying. In particular, it is preferable to use a container-integrated waterproof container as the waterproof flat plate, and laminate and fix the fibrous water-absorbent sheet carrying the culture medium component thereon.
このようにして製造された本発明の簡易培地は、エチレンオキサイドガス等のガス、γ線、電子線等、好ましくはγ線によって滅菌するのが好ましい。好ましい滅菌方法としては、アルミ包材等、ガスバリヤー性と遮光性を兼ね備えた包材で包装した後、γ線、電子線等の放射線照射を行う方法が挙げられ、特にこの包装の際に簡易培地と共に乾燥剤を封入しておくことが最も好ましい。このような方法で滅菌処理を行うことにより、簡易培地の製造の全工程を無菌状態に管理する必要がなくなり、かつ滅菌後の微生物の混入や光及び湿気による培地の経時変化を抑制することが可能となる。 The simple medium of the present invention thus produced is preferably sterilized with a gas such as ethylene oxide gas, γ rays, electron beams, etc., preferably γ rays. As a preferable sterilization method, a method of irradiating with γ rays, electron beams or the like after packaging with a packaging material having both gas barrier properties and light shielding properties such as an aluminum packaging material can be mentioned. Most preferably, a desiccant is enclosed with the medium. By performing sterilization by such a method, it is not necessary to manage all steps of production of a simple medium in a sterile state, and it is possible to suppress aging of microorganisms after sterilization and change with time of the medium due to light and moisture. It becomes possible.
本発明の簡易培地を用いて微生物を検出するには、被検体から調製した被検液を当該培地表面に接種する。本発明にはカテキンを含有する被検体（固体）から調製した被検液や液体被検液も用いることができる。カテキンを含有する被検体（固体、液体）としては、緑茶飲料、食品添加物、特定保健用食品、保健機能食品、栄養食品、健康食品、健康補助食品、サプリメント、栄養ドリンク剤、ペットフ−ド等が挙げられる。そうすると、被検液がメッシュ間の立体的な空洞部の毛細管現象によって容易に拡散し、それに続いて膨潤ゲル化が起こって、被検液中の微生物は捕捉され、自由移動が抑制され、培養によってコロニーが形成される。従って、培地形成面を観察すれば、形成したコロニーが容易に観察できる。また、本発明の簡易培地に乾燥状態で含有されるチオ硫酸塩により、カテキンの微生物増殖抑制作用が中和されるため、被検液がカテキンを含む場合であっても、被検液に含まれる微生物を正確に反映した数のコロニーが形成される。したがって、試料を定量的に簡易培地へ接種すれば、培養後出現したコロニーを計測することにより、容易にかつ精度よく菌数を算定することができる。なお、菌体液の接種は通常ピペット等により一定量を接種する方法が採用されるが、水分の多い個体へスタンプする方法や試料液へ浸す方法でもよい。また、被検液接種後の培養は、静置して行っても、また輸送期間中に行ってもよい。 In order to detect microorganisms using the simple culture medium of the present invention, a test solution prepared from a specimen is inoculated on the surface of the culture medium. In the present invention, a test solution or a liquid test solution prepared from a test sample (solid) containing catechin can also be used. Samples containing catechins (solid, liquid) include green tea drinks, food additives, foods for specified health use, health functional foods, nutritional foods, health foods, health supplements, supplements, energy drinks, pet foods, etc. Is mentioned. Then, the test solution is easily diffused by the capillary action of the three-dimensional cavity between the meshes, followed by swelling gelation, microorganisms in the test solution are captured, free movement is suppressed, and culture is performed. To form a colony. Therefore, if the culture medium formation surface is observed, the formed colonies can be easily observed. Moreover, since the microbial growth inhibitory action of catechin is neutralized by the thiosulfate contained in the dry state in the simple medium of the present invention, even if the test solution contains catechin, it is included in the test solution. The number of colonies accurately reflecting the microorganisms to be formed is formed. Therefore, if the sample is inoculated quantitatively into a simple medium, the number of bacteria can be calculated easily and accurately by measuring the colonies that appear after culturing. Inoculation with a bacterial cell solution usually employs a method of inoculating a fixed amount with a pipette or the like, but a method of stamping an individual with a lot of water or a method of immersing in a sample solution may be used. Further, the culture after inoculation of the test solution may be performed by standing or during the transportation period.
本発明の簡易培地を用いて検出することができる微生物は、特に制限されず、例えば、E.coli、B.subtilis、S.aureus、P.aeruginosa、Candida albicans、B.thuringiensis、B.cereus 、B.thuringiensis 、K.oxytoca等が挙げられるが、カテキン抗菌効果へのチオ硫酸塩による影響が大きい点から、S.aureus、P.aeruginosa、Candida albicans等が好ましい。 Microorganisms that can be detected using the simple medium of the present invention are not particularly limited. For example, E. coli , B. subtilis , S. aureus , P. aeruginosa , Candida albicans , B. thuringiensis , B. cereus , B. thuringiensis , K. oxytoca and the like can be mentioned, and S. aureus , P. aeruginosa , Candida albicans and the like are preferable because thiosulfate has a large influence on the catechin antibacterial effect.
以下に実施例を示し、本発明をさらに詳しく説明する。しかしながら、本発明はこれら実施例に制限されるものではない。 The following examples illustrate the present invention in more detail. However, the present invention is not limited to these examples.
参考例1. カテキン含有茶飲料の抗菌作用の検討
カテキン含有茶飲料を無菌的に９ｍｌずつ、滅菌試験管へ分注した。対照として、滅菌生理食塩水９ｍｌの希釈液を準備した。次に、各菌株について、細菌はトリプトソイブイヨン（日水製薬社製）で35℃、24時間培養し、酵母はブドウ糖ペプトン（日水製薬社製）で35℃、48時間培養し、得られた菌液をカテキン含有茶飲料又は生理食塩水で10-1から10-8まで希釈した。希釈されたそれぞれの菌液を、コンパクトドライＴＣ（フィルム状簡易培地：日水製薬株式会社製、以下同じ）、ＳＰＣ混釈、ＳＰＣ平板塗抹にそれぞれ接種し、35℃で48時間培養後、それぞれの方法により菌数を測定した。
ここで、コンパクトドライTCは、後記の表７における従来のコンパクトドライTCの組成を有する培地組成物が8.11％含まれるアルコール懸濁液を、網目の大きさが30メッシュのセルロ−ス不織布に含浸乾燥させて作製した。
なお、ＳＰＣ混釈は121℃、15分高圧蒸気滅菌し、45℃〜50℃に保持した標準寒天培地を用いて、予め滅菌シャーレに入れた被検液の1ｍｌとよく混和させ固化したのち、35℃、48時間培養後、増殖したコロニー数の計測を行うという方法であり、加温溶解した標準寒天培地（日水製薬社製）に希釈液を懸濁し、かくして得られる増殖コロニー数を計測することにより菌数を求めた。ＳＰＣ平板塗抹は、標準寒天培地を121℃、15分高圧蒸気滅菌したのち、滅菌シャーレに分注し固化させた寒天培地（寒天平板培地）に被検液の0.1ｍｌをコンラージ棒で塗抹し、培養後の増殖したコロニー数の計測を行うという方法であり、寒天平板培地（日水製薬社製）に希釈液を塗抹し、増殖したコロニー数の計測により菌数を測定した。
測定された菌数を菌株ごとに表１に示す。表１において、生食は生理食塩水で希釈した菌液を用いたことを示し、カテキンはカテキン含有茶飲料で希釈した菌液を用いたことを示す。また、＋は、十分数の菌数が測定されたことを示し、−は、菌が検出されなかったことを示す（以下の表２〜表５−１、表５−２、表１０についても同じ）。
Reference Example 1. Examination of antibacterial action of catechin-containing tea beverage 9 ml of catechin-containing tea beverage was aseptically dispensed into sterile test tubes. As a control, a diluted solution of 9 ml of sterile physiological saline was prepared. Next, for each strain, the bacteria were cultured in tryptic soy broth (Nissui Pharmaceutical Co., Ltd.) at 35 ° C. for 24 hours, and the yeast was cultured in glucose peptone (Nissui Pharmaceutical Co., Ltd.) at 35 ° C. for 48 hours. The bacterial solution was diluted from 10 -1 to 10 -8 with a catechin-containing tea beverage or physiological saline. Each diluted bacterial solution was inoculated into compact dry TC (film simple medium: manufactured by Nissui Pharmaceutical Co., Ltd., the same shall apply hereinafter), SPC pour, SPC plate smear, and cultured at 35 ° C. for 48 hours. The number of bacteria was measured by this method.
Here, the compact dry TC is impregnated into a cellulose nonwoven fabric having a mesh size of 30 mesh with an alcohol suspension containing 8.11% of a medium composition having the composition of the conventional compact dry TC in Table 7 below. Made by drying.
The SPC pour was sterilized by autoclaving at 121 ° C. for 15 minutes and mixed with 1 ml of the test solution previously placed in a sterile petri dish using a standard agar medium maintained at 45 ° C. to 50 ° C., and solidified. The method is to measure the number of grown colonies after culturing at 35 ° C for 48 hours. Suspend the diluted solution in a standard agar medium (Nissui Pharmaceutical Co., Ltd.) dissolved by heating, and count the number of grown colonies thus obtained. The number of bacteria was determined. SPC plate smearing is performed by autoclaving a standard agar medium at 121 ° C for 15 minutes, then smearing 0.1 ml of the test solution on agar plate (agar plate medium) dispensed and solidified in a sterilized petri dish with a large stick, In this method, the number of grown colonies after culture was measured. The diluted solution was smeared on an agar plate medium (manufactured by Nissui Pharmaceutical Co., Ltd.), and the number of bacteria was measured by counting the number of grown colonies.
The measured number of bacteria is shown in Table 1 for each strain. In Table 1, it shows that the raw food used the bacterial solution diluted with the physiological saline, and catechin showed that the bacterial solution diluted with the catechin containing tea drink was used. Further, + indicates that a sufficient number of bacteria was measured, and-indicates that no bacteria were detected (also in the following Tables 2 to 5-1, Table 5-2, and Table 10). the same).
表１に示すとおり、平板塗抹を用いた場合においてはカテキンの影響は認められなかった。一方、コンパクトドライＴＣ、混釈培養を用いた場合においてはいずれもカテキンの影響による菌数低下が認められた。また、カテキンの影響の度合いはコンパクトドライＴＣの方がＳＰＣ混釈より大きいことが認められた。このことから、フィルム状簡易培地では、カテキンを含有した被検液を精度よく検出することができないことが明らかになった。 As shown in Table 1, the effect of catechin was not observed when using flat plate smear. On the other hand, in the case of using compact dry TC and pour culture, a decrease in the number of bacteria due to the effect of catechin was observed. Moreover, it was recognized that the degree of the influence of catechin was larger in the compact dry TC than in the SPC pour. From this, it was clarified that the test liquid containing catechin cannot be detected with high accuracy in the film-shaped simple medium.
参考例2. カテキン含有茶飲料の抗菌作用の検討
チオ硫酸ナトリウム、Tween80、レシチンをそれぞれの濃度でカテキン含有茶飲料に溶解し、9mlずつ分注し、100℃で20分間加温溶解後、希釈液として用いた。S.aureus ATCC6538をトリプトソイブイヨンで35℃で24時間培養し、上記の希釈液で10-1から10-8まで希釈しその1ｍｌを摂取し、コンパクトドライＴＣ又はＳＰＣ混釈によって35℃で48時間培養した後、それぞれの方法によって菌数を測定した。
Reference Example 2. Examination of antibacterial action of catechin-containing tea beverages Sodium thiosulfate, Tween 80, and lecithin are dissolved in catechin-containing tea beverages at each concentration, dispensed 9 ml at a time, and heated at 100 ° C for 20 minutes to dissolve and diluted. Used as a liquid. S. aureus ATCC6538 was cultured in trypto soy broth at 35 ° C for 24 hours, diluted from 10 -1 to 10 -8 with the above dilution and ingested 1 ml, and mixed with compact dry TC or SPC at 48 ° C at 35 ° C. After incubation for hours, the number of bacteria was measured by each method.
結果を表２に示す。ｗは増殖したコロニーが微小であったことを示す。生食は生理食塩水を用いて希釈を行った対照を示し、レシチンは、レシチンを1g/L、Tween80を7g/L含有する希釈液で希釈を行った対照を示す。TCは、コンパクトドライＴＣ（日水製薬株式会社製）を用いて、SPCは、SPC混釈を用いて培養を行ったことを示す（以下同じ）。 The results are shown in Table 2. w indicates that the grown colonies were very small. The raw food shows a control diluted with physiological saline, and the lecithin shows a control diluted with a diluent containing 1 g / L of lecithin and 7 g / L of Tween80. TC indicates that culture was performed using compact dry TC (manufactured by Nissui Pharmaceutical Co., Ltd.), and SPC indicates that culture was performed using SPC pour (hereinafter the same).
表２に示されるように、カテキン含有茶飲料にチオ硫酸ナトリウムを加えることにより何も加えないものに比べて数オーダー回収量が回復することが認められた。しかしながら、SPC平板を用いるよりも少ないコロニー数しか検出できなかったことから、精度は不十分であることが明らかになった。一方、抗菌剤へ中和効果が知られていたレシチンを加えても、大きな改善効果は見られなかった。 As shown in Table 2, it was found that the recovery amount of several orders was recovered by adding sodium thiosulfate to the catechin-containing tea beverage as compared with the case where nothing was added. However, since the number of colonies was smaller than that using the SPC plate, it was revealed that the accuracy was insufficient. On the other hand, even if lecithin, which was known to have a neutralizing effect, was added to the antibacterial agent, no significant improvement effect was observed.
実施例１. カテキン含有茶飲料の抗菌作用の検討
0、1、2、3、4又は5％の濃度でチオ硫酸ナトリウムを含むアルコール懸濁液を吸水性シートに含浸させ乾燥させることにより、乾燥状態の培地組成物中にチオ硫酸ナトリウムをそれぞれ13、23、32、38、44重量％含むフィルム状簡易培地を作製した。ここで、チオ硫酸ナトリウムを含む以外の成分は、前記コンパクトドライTCと同様である。作製した培地又はSPC混釈に、各菌株をカテキン含有茶飲料又は生理食塩水にて10-1〜10-8に希釈して得た各希釈液を摂取し、35℃で48時間培養した後、菌数を測定した。結果を表３に示す。
Example 1. Examination of antibacterial action of catechin-containing tea beverage
By impregnating a water-absorbent sheet with an alcohol suspension containing sodium thiosulfate at a concentration of 0, 1, 2, 3, 4 or 5% and drying, 13 thiosulfate was added to the dried medium composition, respectively. , 23, 32, 38 and 44% by weight were prepared as a film-like simple medium. Here, the components other than sodium thiosulfate are the same as those in the compact dry TC. After ingesting each diluted solution obtained by diluting each strain to 10 -1 to 10 -8 with a catechin-containing tea beverage or physiological saline in the prepared medium or SPC pour, and culturing at 35 ° C for 48 hours The number of bacteria was measured. The results are shown in Table 3.
表3に示したように、あらかじめチオ硫酸ナトリウムが乾燥状態で含まれているフィルム状簡易培地を用いることにより、回収菌数がＳＰＣ混釈とほぼ同等までに回復することが認められた。また、4％を越えるとチオ硫酸塩の効果が低下する傾向が見られたため、3％の添加がもっとも良好であると考えられた。 As shown in Table 3, it was confirmed that the number of recovered bacteria recovered to almost the same as that of the SPC pour by using a simple film medium containing sodium thiosulfate in a dry state in advance. In addition, when it exceeded 4%, the effect of thiosulfate tended to decrease, so 3% addition was considered the best.
実施例２. カテキン含有茶飲料の抗菌作用の検討
コンパクトドライは、通常γ線滅菌して流通しているため、チオ硫酸ナトリウムを添加したコンパクトドライＴＣでのγ線滅菌の影響を検討した。まず、チオ硫酸ナトリウムのγ線滅菌の影響を確認するために、チオ硫酸ナトリウムを乾燥状態の培地組成物中に３％含むフィルム状簡易培地を作製し、γ線滅菌を施した。次に、各菌株を各茶飲料又は生理食塩水（分注後100℃で20分間加温溶解後使用）によって10-1から10-8にまで希釈した菌液を、上記のγ線滅菌を施したコンパクトドライに接種し、35℃、48時間培養後の菌数を確認した。対照としては、γ線滅菌を施したフィルム状簡易培地（滅菌チオTC）の代わりに、コンパクトドライＴＣ（製品TC）、チオ硫酸ナトリウム3%を含む実験３の培地で未滅菌のもの（未滅菌チオTC）、SPC混釈（SPC）を用いた。結果を表４に示す。
Example 2. Examination of antibacterial action of catechin-containing tea drink Since compact dry is normally distributed after γ-ray sterilization, the effect of γ-ray sterilization in compact dry TC added with sodium thiosulfate was examined. First, in order to confirm the influence of γ-ray sterilization of sodium thiosulfate, a film-like simple medium containing 3% of sodium thiosulfate in a dry medium composition was prepared and subjected to γ-ray sterilization. Next, the bacterial solution obtained by diluting each strain from 10 -1 to 10 -8 with each tea drink or physiological saline (dissolved by heating at 100 ° C for 20 minutes after dispensing) is subjected to the above-mentioned γ-ray sterilization. The inoculated compact dry was inoculated and the number of bacteria after culturing at 35 ° C. for 48 hours was confirmed. As a control, instead of a film-like simple medium (sterilized thio TC) subjected to γ-ray sterilization, the medium of Experiment 3 containing compact dry TC (product TC) and sodium thiosulfate 3% was not sterilized (unsterilized) Thio TC) and SPC pour (SPC) were used. The results are shown in Table 4.
表４に示されるとおり、γ線滅菌によるチオ硫酸ナトリウムへの影響は認められなかった。また、カテキン含有茶飲料を検体とした場合、チオ硫酸ナトリウムを含むフィルム状簡易培地を用いることによって、ＳＰＣ混釈と同等の検出精度で菌数が検出できることが明らかになった。 As shown in Table 4, no effect on sodium thiosulfate by γ-ray sterilization was observed. In addition, when a catechin-containing tea beverage was used as a specimen, it was revealed that the number of bacteria could be detected with the same detection accuracy as that of SPC pour by using a film-like simple medium containing sodium thiosulfate.
実施例３. 微生物のコロニー拡散の程度の検討
キサンタンガム濃度を30、40、50、60、70、80g/L含むアルコール懸濁液を吸水性シートに含浸させ乾燥させることにより、乾燥状態の培地組成物中キサンタンガムをそれぞれ46、53、59、63、67、70重量％含むフィルム状簡易培地を作製した。作製した培地に、参考例１と同様の方法によって生理食塩水で10-4から10-7まで希釈した各菌株の希釈液を接種し、35℃で48時間培養した後、菌数を確認した。SPC混釈を用いたものを対照とした（SPC）。結果を表５−１及び表５−２に示す。
Example 3. Examination of the degree of microbial colony diffusion A dry medium composition by impregnating a water-absorbent sheet with an alcohol suspension containing xanthan gum concentration of 30, 40, 50, 60, 70, 80 g / L. A simple film-like medium containing 46, 53, 59, 63, 67, and 70% by weight of xanthan gum was prepared. The prepared medium was inoculated with a diluted solution of each strain diluted with physiological saline from 10 −4 to 10 −7 in the same manner as in Reference Example 1, and after culturing at 35 ° C. for 48 hours, the number of bacteria was confirmed. . A control using SPC pour was used as a control (SPC). The results are shown in Tables 5-1 and 5-2.
表５−１及び表５−２に示すとおり、キサンタンガムの濃度を30g/Lから80g/Lへ増やしても微生物の増殖抑制は見られなかった。また、視認の結果、B.cereus GH-2、B.cereus GH-3、B.thuringiensis NBRC 13866、P.aeruginosa ATCC 9027の場合には、それぞれキサンタンガム60g/L、50 g/L、60 g/L、50 g/L以上の濃度の培地を用いることにより、コロニーの形状が良好になることが認められた。 As shown in Tables 5-1 and 5-2, even if the concentration of xanthan gum was increased from 30 g / L to 80 g / L, no growth inhibition of microorganisms was observed. In addition, as a result of visual recognition, in the case of B. cereus GH-2, B. cereus GH-3, B. thuringiensis NBRC 13866, P. aeruginosa ATCC 9027, xanthan gum 60 g / L, 50 g / L, 60 g / It was confirmed that the colony shape was improved by using a medium having a concentration of L, 50 g / L or more.
次に、不織布へ生理食塩水を注加した時から不織布全面へ生理食塩水が拡散する時間をストップウオッチにより測定した。結果を表６に示す。 Next, the time for the physiological saline to diffuse over the entire surface of the nonwoven fabric from the time when the physiological saline was poured into the nonwoven fabric was measured with a stopwatch. The results are shown in Table 6.
表６に示すとおり、キサンタンガムを増加させるに従い拡散が小さくなることが明らかになった。 As shown in Table 6, it became clear that diffusion became small as xanthan gum was increased.
実施例４. カテキンおよびバチルス対策コンパクトドライＴＣの検討
表７に示す組成からなる培地組成物を用いて、表８に示す組成からなるアルコール懸濁液を用意し、これを用いてフィルム状簡易培地を作製した。各菌株を表９に示した各希釈液にて10-1から10-8まで希釈した菌液を用意し、かかる菌液を作製した培地に接種し、35℃で48時間培養した後、菌数を確認した。供試菌株の初発菌数について滅菌生理食塩水を用いて希釈しＳＰＣ混釈にて確認した菌数を対照とした（SPC）。結果を表１０及び表１１に示す。なお、表１０及び表１１において、従来品TCとは、コンパクトドライTCを用いて行ったものを示し、改良品TCとは、本発明のフィルム状簡易培地を用いて行ったものを示す。
Example 4. Examination of catechin and Bacillus countermeasure compact dry TC Using a culture medium composition having the composition shown in Table 7, an alcohol suspension having the composition shown in Table 8 was prepared, and using this, a film-like simple culture medium was prepared. Was made. Prepare a bacterial solution obtained by diluting each strain from 10 -1 to 10 -8 with each dilution shown in Table 9, inoculate the prepared bacterial solution, and incubate at 35 ° C for 48 hours. Check the number. The initial bacterial count of the test strain was diluted with sterilized physiological saline and confirmed by SPC pour control as a control (SPC). The results are shown in Table 10 and Table 11. In Tables 10 and 11, the conventional product TC indicates that performed using compact dry TC, and the improved product TC indicates that performed using the film-like simple medium of the present invention.
表１０及び表１１に示すとおり、本発明の培地を用いた場合にはすべての茶飲料の希釈液においてコンパクトドライTC（従来品TC）よりも回収菌数が回復し、ほぼ滅菌生理食塩水を用いた菌数と同等であることが認められた。また視認の結果、コンパクトドライTC（従来品TC）と比較してコロニーの拡散はほとんど見られないことも認められた。 As shown in Table 10 and Table 11, when the culture medium of the present invention was used, the number of recovered bacteria recovered in all tea beverage dilutions compared to Compact Dry TC (conventional TC), and almost sterile physiological saline was used. It was found to be equivalent to the number of bacteria used. In addition, as a result of visual inspection, it was confirmed that almost no colony was diffused compared to compact dry TC (conventional TC).
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| JPH0919282A (en) * | 1995-07-07 | 1997-01-21 | Nissui Pharm Co Ltd | Simple medium and detection of microorganism |
| JP2000325072A (en) * | 1999-05-19 | 2000-11-28 | Nissui Pharm Co Ltd | Simplified culture medium and its production |
| JP2002355091A (en) * | 2001-05-31 | 2002-12-10 | Nissui Pharm Co Ltd | Culture medium for detecting enteritis vibrio |
| JP2004057054A (en) * | 2002-07-26 | 2004-02-26 | Nissui Pharm Co Ltd | Method for simply detecting salmonella |
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| JPH0919282A (en) * | 1995-07-07 | 1997-01-21 | Nissui Pharm Co Ltd | Simple medium and detection of microorganism |
| JP2000325072A (en) * | 1999-05-19 | 2000-11-28 | Nissui Pharm Co Ltd | Simplified culture medium and its production |
| JP2002355091A (en) * | 2001-05-31 | 2002-12-10 | Nissui Pharm Co Ltd | Culture medium for detecting enteritis vibrio |
| JP2004057054A (en) * | 2002-07-26 | 2004-02-26 | Nissui Pharm Co Ltd | Method for simply detecting salmonella |
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