JP2013236553A - Simple medium - Google Patents
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Abstract
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本発明は、被検液を滴下し、そのまま培養することにより簡易に微生物の検出、固定等が可能な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 capable of easily detecting and immobilizing microorganisms by dropping a test solution and culturing it as it is, and a microorganism detecting method using the same.
種々の微生物を検出、同定するための培地として、被検液を培地上に滴下し、該培地上で培養して検出できる簡易培地が数多く報告されている。例えば、防水性基体の上面部に、接着剤層、水可溶性ゲル化剤粉末層、繊維質吸水性シート等を順次積層した培地（特許文献１〜３）、濾紙等に菌体栄養成分を含浸させた培地（特許文献４）等がある。しかし、これらの培地には、製造上の問題や、コロニーの観察が容易にできない等の問題があった。 As a medium for detecting and identifying various microorganisms, many simple media that can be detected by dropping a test solution onto the medium and culturing on the medium have been reported. For example, a culture medium (Patent Documents 1 to 3) in which an adhesive layer, a water-soluble gelling agent powder layer, a fibrous water-absorbing sheet, etc. are sequentially laminated on the upper surface of a waterproof substrate, filter paper, etc. are impregnated with bacterial nutrients Medium (Patent Document 4) and the like. However, these media have problems such as manufacturing problems and inability to easily observe colonies.
これに対し、（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、及び（ｃ）菌体栄養成分を含有する培地組成物を、該ゲル化剤により大きいメッシュを有する繊維状吸水性シートに担持させた簡易培地（特許文献５）、多孔質マトリックス層と水溶性高分子化合物層を積層した積層物を有するシート状培地（特許文献６）が報告された。この培地は、被検液を滴下し、そのまま培養するだけで微生物のコロニーが容易に観察できることから、広く使用されている。 In contrast, (a) an adhesive that is soluble in water and alcohol, (b) a gelling agent that is soluble in water and insoluble in alcohol, and (c) a medium composition containing a cell nutrient component, A simple culture medium (Patent Document 5) supported on a fibrous water-absorbent sheet having a larger mesh as a gelling agent, and a sheet-shaped medium having a laminate in which a porous matrix layer and a water-soluble polymer compound layer are laminated (Patent Document 6) ) Was reported. This medium is widely used because a colony of microorganisms can be easily observed just by dropping a test solution and culturing it as it is.
しかし、前記特許文献５及び６の簡易培地は、多孔質マトリックス層や繊維状吸水性シートを有するため、不透明であり、白色のコロニーの検出性が悪い、シート内のコロニーの検出性が悪い等の問題があった。
従って、本発明の課題は、簡易培地でありながら、透明で寒天培地と同様の使用感、コロニーの検出性を有する簡易培地を提供することにある。
However, since the simple culture media of Patent Documents 5 and 6 have a porous matrix layer and a fibrous water-absorbent sheet, they are opaque, have poor white colony detectability, poor colony detectability in the sheet, etc. There was a problem.
Therefore, an object of the present invention is to provide a simple medium that is transparent and transparent and has the same feeling of use and colony detection as that of an agar medium.
そこで本発明者は、簡易培地の成分について種々検討した結果、防水性平板上に、水溶性高分子と菌体栄養成分と水溶性繊維片とを実質的に均一に含有する薄膜を形成すれば、被検水性液を滴下した時、該被検水性液が薄膜中に速やかに拡散するとともに、薄膜が透明になり、寒天培地と同様の使用性、検出性が得られることを見出し、本発明を完成した。 Therefore, as a result of various studies on the components of the simple medium, the present inventor has formed a thin film that substantially contains the water-soluble polymer, the microbial nutrients, and the water-soluble fiber pieces on the waterproof flat plate. In addition, when the test aqueous solution is dropped, the test aqueous solution quickly diffuses into the thin film, and the thin film becomes transparent, and the same usability and detectability as the agar medium can be obtained. Was completed.
すなわち、本発明は、防水性平板の表面の少なくとも一部に、（ａ）水溶性高分子、（ｂ）水溶性繊維片及び（ｃ）菌体培養成分を実質的に均一に含有する薄膜を形成してなる簡易培地を提供するものである。
また、本発明は、上記の簡易培地の薄膜上に被検水性液を滴下した後培養し、該薄膜上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。
That is, the present invention provides a thin film containing (a) a water-soluble polymer, (b) a water-soluble fiber piece and (c) a cell culture component substantially uniformly on at least a part of the surface of the waterproof flat plate. A simple medium formed is provided.
In addition, the present invention provides a method for detecting a microorganism, which comprises culturing after dropping a test aqueous solution on a thin film of the simple medium and detecting colonies formed on the thin film. is there.
本発明の簡易培地を用いれば、被検水性液を培地の薄膜上に滴下するだけで、透明な薄膜培地となり、そのまま培養後に生成したコロニーを観察することが可能であり、コロニー検出、釣菌が容易である。また、コロニーの観察が容易となるため、検出精度も向上する。 If the simple culture medium of the present invention is used, it is possible to observe a colony produced after culturing as it is by simply dropping the aqueous test solution on the thin film of the medium, and observing colonies generated after culturing. Is easy. Moreover, since observation of a colony becomes easy, a detection accuracy improves.
本発明の簡易培地は、防水性平板の表面の少なくとも一部に、（ａ）水溶性高分子、（ｂ）水溶性繊維片及び（ｃ）菌体栄養成分を実質的に均一に含有する薄膜を形成してなる。 The simple culture medium of the present invention is a thin film containing substantially uniformly (a) a water-soluble polymer, (b) a water-soluble fiber piece, and (c) a cell nutrient component on at least a part of the surface of the waterproof flat plate. Formed.
防水性平板は、プラスチック、ガラス等の防水性の材質の平板である。当該平板は、プラスチックシャーレであるのが好ましく、無色透明なプラスチックシャーレであるのが特に好ましい。 The waterproof flat plate is a flat plate made of a waterproof material such as plastic or glass. The flat plate is preferably a plastic petri dish, particularly preferably a colorless and transparent plastic petri dish.
薄膜に含まれる（ａ）水溶性高分子としては、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、ポリアクリル酸、ポリアルギン酸等の（ａ１）水及びアルコールに可溶な高分子；キサンタンガム、ローカストビーンガム、グアーガム、カラギーナン等の水に可溶でアルコールに不溶な高分子が挙げられる。これらの水溶性高分子のうち、（ａ１）水及びアルコールに可溶な高分子と、（ａ２）水に可溶でアルコールに不溶な高分子を併用するのが、製造が簡便である点、培地性能保持の点から好ましい。（ａ１）水及びアルコールに可溶な高分子としては、ヒドロキシプロピルセルロース、ポリビニルピロリドン及びポリエチレンオキサイドが好ましく、特にヒドロキシプロピルセルロースが好ましい。（ａ２）水に可溶でアルコールに不溶な高分子としては、キサンタンガムが特に好ましい。ここでアルコールは、低級アルコールを意味し、特にメタノール、エタノール、イソプロパノールが好ましい。また水に可溶とは、１質量％以上水に溶解することを意味し、アルコールに可溶とは、１質量％以上アルコールに溶解することをいう。 (A) Water-soluble polymers contained in the thin film include (a1) water- and alcohol-soluble polymers such as hydroxypropylcellulose, polyvinylpyrrolidone, polyethylene oxide, polyacrylic acid, polyalginic acid; xanthan gum, locust bean gum , Guar gum, carrageenan and other water-soluble and alcohol-insoluble polymers. Among these water-soluble polymers, (a1) a polymer soluble in water and alcohol and (a2) a combination of a polymer soluble in water and insoluble in alcohol are easy to manufacture, It is preferable from the viewpoint of maintaining the medium performance. (A1) As a polymer soluble in water and alcohol, hydroxypropylcellulose, polyvinylpyrrolidone and polyethylene oxide are preferable, and hydroxypropylcellulose is particularly preferable. (A2) Xanthan gum is particularly preferable as the polymer that is soluble in water and insoluble in alcohol. Here, the alcohol means a lower alcohol, and methanol, ethanol, and isopropanol are particularly preferable. Moreover, being soluble in water means dissolving in water by 1% by mass or more, and being soluble in alcohol means dissolving in alcohol by 1% by mass or more.
（ａ）水溶性高分子の薄膜中の含有量は、被検水性液の薄膜への分散性、透明性、コロニー形成能の点から、測定時の濃度として０．１〜２０質量％が好ましく、２〜７質量％がより好ましい。このうち、（ａ１）水及びアルコールに可溶な高分子の含有量は、薄膜中に測定時の濃度として０．０５〜５質量％が好ましく、０．０５〜２質量％がより好ましい。また、（ａ２）水に可溶でアルコールに不溶な高分子の含有量は、薄膜中に測定時の濃度として０．１〜２０質量％が好ましく、２〜７質量％がより好ましい。ここで測定時の濃度とは、簡易培地に所定量の被検水性液を添加したときの濃度をいう。 (A) The content of the water-soluble polymer in the thin film is preferably 0.1 to 20% by mass as the concentration at the time of measurement from the viewpoint of dispersibility of the aqueous test liquid in the thin film, transparency, and colony forming ability. 2 to 7% by mass is more preferable. Among these, (a1) The content of the polymer soluble in water and alcohol is preferably 0.05 to 5% by mass, more preferably 0.05 to 2% by mass as the concentration at the time of measurement in the thin film. Moreover, (a2) 0.1-20 mass% is preferable as a density | concentration at the time of a measurement in a thin film, and, as for content of the polymer insoluble in alcohol which is soluble in water, 2-7 mass% is more preferable. Here, the concentration at the time of measurement refers to the concentration when a predetermined amount of the test aqueous solution is added to the simple medium.
（ｂ）水溶性繊維片としては、水溶性ポリマーの繊維片が好ましく、特にポリビニルアルコール系繊維片が、被検水性液滴下時の透明性、拡散性の点から好ましい。（ｂ）水溶性繊維片は、被検水性液滴下前は薄膜中に均一に分散しているが、被検水性液滴下時に徐々に水に溶解して薄膜を透明にし、かつ薄膜中に被検水性液を毛細管現象によって速やかに拡散させる作用を有し、さらに被検水性液中に存在する微生物が増殖する際の足場となる。当該（ｂ）水溶性繊維片が薄膜中に均一に分散していることにより、被検水性液を滴下した時に、被検水性液が薄膜に速やかに拡散し、かつ薄膜が透明となり、被検水性液中の微生物が増殖した時に良好なコロニーが形成されやすくなる。ここで、透明とは、約４００〜７００ｎｍの波長の光に対する無色透明のガラスの光透過率を１００％としたときに、８０％以上が透過することをいう。 (B) As a water-soluble fiber piece, the fiber piece of a water-soluble polymer is preferable, and especially the polyvinyl alcohol-type fiber piece is preferable from the point of the transparency at the time of a test water droplet, and a diffusibility. (B) Although the water-soluble fiber pieces are uniformly dispersed in the thin film before dropping the test aqueous droplet, the water-soluble fiber pieces are gradually dissolved in water when the test aqueous droplet is dropped to make the thin film transparent. It has the effect of rapidly diffusing the aqueous test solution by capillary action, and further serves as a scaffold when microorganisms present in the aqueous test solution grow. (B) Since the water-soluble fiber pieces are uniformly dispersed in the thin film, when the test aqueous solution is dropped, the test aqueous solution quickly diffuses into the thin film, and the thin film becomes transparent. Good colonies are easily formed when microorganisms in the aqueous liquid grow. Here, the term “transparent” means that 80% or more is transmitted when the light transmittance of the colorless and transparent glass with respect to light having a wavelength of about 400 to 700 nm is 100%.
（ｂ）水溶性繊維片は、被検水性液滴下時の透明性の点から、直径１〜１００μｍ、長さ０．１〜５ｍｍ、アスペクト比１０〜１５０の水溶性ポリマー繊維であるのが好ましい。さらに直径１〜５０μｍ、長さ０．１〜３ｍｍ、アスペクト比１０〜１５０が好ましく、さらに直径１〜５０μｍ、長さ０．１〜２ｍｍ、アスペクト比２０〜１００が好ましい。さらに好ましくは、直径１〜５０μｍ、長さ０．２〜０．８ｍｍ、アスペクト比２０〜１００のポリビニルアルコール系繊維片である。 (B) The water-soluble fiber piece is preferably a water-soluble polymer fiber having a diameter of 1 to 100 μm, a length of 0.1 to 5 mm, and an aspect ratio of 10 to 150 from the viewpoint of transparency when the test water droplet is dropped. . Further, a diameter of 1 to 50 μm, a length of 0.1 to 3 mm, and an aspect ratio of 10 to 150 are preferable, and a diameter of 1 to 50 μm, a length of 0.1 to 2 mm, and an aspect ratio of 20 to 100 are preferable. More preferably, it is a polyvinyl alcohol fiber piece having a diameter of 1 to 50 μm, a length of 0.2 to 0.8 mm, and an aspect ratio of 20 to 100.
（ｂ）水溶性繊維片の含有量は、検出しようとする微生物に対する栄養成分量が異なるため大きく異なるが、被検水性液滴下時の透明性及び拡散性の点から、薄膜中に測定時の濃度として０．１〜１０質量％が好ましく、０．１〜５質量％がより好ましく、０．１〜４質量％がさらに好ましい。 (B) The content of the water-soluble fiber pieces varies greatly because the amount of nutrient components for the microorganism to be detected is different, but from the point of transparency and diffusibility under the test aqueous droplet, As a density | concentration, 0.1-10 mass% is preferable, 0.1-5 mass% is more preferable, 0.1-4 mass% is further more preferable.
（ｃ）菌体栄養成分としては、検出しようとする微生物の生育に適し、水可溶性の成分であれば特に限定されないが、一般的な栄養培地成分が用いられる。そのような栄養成分としては、ペプトン、酵母エキス、ブドウ糖、無機塩等が挙げられる。 (C) The cell nutrient component is not particularly limited as long as it is suitable for the growth of the microorganism to be detected and is water-soluble, but a general nutrient medium component is used. Examples of such nutrient components include peptone, yeast extract, glucose, and inorganic salts.
前記薄膜中には、前記成分（ａ）〜（ｃ）が実質的に均一に分散しているのが、被検水性液滴下時の透明性の確保及び拡散性の点から好ましい。ここで実質的に均一に分散しているとは薄膜中に、前記成分が層を形成したりせずに、略均一に分散して含まれていることをいう。 In the thin film, it is preferable that the components (a) to (c) are substantially uniformly dispersed from the viewpoint of ensuring transparency and diffusibility when the aqueous droplet is to be tested. Here, “substantially uniformly dispersed” means that the component is contained in the thin film substantially uniformly without forming a layer.
本発明の簡易培地は、例えば成分（ａ）〜（ｃ）を含有するアルコール懸濁液を作製し、これを防水性平板上に分注し、均一に拡散、乾燥後、滅菌することにより製造することができる。アルコール懸濁液中の各成分の濃度は、各成分の薄膜中の濃度を考慮して決定すればよい。得られた簡易培地の表面は、フイルムで覆うか、フタをするのが好ましい。 The simple culture medium of the present invention is produced, for example, by preparing an alcohol suspension containing components (a) to (c), dispensing the suspension on a waterproof flat plate, uniformly diffusing, drying, and sterilizing. can do. The concentration of each component in the alcohol suspension may be determined in consideration of the concentration of each component in the thin film. The surface of the obtained simple medium is preferably covered with a film or covered.
本発明の簡易培地を用いて微生物を検出するには、薄膜上に被検水性液を滴下後培養し、該薄膜上に形成されるコロニーを検出すればよい。被検水性液を滴下すると、被検水性液が毛細管現象によって容易に拡散し、薄膜は透明になり、それに続いて水溶性高分子のゲル化が起こって、被検水性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、薄膜を観察すれば、形成したコロニーが容易に観察できる。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 medium of the present invention, a test aqueous solution is dropped on a thin film and cultured, and colonies formed on the thin film are detected. When the test aqueous solution is dropped, the test aqueous solution diffuses easily by capillary action, the thin film becomes transparent, and then gelation of the water-soluble polymer occurs, and microorganisms in the test aqueous solution are captured. Thus, free movement is suppressed and colonies are formed by culturing. Therefore, if the thin film is observed, the formed colonies can be easily observed. If a sample is inoculated quantitatively into a simple medium, the number of bacteria can be easily calculated by measuring the colonies that appear after the culture.
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.
実施例１
ペプトン１０ｇ、エキス末１０ｇ、ブドウ糖２ｇ、ピルビン酸ナトリウム２．５ｇ、塩化ナトリウム８ｇ、リン酸２水素カリウム３．６ｇ、リン酸水素２ナトリウム７．２ｇ、キサンタンガム７０ｇ、ポリビニルアルコール系繊維片（直径１３〜１５μｍ、長さ約０．５ｍｍ、アスペクト比３３〜３８）６．６ｇ及びヒドロキシプロピルセルロース２ｇをエタノール１．０Ｌに懸濁し良く攪拌した。このエタノール懸濁液を１．０ｍＬずつプラスチック製防水性平板（５０ｍｍφ）に分注し、均一に拡散・乾燥後、γ線で滅菌し簡易培地を調製した。
Example 1
Peptone 10 g, extract powder 10 g, glucose 2 g, sodium pyruvate 2.5 g, sodium chloride 8 g, potassium dihydrogen phosphate 3.6 g, disodium hydrogen phosphate 7.2 g, xanthan gum 70 g, polyvinyl alcohol fiber fragment (diameter 13 ˜15 μm, length of about 0.5 mm, aspect ratio 33-38) 6.6 g and hydroxypropylcellulose 2 g were suspended in 1.0 L of ethanol and stirred well. 1.0 ml of this ethanol suspension was dispensed onto a plastic waterproof flat plate (50 mmφ), uniformly spread and dried, and then sterilized with γ rays to prepare a simple medium.
大腸菌及び黄色ブドウ球菌をＳＣＤ寒天培地にて３５℃で２０時間培養後、滅菌生理食塩水にマックファーランド０．５（１．５×１０8ｃｆｕ／ｍＬ相当）に菌液を調製した。滅菌生理食塩水でマックファーランド０．５菌液より１０倍希釈系列を作製し、その１０-5〜１０-8希釈液のそれぞれ１ｍＬずつを本簡易培地に接種した。対照法として、１０-5〜１０-8希釈液のそれぞれ０．１ｍＬずつをＳＣＤ寒天培地に滴下し滅菌コンラージ棒にて培地全体に菌液を分散接種した。３５℃で２４時間培養後、それぞれの希釈段階における菌数を測定し、対照法については得られたコロニー数に１０を乗じて菌数とした。対照と比較したところ同等の発育性能を有していることが確認された。 Escherichia coli and Staphylococcus aureus were cultured on an SCD agar medium at 35 ° C. for 20 hours, and then a bacterial solution was prepared in a sterilized physiological saline to Macfarland 0.5 (equivalent to 1.5 × 10 8 cfu / mL). A 10-fold dilution series was prepared from a Macfarland 0.5 bacterial solution with sterile physiological saline, and 1 mL each of the 10 −5 to 10 −8 dilution was inoculated into this simple medium. As a control method, 0.1 mL of each of 10 −5 to 10 −8 dilutions was dropped onto an SCD agar medium, and the whole culture medium was dispersed and inoculated with a sterile conage rod. After culturing at 35 ° C. for 24 hours, the number of bacteria at each dilution stage was measured, and for the control method, the number of colonies obtained was multiplied by 10 to obtain the number of bacteria. Compared with the control, it was confirmed to have the same growth performance.
実施例２
実施例１における培地調製に準じ、ポリビニルアルコール系繊維片（直径１３〜１５μｍ）の繊維長及び添加量を変えて調製し、影響について検討した。
繊維長約０．５ｍｍ（アスペクト比３３〜３８）、約１ｍｍ（アスペクト比６７〜７７）、約３ｍｍ（アスペクト比２００〜２３０）の繊維片それぞれについて添加量を測定時濃度として１．７質量％、３．３質量％、６．６質量％になるように培地を調製した。
培地調製の可否、培地分注時培地液拡散性、乾燥後の生理食塩水の拡散性を評価した結果、アスペクト比１５０以下、好ましくはアスペクト比１００以下で培地調製が可能であった。
繊維長０．５ｍｍで繊維片添加量６．６質量％では懸濁物質が全体に行き渡らず、液性成分のみが周囲に拡散した。添加量が５質量％以下になると全体へ拡散した。
Example 2
According to the culture medium preparation in Example 1, it prepared by changing the fiber length and addition amount of a polyvinyl alcohol-type fiber piece (diameter 13-15 micrometers), and examined the influence.
The addition amount of each fiber piece having a fiber length of about 0.5 mm (aspect ratio of 33 to 38), about 1 mm (aspect ratio of 67 to 77), and about 3 mm (aspect ratio of 200 to 230) is 1.7% by mass. The culture medium was prepared so that it might become 3.3 mass% and 6.6 mass%.
As a result of evaluating the feasibility of medium preparation, medium liquid diffusibility during medium dispensing, and physiological saline after drying, medium preparation was possible with an aspect ratio of 150 or less, preferably an aspect ratio of 100 or less.
When the fiber length was 0.5 mm and the added amount of fiber pieces was 6.6% by mass, the suspended substance did not spread throughout, and only the liquid component diffused around. When the added amount was 5% by mass or less, it diffused throughout.
実施例３
実施例１における培地調製に準じ、ヒドロキシプロピルセルロースの濃度０．１質量％及び０．２質量％、培地液分注量を０．９ｍＬ、１ｍＬ、１．２ｍＬ、１．４ｍＬと変えた培地を調製し、培地液拡散性及び乾燥後の生理食塩水の拡散性についてへの影響について検討した。
ヒドロキシプロピルセルロースの濃度０．２質量％、培地分注量は１．２〜１．４ｍＬで良好に拡散した。
Example 3
According to the medium preparation in Example 1, the medium was changed to hydroxypropylcellulose concentrations of 0.1% by mass and 0.2% by mass, and the medium liquid dispensing amount was changed to 0.9 mL, 1 mL, 1.2 mL, and 1.4 mL. The effects on the diffusibility of the medium solution diffusivity and the physiological saline after drying were examined.
The concentration of hydroxypropylcellulose was 0.2% by mass, and the amount of medium dispensed was 1.2 to 1.4 mL.
比較例１
実施例１における培地調製に準じ、ポリビニルアルコール系繊維片を添加せずに培地を調製し、分注、乾燥後、γ線滅菌した。
得られた簡易培地に、生理食塩水を滴下したところ拡散性が悪く、全面に均一に拡散しなかった（図３）。
Comparative Example 1
In accordance with the medium preparation in Example 1, a medium was prepared without adding polyvinyl alcohol fiber pieces, dispensed, dried, and sterilized with γ rays.
When physiological saline was added dropwise to the obtained simple medium, the diffusibility was poor and it did not diffuse uniformly over the entire surface (FIG. 3).
Claims (8)
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Citations (5)
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|---|---|---|---|---|
| JPS55127985A (en) * | 1979-03-22 | 1980-10-03 | Yoshitomi Pharmaceut Ind Ltd | Thin layer culture medium |
| JPH08286A (en) * | 1994-04-20 | 1996-01-09 | Nissui Pharm Co Ltd | Filmy medium and detection of microorganism using the medium |
| JPH08140664A (en) * | 1994-11-18 | 1996-06-04 | Nissui Pharm Co Ltd | Simple culture medium |
| JP3148250B2 (en) * | 1995-12-27 | 2001-03-19 | チッソ株式会社 | Microbial culture equipment and medium |
| JP2001275651A (en) * | 2000-04-03 | 2001-10-09 | Chisso Corp | Sheet-like microorganism incubator and culture medium using the same |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS55127985A (en) * | 1979-03-22 | 1980-10-03 | Yoshitomi Pharmaceut Ind Ltd | Thin layer culture medium |
| JPH08286A (en) * | 1994-04-20 | 1996-01-09 | Nissui Pharm Co Ltd | Filmy medium and detection of microorganism using the medium |
| JPH08140664A (en) * | 1994-11-18 | 1996-06-04 | Nissui Pharm Co Ltd | Simple culture medium |
| JP3148250B2 (en) * | 1995-12-27 | 2001-03-19 | チッソ株式会社 | Microbial culture equipment and medium |
| JP2001275651A (en) * | 2000-04-03 | 2001-10-09 | Chisso Corp | Sheet-like microorganism incubator and culture medium using the same |
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| JPN6016005774; 繊維学会誌， １９８７，Ｖｏｌ． ４３， Ｎｏ． １０， Ｐ-４０１ * |
| JPN6016005776; 繊維学会誌， １９８７， Ｖｏｌ． ４３， Ｎｏ． ５， ｐｐ． ２７１-２７４ * |
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