JP3813410B2 - Liquid culture medium for microbial culture containing coloring indicator and method for producing the same - Google Patents
Liquid culture medium for microbial culture containing coloring indicator and method for producing the same Download PDFInfo
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Description
【０００１】
【発明の属する技術分野】
この発明は主として、臨床微生物検査で培養した微生物の存在の有無を他の夾雑物から明確に分離可能な液体培地及びその液体培地の製法を市場に提供することである。
【０００２】
【従来の技術】
従来臨床微生物検査、特に抗酸菌検出のための分離培地としては、液体培地はその有用性が知られていた。
しかし、液体培地での微生物増殖確認の方法は、目視的で微生物増殖による濁度の増加や沈殿の増加で判断していた。しかし、臨床検体は夾雑物が多く、これらが目的の微生物の増殖との区別がつかず、なかなか実用にならなかった。
近年これらを解決するため、培地容器の底に酸素に鋭敏な蛍光色素をコ−ティングしておき、微生物の増殖により培地内の酸素が消費されることにより、蛍光を発し微生物増殖を検出する方法も知られている。
他方、炭酸ガスに反応する色素を特殊な隔壁で培地に接触させておき、微生物の増殖により排出される炭酸ガスを検知して、その目的を果たせる方法等が発表されている。
これらの方法は微生物そのものを捕らえる方法ではなく、微生物の増殖による培地の変化で間接的に検出する方法である。
また、増殖微生物中より目的の微生物を他の夾雑物から分離して検出する方法として、結核菌に対して増殖抑制的に作用する酸化還元色素を用い、特殊な栄養素を加える方法により、その抑制的作用を減殺させ、前記色素を微生物に取り込ませ、微生物中の還元酵素の作用で還元させて、赤黒く発色させ、微生物の繁殖に伴い、発色した微生物が多くなることで、増殖確認をする方法等が発表されている。
【０００３】
【発明が解決しようとする課題】
前述の先行技術の方法はそれなりに評価されているが、増殖確認のために蛍光色素には紫外線発光器が必要であり、炭酸ガス検出では微細な変化を読み取るための特殊な機器類が必要である。
また酸化還元色素を用いた方法は目視検査が可能ではあるが、発色が黒赤色であるため、初期段階では微生物として確認しづらく、更に培地に特殊栄養素を添加するため、培地の色が褐色となっており、増殖判定がしづらい欠点があった。従って、液体培地を分離培地として活用するには、特殊な機器を必要とせず、かつ酸化還元色素或いは胆汁酸を用いても、菌増殖抑制作用が少なく、増殖の初期段階から明確に微生物の増殖が認識できるインジケータ付きの液体培地及びその製造方法を市場に提供することである。
【０００４】
【課題を解決するための手段】
前記の課題を達成するために、この発明は微生物培養用液体培地４ミリリットルに対し、酸化還元色素を５乃至４０マイクログラム／ミリリットルと吸着剤を０．０２乃至５グラムが混合乃至敷き込んであることを特徴とする着色インジケータを含有する微生物培養用液体培地とする。
【０００５】
また、前記の課題を達成するために、この発明の着色インジケータを含有する微生物培養用液体培地の前記酸化還元色素としては、ＳＴＣ、β−ＳＴＣ、ＯＴＣ、β−ＯＴＣ、α−ＰＴＣ、β−ＰＴＣ、γ−ＰＴＣ、ＭＤＴ及びＴＴＣから選択される少なくとも１種の色素であることを特徴とすることが好ましい。
【０００６】
また、前記の課題を達成するために、この発明は微生物培養用液体培地４ミリリットルに対し、ニュ−トラルレッド、メチレンブル−から選択される少なくとも１種の胆汁酸結合沈着色素５乃至４０マイクログラム／ミリリットル及び胆汁酸、胆汁酸塩のうちの一種の添加剤を胆汁酸結合沈着色素に対し０．１乃至１％、並びに吸着剤を０．０２乃至０．１５グラムが混合してあることを特徴とする請求項１記載の着色インジケータを含有する微生物培養用液体培地する場合もある。
【０００７】
また、前記の課題を達成するために、この発明の着色インジケータを含有する微生物培養用液体培地の前記吸着剤としてはシリカゲル、アルミナ、活性炭粉末、濾紙、変性動物性蛋白質、有機化合物系吸収剤のうちの一種であることを特徴とすることが好ましい。
【０００８】
また、前記の課題を達成するために、関連の方法発明として培養容器中の微生物培養用液体培地４ミリリットルに対し、酸化還元色素を５乃至４０マイクログラム／ミリリットルを加え、これと同時乃至順次、前記色素で着色される微生物の色彩と明確に識別し得る色彩の吸着剤を０．０２乃至５グラム加えることを特徴とする着色インジケ−タを含有する微生物培養用液体培地の製造方法とする。
【０００９】
また、前記の課題を達成するために、関連の方法発明として培養容器中の微生物培養用液体培地４ミリリットルに対し、ニュ−トラルレッド、メチレンブル−から選択される少なくとも１種の胆汁酸結合沈着色素５乃至４０マイクログラム／ミリリットル及び胆汁酸、胆汁酸塩のうちの一種の添加剤を胆汁酸結合沈着色素に対し０．１乃至１％、並びに吸着剤を０．０２乃至０．１５グラムを加えることを特徴とする場合もある。
【００１０】
また、前記の課題を達成するために、関連の方法発明として、培養容器中の底に白色乃至乳白色の吸着剤たる濾紙を適当な接着剤で貼付させ、その後、微生物培養用液体培地４ミリリットルに対し、酸化還元色素を５乃至４０マイクログラム／ミリリットルを順次乃至同時に注入することを特徴とする場合も有る。
【００１１】
また、前記の課題を達成するために、関連の方法発明として培養容器中の底に白色乃至乳白色の吸着剤よりなる濾紙を適当な接着剤で貼付させ、その後微生物培養用液体培地４ミリリットルに対し、ニュ−トラルレッド、メチレンブル−から選択される少なくとも１種の胆汁酸結合沈着色素５乃至４０マイクログラム／ミリリットル及び胆汁酸、胆汁酸塩のうちの一種の添加剤を胆汁酸結合沈着色素に対し０．１乃至１％を順次乃至同時に注入することを特徴とする着色インジケータを含有する微生物培養用液体培地の製造方法とする場合もある。
【００１２】
また、前記の課題を達成するために、関連の方法発明として、培養容器中の底に全卵乃至動物血清を前記容器底面が充分に覆われる程度に注入し、これを加熱して、変性反蛋白質として吸着剤とし、その後微生物培養用液体培地４ミリリットルに対し、ニュ−トラルレッド、メチレンブル−から選択される少なくとも1種の胆汁酸結合沈着色素５乃至４０マイクログラム／ミリリットル及び胆汁酸、胆汁酸塩のうちの一種の添加剤を胆汁酸結合沈着色素に対し０．１乃至１％を順次乃至同時に注入することを特徴とする着色インジケータを含有する微生物培養用液体培地の製造方法とする。
【００１３】
発明の作用
先ず、請求項５、６、７、８又は９記載の発明の製造方法により、請求項１、２、３又は４記載の発明の培地を製造する。
検体としては、還元酵素を含む菌、代表例としては抗酸菌であり、これをを含むかどうかの判定をするため、臨床微生物検査においては、喀痰に蛋白質均等化剤を含む２％水酸化ナトリウム（ＮＡＬＣ−Ｎａ０Ｈまたはチェックスクリア［出願人会社所有登録商標］）を当量以上加え、喀痰を均質化すると共に、終末１％の水酸化ナトリウムで、前記喀痰中に存在する抗酸菌以外の微生物を殺菌する。
これをｐＨ７のリン酸緩衝液で１０倍程度に稀釈し、水酸化ナトリウムの強アルカリを中和する。
これを３０００Ｇの遠心力分離機にかけ、沈渣物上澄液とを分離し、沈渣物に当量のリン酸緩衝液を加え懸濁液を作り、これを検体とする。
【００１４】
次に、この検体をこの発明の請求項１、２、３または４記載の発明の培地の上に接種し、公知の手法により培養を行うと、検体中に目的の菌が存在すれば、前記の培地は通常の液体培地と同等の増殖支持能を持ち、前記白色または黒色の吸着剤とは異なる色彩に着色されて、培養容器の底に沈殿する。前記吸着剤の上に沈殿し、微細な発育も周囲とは歴然と異なる色で発色した集落が形成され、早目に目的の菌の確認ができる。
【００１５】
請求項３記載の発明に於いては、培地自体は色素の色彩で淡く着色されているが、培地中において、菌が増殖されると、その菌体内に前記色素及び胆汁酸、または胆汁酸塩が取り込まれ、菌体内でこれらが反応して、色素が固定され、菌が色素色を呈し、沈殿して集落を形成した場合、確実に周囲と識別可能な色彩乃至濃度となり、判別可能となる。
【００１６】
請求項４記載の発明においては、菌の増殖支持能の維持は勿論の事、吸着剤として、前記のものが用いられ、殊に活性炭を用いたものは培養容器の底にこれが沈殿して黒色となり、菌体に取り込まれた色と異なる色彩となり、菌体の集落と明確に判別できる。
また他の吸着剤のときは同様に培養容器の底が白色乃至淡黄色となるため、菌の集落とのコントラストが鮮明となり、この菌の増殖の判別が容易となる。尚、吸着剤として、活性炭を用いる場合は濾紙などで表面を覆って用いれば、白色の吸着剤を用いたときと同様に菌の増殖が判別し易い。
【００１７】
また請求項５記載の方法においては、請求項１、２又は４記載の発明の着色インジケ−タを含有する微生物培養用液体培地が製造できる。
また請求項６記載の方法においては、請求項３、４記載の発明の着色インジケ−タを含有する微生物培養用液体培地が製造できる。
【００１８】
また請求項７、８又は９記載の方法においては、請求項１、２又は４記載の発明一種若しくは請求項３又は４記載の発明のの着色インジケータを含有する微生物培養用液体培地が製造でき、特に培養容器内の濾紙又は、変性動物蛋白質は移動せず、反応が見やすいものが製造できる。
これら請求項１乃至４記載の発明のうちの、一種の培地で培養した前記集落を必要に応じて取り出し、抗酸菌性染色を行い、染色されれば抗酸菌検出となる。抗酸菌が存在しなければ、前記着色集落もなく、反応はマイナスとなる。
【００１９】
【実施の形態】
実施の形態１
請求項１、２及び４記載の発明を含む実施の形態である。
１．培地
培地としては、ブイヨンなどで代表される液体培地であれば特に制限はない。
２．色素
酸化還元色素、
略記号 化学名
ＳＴＣ、 2.3-ジフェニル-5-チエニル-(2)-テトラゾリウム クロライド
(2,3-Diphenyl-5-thienyl-(2)-tetrazolium Chloride)
β−ＳＴＣ、2.3-ジフェニル-5-チエニル-(3)-テトラゾリウム クロライド
(2,3-Diphenyl-5-thienyl-(3)-tetrazolium Chloride)
ＯＴＣ、 2.3-ジフェニルー5-フロイル-(2)-テトラゾリウム クロライド
(2,3-Diphenyl-5-furoyl-(2)-tetorazolium Chloride)
β−ＯＴＣ、2.3-ジフェニル-5-フロイル-(3)-テトラゾリウム クロライド
(2,3-Diphenyl-5-furoyl-(3)-tetrazolium Chloride)
α−ＰＴＣ、2.3-ジフェニル-5-ピリジル-(2)-テトラゾリウム クロライド
(2,3-Diphenyl-5-pyridyl-(2)-tetrazolium Chloride)
β−ＰＴＣ、2.3-ジフェニル-5-ピリジル-(3)-テトラゾリウム クロライド
(2,3-Diphenyl-5-pyridyl-(3)-tetrazolium Chloride)
γ−ＰＴＣ、2.3-ジフェニル-5-ピリジル-(4)-テトラゾリウム クロライド
(2,3-Diphenyl-5-pyridyl-(4)-tetrazolium Chjoride)
ＭＤＴ 2.3-ジフェニル-5-メチル テトラゾリウム クロライド
(2,3-Diphenyl-5-methyl tetrazolium Chloride)
ＴＴＣ 2,3,5-トリフェニル テトラゾリウム クロライド
(2,3,5-triphenyl tetrazolium Chloride)
など、
以上の色素より適宜一種の色素を選択する。
３． 吸着剤
シリカゲル
アルミナ
活性炭粉末
微生物培養用液体培地４ミリリットルに対し、前記の酸化還元色素のうち一種を選択し、これを５乃至４０マイクログラム／ミリリットルと前記吸着剤のうちの一種を０．０２乃至０．１５グラムが混合して目的の着色インジケータを含有する微生物培養用液体培地としたものである。
【００２０】
実施の形態２
請求項３および４記載の発明を含む実施の形態である。
１． 培地としては、実施の形態１と同じものを用いる。
２． 色素及び添加剤
１）胆汁酸結合沈着色素
ニュ−トラルレッド
メチレンブル−
のうちの一種を選択する。
２） 添加剤
胆汁酸、胆汁酸塩のうちの一種を用いる。
３． 吸着剤
実施の形態１と同じ吸着剤より、一種を選択して用いる。
前記の微生物培養用液体培地４ミリリットルに対し、前記色素及び添加剤のうちより、ニュ−トラルレッド、メチレンブル−から選択される少なくとも1種の胆汁酸結合沈着色素５乃至４０マイクログラム／ミリリットル及び胆汁酸、胆汁酸塩のうちの一種の添加剤を胆汁酸結合沈着色素に対し０．１乃至１％、並びに吸着剤を０．０２乃至０．１５グラムを混合して目的の着色インジケータを含有する微生物培養用液体培地としてある。
実施の形態1及び２において、吸着剤として、シリカゲル、アルミナを用いる場合は、予め粉末乃至顆粒物であることを前提として説明したが、これら材料よりなる吸着剤においては、予め板状に加工してあるものを敷き込んで、この後上記の培地及び色素と添加剤を注入したものも、この発明の実施の態様に含まれる。
【００２１】
実施の形態３
請求項１、２、４記載の発明を含む実施の形態である。吸着剤として、濾紙
を用い、これにが培養容器の底に貼付してあり、この上に実施の形態１の色素の一種と液体培地を、前記と同様の割合で加えて、目的の着色インジケータを含有する微生物培養用液体培地としたものである。
或いは、前記濾紙に予め還元色素を前記の割合でしみ込ませた後、これを培養容器内の底に貼り付け、この上に実施の形態１に用いた液体培地を注入したものもこの実施の形態の範囲である。或いは濾紙を貼付後、酸化還元色素を先ず濾紙にしみ込ませ、後、液体培地を注入しても、この発明の実施の形態に含まれる。前記濾紙の乾燥状態の重量は、培養容器の底の面積にもよるが、０．０２乃至０．１グラムである。
【００２２】
実施の形態４
請求項３及び４記載の発明を含むの実施の形態である。吸着剤として、濾紙を用い、これに培養容器の底に貼付してあり、この上に実施の形態２の色素及び添加剤のそれぞれ一種と液体培地を加えて、目的の着色インジケータを含有する微生物培養用液体培地としたものである。
この場合も予め濾紙に胆汁酸結合沈着色素及び胆汁酸若しくは胆汁酸塩をしみ込ませたものを培養容器内の底に貼付し、この上より液体培地を注入したものもこの実施の形態に含まれる。或いは濾紙を貼付後、これに胆汁酸結合沈着色素と添加剤を先ず濾紙にしみ込ませ、後液体培地を注入してもこの実施の形態に含まれる。
実施の形態３及び４においては、濾紙の間に活性炭をサンドイッチ状に挟みこんだものも、これらの実施の形態に含まれる。
【００２３】
製造方法の実施の形態
実施の形態５
請求項５記載の製造方法の実施の形態であり、実施の形態１のものを製造する方法であり、実施の形態１の組成の各材料を任意の順序で順次乃至同時に培養容器に入れ、混合して着色インジケータを含有する微生物培養用液体培地を製造する。
この方法において、培養容器以外の容器で予め前記の材料を混合しておき、或いは混合液を予め、他の容器に密封して置いたものを、一気に培養容器に注入しても、請求項５記載の製造方法の実施の形態である。
【００２４】
実施の形態６
請求項６記載の製造方法の実施の形態であり、実施の形態２のものを製造する方法であり、実施の形態２の組成の各材料を任意の順序で順次乃至同時に培養容器に入れ、混合して着色インジケータを含有する微生物培養用液体培地を製造する。
この方法において、培養容器以外の容器で予め前記の材料を混合しておき、これを一気に培養容器に注入しても、請求項６記載の製造方法の実施の形態である。
【００２５】
実施の形態７
請求項７記載の製造方法の実施の形態であり、実施の形態３のものを製造する方法であり、先ず培養容器１内の底全面に、白色乃至乳白色の濾紙をシリコン系接着剤、アクリル系乃至シリコン系粘着剤で貼り付ける。次いで実施の形態１の組成から、吸着剤を除いたものと同じ組成のものを、この濾紙の上に注入する。この実施の形態において、予め前記濾紙の酸化還元色素をしみ込ませておき、これを培養容器内の底に貼付け、或いは濾紙を貼付後、先ず酸化還元色素をこの濾紙にしみ込ませ、その後液体培地のみを注入する方法もこの発明の実施の形態に含まれる。
【００２６】
実施の形態８
請求項８記載の製造方法の実施の形態であり、実施の形態４のものを製造する方法であり、先ず培養容器内の底全面に、実施の形態７と同様に白色乃至乳白色の濾紙をシリコン系接着剤、アクリル系乃至シリコン系粘着剤で濾紙を貼り付ける。次いで実施の形態２の組成から、吸着剤を除いたものと同じ組成のものを、この濾紙の上に注入する。
この実施の形態８において、濾紙を培養容器内の底に貼付する前に胆汁酸結合沈着色素及び前記添加剤をしみ込ませたものを用い、或いは濾紙を貼付後、先ず胆汁酸結合沈着色素及び前記添加剤をしみ込ませた後、液体培地のみを注入する方法もこの実施の形態に含まれる。
【００２７】
実施形態９
請求項9記載の発明の実施の形態であり、先ず、吸着剤として使用する鶏卵の全卵をよく攪拌し、培養容器の底全面を覆うに充分な量注入する。通常その深さは１ｍｍ乃至３ｍｍとなるが、この深さ自体に制限はない。卵蛋白質の場合使用される量は培養容器の底の面積にもよるが、０．１０乃至１．５グラムあれば通常は充分であるが、５グラム以上用いても、この発明の方法の実施の形態に含まれる。
次に、この培養容器の底を水平にしたまま加熱し、前記卵を変性蛋白質とする。つまり固化させ、吸着剤層を形成する。
この上に、前記実施の形態８と同様に実施の形態２の組成から吸着剤を除いたものと同じ組成のものを、この変性蛋白質層の上に注入する。
前記の全卵では吸着剤層が黄色になり、酸化還元色素又は胆汁酸結合沈着色素との識別が困難なときは、吸着剤として、卵白のみを用いても、この方法発明の実施の形態に含まれる。
また、吸着剤として、動物血清を用いこれを卵を用いたときと同様に注入し、過熱して吸着剤層を形成する方法も、請求項9記載の発明の実施の形態である。
これらの方法により製造されたものは、請求項4記載の発明の培地である。
【００２８】
実施の形態１０
吸着剤として、有機化合物系吸着剤として、疎水性クロマトの担体、イオン交換樹脂のうちの一種を培養容器に敷き込み、吸着材層を形成する。
イオン交換樹脂は通常顆粒状であるから、培養容器の底が平坦な場合は、前記顆粒物が、２乃至３粒層になる程度、培養容器が試験管の場合は、底の半球状の部分が埋まる程度の量とする。
この上に実施の形態２の組成から、吸着剤を除いたものと同じ組成のものを前記の吸着剤の上に注入する。
この方法により製造された培地は請求項４記載の発明の実施の形態となる。
【００２９】
【発明の効果】
請求項１記載の発明の培地は前記の組成としたために、酸化還元色素が混入してあっても、菌の増殖に対する抑制作用が従来の培地と比較し極端に低下し、その増殖支持能は従来の酸化還元色素剤の混入していない微生物培養用液体培地と同等であり、菌の増殖と共に酸化還元色素は菌体に取り込まれ、菌体内の還元酵素と反応し、菌体は確実に発色し、微細な発育も培地容器の底部に沈殿する吸着剤と歴然と異なる色で発色し、早目に確認できる効果を奏する。
前記請求項２記載の発明の培地には前記酸化還元色素としては、ＳＴＣ、β−ＳＴＣ、ＯＴＣ、β−ＯＴＣ、α−ＰＴＣ、β−ＰＴＣ、γ−ＰＴＣ、ＭＤＴ及びＴＴＣから選択される少なくとも１種の色素が混入してあるから、菌の増殖に従いこれら色素は還元酵素を持っている菌体内に取り込まれ、確実に赤発色する効果を奏する。
【００３０】
前記請求項３記載の発明の培地のうちニュ−トラルレッドが混合してある場合は請求項２記載の発明と同様に菌は増殖と共に赤色に発色する効果を奏する。
前記請求項３記載の発明の培地のうちメチレンブル−が混入してあるものは、菌は増殖と共に青色に発色する効果を奏する。この場合も増殖支持能は何ら低下しない。
殊に、添加剤として、胆汁酸、胆汁酸塩の一種が混入してあるから、菌体が繁殖し、前記胆汁酸結合沈着色素と共に、菌体に取り込まれたとき、菌体内において、前記胆汁酸結合沈着色素は固定され、再び菌体外に溶出せず、確実に菌体を着色し、菌体が繁殖し集落を形成したとき、容易にその繁殖を認識できる。
【００３１】
請求項４記載の発明の培地は、前記の請求項１、２又は３記載の発明の効果の他、吸着剤としてはシリカゲル、アルミナ、混入してあるものは容器の底にこれらが沈殿し、若しくは白色乃至乳白色の濾紙を培地容器内の底に沈設乃至貼付けしたものは、この上で増殖する菌を確実に増殖させ、かつその色彩が白色であるため、色素が赤若しくは青と歴然とし、その増殖の初期段階から観測できる効果を奏する。
吸着剤が活性炭で或る場合は、培養容器底面部は黒色となり、菌の繁殖による前記色彩とは歴然と異なり、容易に判別できる効果を奏する。前記活性炭を用いこれを濾紙で覆うようにして、２種吸着剤を用いた場合は白色の吸着剤を用いた場合と同様に菌の集落とのコントラストがよく、判別し易い。
【００３２】
請求項５記載の製造方法の発明としては、前記請求項１又は記載の着色インジケ−タ付きの微生物培養用液体培地の組成要素を任意の順序で順次乃至一時に混合すれば、前記請求項１記載の着色インジケ−タ付きの微生物培養用液体培地が製造でき、検査現場での製造が簡単にできる。
請求項６記載の製造方法の発明としては、前記請求項３又は４記載の着色インジケ−タ付きの微生物培養用液体培地の組成要素を任意の順序で順次乃至一時に嵌合すれば、前記請求項３又は４記載の着色インジケ−タ付きの微生物培養用液体培地が製造でき、検査現場での製造が簡単にできる。
【００３３】
請求項７又は８記載の方法発明においては、吸着剤たる濾紙を予め培養容器内の底面に貼付る方法であり、簡単であると共に、吸着剤が底面全体に確実に位置したものが製造できるから、これを使用して菌の培養をすると、菌の繁殖時の繁殖抑制効果が菌体の近傍で作用し、その効果をより確実とし、染色された菌とのコントラストにより、菌の繁殖の初期段階から明確に認識できる着色インジケ−タ付きの微生物培養用液体培地が製造できる。
請求項9記載の方法においては、吸着剤が培養容器の底部に固化安定し、反応像が滲まず、安定するものが得られる。
【００３４】
実験例
液体培地として、抗酸菌培養用の液体培地としてミドルブルック７Ｈ９を４ミリリットルに対して、インジケ−タとなる酸化還元色素として前記ＳＴＣを２０マイクログラム／ミリリットル入れる。更にシリカゲル０．１０グラム入れたものを本件実施例とする。対照培地として通常の液体培地としてミドルブルック７Ｈ９を用い、比較例として本件発明の構成要素の一つたる前記シリカゲルを除いたもの及び、前記ミドルブルック７Ｈ９に前記ＳＴＣを２０マイクログラム／ミリリットルと栄養素を加えたものを、菌液濃度は１０-6ミリグラム/ミリリットルにおいて、それぞれ２週間培養の実験をした。その結果は下記の通りである。
【００３５】
【表１】
本件発明の実施例と対照のミドルブルック７Ｈ９のみとは１０-6ミリグラム／ミリリットルの菌の接種した培地まで同程度の日数（２週間）で、同程度の菌の増殖が見られたが、シリカゲルを添加しなかった例においては、菌の増殖は見られなかった。
また本件発明の実施品においては、菌の増殖は鮮明な赤色を呈し、沈殿した白色のシリカゲルと明確に識別され、充分なインジケ−タの機能を発揮することが確認できた。
【００３７】
その他、酸化還元色素及び胆汁酸結合沈着色素が前記範囲以下の場合は着色が不充分であり、その範囲を越えるに従い、菌の発育抑制効果が生じる。
また吸着剤として、前記の範囲内の量が好ましく、この範囲より少ないと、菌増殖支持能が充分に得られないし、培養容器の底は全面を覆う量として少なく、前記範囲を越えると無駄な量となる。
また胆汁酸、胆汁酸塩の量も前記範囲以下の場合は、着色効果が充分ではないし、範囲を越える場合は菌の発育抑制作用を為し、好ましくない。[0001]
BACKGROUND OF THE INVENTION
This invention mainly provides the market with a liquid medium capable of clearly separating the presence or absence of microorganisms cultured in a clinical microorganism test from other contaminants and a method for producing the liquid medium.
[0002]
[Prior art]
Conventionally, the usefulness of a liquid medium has been known as a separation medium for clinical microbiological examination, particularly for detection of acid-fast bacteria.
However, the method for confirming the growth of microorganisms in a liquid medium is visually judged based on an increase in turbidity and an increase in precipitation due to the growth of microorganisms. However, clinical specimens are often contaminated, and these are indistinguishable from the growth of the target microorganisms, so it has not been practical.
In order to solve these problems in recent years, a method of detecting fluorescence by emitting fluorescence by coating a fluorescent dye sensitive to oxygen on the bottom of a medium container and consuming oxygen in the medium due to the growth of microorganisms. Is also known.
On the other hand, a method has been announced in which a pigment that reacts with carbon dioxide gas is brought into contact with the culture medium through a special partition, and the carbon dioxide gas discharged by the growth of microorganisms is detected to achieve its purpose.
These methods are not a method of capturing microorganisms themselves, but a method of detecting indirectly by a change in the medium due to the growth of microorganisms.
In addition, as a method for detecting the target microorganism from other microorganisms in the growing microorganism, it is possible to suppress it by adding a special nutrient using a redox dye that acts to inhibit the growth of Mycobacterium tuberculosis. Method of confirming growth by reducing the natural action, incorporating the dye into the microorganism, reducing it by the action of the reductase in the microorganism, causing the color to turn red and black, and the number of the colored microorganism increases as the microorganism propagates Etc. have been announced.
[0003]
[Problems to be solved by the invention]
Although the above prior art methods have been evaluated as such, fluorescent dyes require ultraviolet light emitters for growth confirmation, and carbon dioxide gas detection requires special equipment to read minute changes. is there.
In addition, the method using redox dyes can be visually inspected, but since the color development is black-red, it is difficult to confirm as microorganisms at the initial stage, and since the special nutrient is added to the medium, the color of the medium is brown. Therefore, there is a drawback that it is difficult to determine proliferation. Therefore, in order to use a liquid medium as a separation medium, no special equipment is required, and even when redox dyes or bile acids are used, the growth of microorganisms is clearly increased from the initial stage of growth with little effect on inhibiting bacterial growth. Provides a liquid medium with an indicator that can be recognized by the market and a method for producing the same.
[0004]
[Means for Solving the Problems]
In order to achieve the above object, according to the present invention, 5 to 40 micrograms / milliliter of redox dye and 0.02 to 5 grams of adsorbent are mixed or spread with respect to 4 milliliters of a liquid culture medium for microbial culture. A liquid culture medium for microbial culture containing a colored indicator is provided.
[0005]
In order to achieve the above object, as the redox dye of the liquid culture medium for microorganism culture containing the coloring indicator of the present invention, STC, β-STC, OTC, β-OTC, α-PTC, β- It is preferably characterized by being at least one dye selected from PTC, γ-PTC, MDT and TTC.
[0006]
In order to achieve the above object, according to the present invention, 4 ml of a liquid culture medium for microbial culture is used, and at least one bile acid bond-deposited pigment selected from neutral red and methylene blue is 5 to 40 microgram / 0.1 to 1% of an additive of milliliters and bile acids and bile salts with respect to bile acid-binding pigments and 0.02 to 0.15 grams of adsorbent are mixed. In some cases, a liquid medium for culturing microorganisms containing the colored indicator according to claim 1 is used.
[0007]
In order to achieve the above object, the adsorbent of the liquid culture medium for microorganism cultivation containing the colored indicator of the present invention includes silica gel, alumina, activated carbon powder, filter paper, denatured animal protein, and organic compound-based absorbent. It is preferable that it is one of them.
[0008]
In order to achieve the above-mentioned problem, as a related method invention, 5 to 40 microgram / ml of redox dye is added to 4 ml of the liquid culture medium for culturing microorganisms in the culture vessel, and simultaneously or sequentially, A method for producing a liquid medium for culturing microorganisms containing a colored indicator, characterized in that 0.02 to 5 grams of an adsorbent having a color that can be clearly distinguished from the color of microorganisms colored with the pigment is added.
[0009]
In order to achieve the above-mentioned object, as a related method invention, at least one bile acid-binding pigment selected from neutral red and methylene blue is used per 4 ml of a liquid culture medium for culturing microorganisms in a culture vessel. Add 5 to 40 micrograms / milliliter and bile acid, one of bile salts, 0.1 to 1% of bile acid binding pigment, and 0.02 to 0.15 grams of adsorbent. It may be characterized by this.
[0010]
In order to achieve the above-mentioned problem, as a related method invention, a filter paper as a white or milky white adsorbent is attached to the bottom of a culture vessel with an appropriate adhesive, and then the liquid culture medium for microbial culture is added to 4 ml. On the other hand, there may be a case where 5 to 40 micrograms / milliliter of redox dye is injected sequentially or simultaneously.
[0011]
In order to achieve the above object, as a related method invention, a filter paper made of a white or milky white adsorbent is attached to the bottom of a culture vessel with an appropriate adhesive, and thereafter, to 4 ml of a liquid culture medium for microbial culture. 5 to 40 micrograms / milliliter of at least one bile acid-binding pigment selected from neutral red and methylene blue, and one additive of bile acid and bile salt for bile acid-binding pigment In some cases, the method may be a method for producing a liquid culture medium for microbial culture containing a colored indicator, wherein 0.1 to 1% is injected sequentially or simultaneously.
[0012]
In order to achieve the above-mentioned problem, as a related method invention, whole egg or animal serum is injected into the bottom of the culture container to such an extent that the bottom of the container is sufficiently covered, and heated to Adsorbent as protein, then 4 ml of liquid culture medium for microorganism culture, 5 to 40 microgram / ml of bile acid-binding pigment selected from neutral red and methylene blue, bile acid, bile acid A method for producing a liquid medium for cultivating a microorganism comprising a color indicator, wherein one to one of salts is sequentially or simultaneously injected at 0.1 to 1% of a bile acid-binding pigment.
[0013]
First, the medium of the invention of claim 1, 2, 3, or 4 is produced by the production method of the invention of claim 5, 6, 7, 8, or 9.
The specimen is a bacterium containing a reductase, a representative example is an acid-fast bacterium, and in order to determine whether or not it contains a 2% hydroxylated hydrate containing a protein equalizing agent in the sputum Sodium (NALC-Na0H or Check Clear (registered trademark owned by the applicant company)) is added in an equivalent amount to homogenize the soot, and with 1% sodium hydroxide at the end, other than the acid-fast bacteria present in the soot Sterilize microorganisms.
This is diluted about 10 times with a pH 7 phosphate buffer to neutralize the strong alkali of sodium hydroxide.
This is subjected to a 3000 G centrifugal separator to separate the sediment supernatant, and an equivalent phosphate buffer is added to the sediment to form a suspension, which is used as a specimen.
[0014]
Next, when this specimen is inoculated on the medium of the invention according to claim 1, 2, 3 or 4 of the present invention and cultured by a known technique, if the target bacteria are present in the specimen, The medium has a growth supporting ability equivalent to that of a normal liquid medium, is colored in a color different from that of the white or black adsorbent, and settles on the bottom of the culture vessel. A settlement that settles on the adsorbent and develops a fine growth with a color that is clearly different from the surroundings is formed, so that the target bacteria can be confirmed quickly.
[0015]
In the invention of claim 3, the medium itself is lightly colored with the color of the pigment. However, when the bacteria are grown in the medium, the pigment and bile acids or bile salts are contained in the cells. Is taken up and reacts in the cells, the pigment is fixed, and the bacteria exhibit a pigment color and settles to form a colony. .
[0016]
In the invention of claim 4, the above-mentioned adsorbent is used as well as maintaining the ability to support the growth of bacteria, and in particular, the one using activated charcoal is precipitated on the bottom of the culture vessel and becomes black. Thus, it becomes a color different from the color taken in by the cells and can be clearly distinguished from the colonies of the cells.
Similarly, in the case of other adsorbents, the bottom of the culture vessel becomes white to light yellow, so that the contrast with the colonies of the bacteria becomes clear, and the growth of the bacteria is easily discriminated. When activated carbon is used as the adsorbent, if the surface is covered with a filter paper or the like, the growth of bacteria can be easily discriminated as in the case of using the white adsorbent.
[0017]
Moreover, in the method of Claim 5, the liquid culture medium for microorganism culture | cultivation containing the colored indicator of invention of Claim 1, 2 or 4 can be manufactured.
Moreover, in the method of Claim 6, the liquid culture medium for microorganisms culture | cultivation containing the colored indicator of the invention of Claims 3 and 4 can be manufactured.
[0018]
Moreover, in the method of Claim 7, 8 or 9, the liquid culture medium for microbial culture containing the coloring indicator of the invention of Claim 1, 2 or 4 or the invention of Claim 3 or 4 can be produced, In particular, the filter paper in the culture container or the denatured animal protein does not move, and can easily produce a reaction.
Of these inventions according to claims 1 to 4, the colony cultured in a kind of medium is taken out as necessary, and acid-fast bacteriostatic staining is performed. If no acid-fast bacteria are present, there will be no colored settlement and the reaction will be negative.
[0019]
Embodiment
Embodiment 1
Embodiments including the inventions of claims 1, 2 and 4.
1. The medium is not particularly limited as long as it is a liquid medium represented by bouillon or the like.
2. Dye redox dye,
Abbreviations chemical name STC, 2.3-diphenyl-5-thienyl- (2) -tetrazolium chloride
(2,3-Diphenyl-5-thienyl- (2) -tetrazolium Chloride)
β-STC, 2.3-diphenyl-5-thienyl- (3) -tetrazolium chloride
(2,3-Diphenyl-5-thienyl- (3) -tetrazolium Chloride)
OTC, 2.3-Diphenyl-5-furoyl- (2) -tetrazolium chloride
(2,3-Diphenyl-5-furoyl- (2) -tetorazolium Chloride)
β-OTC, 2.3-Diphenyl-5-furoyl- (3) -tetrazolium chloride
(2,3-Diphenyl-5-furoyl- (3) -tetrazolium Chloride)
α-PTC, 2.3-diphenyl-5-pyridyl- (2) -tetrazolium chloride
(2,3-Diphenyl-5-pyridyl- (2) -tetrazolium Chloride)
β-PTC, 2.3-diphenyl-5-pyridyl- (3) -tetrazolium chloride
(2,3-Diphenyl-5-pyridyl- (3) -tetrazolium Chloride)
γ-PTC, 2.3-diphenyl-5-pyridyl- (4) -tetrazolium chloride
(2,3-Diphenyl-5-pyridyl- (4) -tetrazolium Chjoride)
MDT 2.3-Diphenyl-5-methyl tetrazolium chloride
(2,3-Diphenyl-5-methyl tetrazolium Chloride)
TTC 2,3,5-Triphenyl tetrazolium chloride
(2,3,5-triphenyl tetrazolium Chloride)
Such,
One kind of dye is appropriately selected from the above dyes.
3. Adsorbent Silica Gel Alumina Activated Carbon Powder For 4 ml of liquid culture medium for microorganism culture, one of the redox dyes is selected, and this is 5 to 40 micrograms / milliliter, and one of the adsorbents is 0.02 to A liquid culture medium for microbial culture containing 0.15 grams mixed and containing the desired colored indicator.
[0020]
Embodiment 2
An embodiment including the inventions of claims 3 and 4.
1. As the medium, the same medium as in the first embodiment is used.
2. Dyes and additives
1) Bile acid-bonded pigments Neutral red Methylene blue
Choose one of them.
2) Additive Use one of bile acids and bile salts.
3. Adsorbent One type is selected from the same adsorbent as in the first embodiment.
5 to 40 micrograms / milliliter of at least one bile acid-binding deposition pigment selected from neutral red and methylene blue among the pigments and additives and 4 ml of the liquid medium for microbial culture and bile One kind of additive of acid or bile salt is mixed with 0.1 to 1% of bile acid-bonded pigment, and 0.02 to 0.15 gram of adsorbent is mixed to contain the desired colored indicator. It is a liquid medium for culturing microorganisms.
In Embodiments 1 and 2, when silica gel or alumina is used as the adsorbent, it has been described on the premise that the adsorbent is a powder or a granule. However, in the adsorbent made of these materials, it is processed into a plate shape in advance. An embodiment in which a certain material is laid and the above medium, pigment, and additive are injected is also included in the embodiment of the present invention.
[0021]
Embodiment 3
Embodiments including the inventions according to claims 1, 2, and 4. A filter paper is used as an adsorbent, which is attached to the bottom of the culture vessel, and a kind of the pigment of the first embodiment and a liquid medium are added thereto at a ratio similar to the above, and a target coloring indicator A liquid culture medium for culturing microorganisms containing
Alternatively, the filter paper is preliminarily impregnated with the above-mentioned ratio and then pasted on the bottom of the culture vessel, and the liquid medium used in the first embodiment is injected thereon. Range. Alternatively, after attaching the filter paper, the oxidation-reduction pigment is first impregnated into the filter paper and then the liquid medium is injected, which is included in the embodiment of the present invention. The dry weight of the filter paper is 0.02 to 0.1 gram, depending on the area of the bottom of the culture vessel.
[0022]
Embodiment 4
An embodiment including the inventions of claims 3 and 4. A filter paper is used as an adsorbent, which is affixed to the bottom of the culture vessel, and a microorganism containing a target color indicator by adding a liquid medium and each of the dye and additive of the second embodiment. A liquid medium for culture is used.
In this case as well, the filter paper previously impregnated with bile acid-binding pigment and bile acid or bile salt is attached to the bottom of the culture vessel, and a liquid medium is injected from above, which is also included in this embodiment. . Alternatively, after attaching the filter paper, the bile acid-binding pigment and the additive are first soaked in the filter paper, and then the liquid medium is injected, and this embodiment is included.
In Embodiments 3 and 4, those in which activated carbon is sandwiched between filter papers are also included in these embodiments.
[0023]
Embodiment 5 of Manufacturing Method Embodiment 5
An embodiment of the manufacturing method according to claim 5, wherein the material according to the first embodiment is manufactured. The materials having the composition according to the first embodiment are sequentially and simultaneously placed in a culture vessel in any order and mixed. Thus, a liquid medium for culturing microorganisms containing a colored indicator is produced.
In this method, even if the above-mentioned materials are mixed in advance in a container other than the culture container or the mixture is previously sealed in another container and poured into the culture container at once. It is an embodiment of the described manufacturing method.
[0024]
Embodiment 6
An embodiment of the manufacturing method according to claim 6, which is a method for manufacturing the second embodiment, wherein the materials of the composition of the second embodiment are sequentially and simultaneously placed in a culture vessel in any order and mixed. Thus, a liquid medium for culturing microorganisms containing a colored indicator is produced.
In this method, even if it mixes the said material beforehand with containers other than a culture container, and injects this to a culture container at a stretch, it is embodiment of the manufacturing method of Claim 6.
[0025]
Embodiment 7
It is an embodiment of the manufacturing method according to claim 7, and is a method for manufacturing the one according to Embodiment 3. First, white or milky white filter paper is applied to the entire bottom surface in the culture vessel 1 with a silicon-based adhesive, acrylic Paste with silicon adhesive. Next, the same composition as that obtained by removing the adsorbent from the composition of Embodiment 1 is poured onto the filter paper. In this embodiment, the filter paper is preliminarily impregnated with the oxidation-reduction dye, and this is attached to the bottom of the culture vessel, or after attaching the filter paper, the oxidation-reduction dye is first impregnated into the filter paper, and then only the liquid medium. The method of injecting the liquid is also included in the embodiment of the present invention.
[0026]
Embodiment 8
An embodiment of the manufacturing method according to claim 8, which is a method for manufacturing the fourth embodiment. First, white or milky white filter paper is applied to the entire bottom surface in the culture vessel in the same manner as in the seventh embodiment. Affix the filter paper with an adhesive or acrylic or silicone adhesive. Next, the same composition as that obtained by removing the adsorbent from the composition of Embodiment 2 is poured onto the filter paper.
In this Embodiment 8, using a solution soaked with bile acid-binding pigment and the additive before affixing the filter paper to the bottom of the culture vessel, or after affixing the filter paper, first bile acid-binding pigment and the above-mentioned A method of injecting only the liquid medium after impregnating the additive is also included in this embodiment.
[0027]
Embodiment 9
In an embodiment of the invention as set forth in claim 9, first, the whole egg of the chicken egg used as the adsorbent is thoroughly stirred and injected in an amount sufficient to cover the entire bottom surface of the culture vessel. Usually, the depth is 1 mm to 3 mm, but the depth itself is not limited. In the case of egg protein, the amount used depends on the area of the bottom of the culture vessel, but 0.10 to 1.5 grams is usually sufficient, but even when 5 grams or more are used, the method of the present invention is carried out. It is included in the form.
Next, it heats with the bottom of this culture container horizontal, and uses the said egg as a denatured protein. That is, it is solidified to form an adsorbent layer.
Further, the same composition as that obtained by removing the adsorbent from the composition of the second embodiment is injected onto the denatured protein layer as in the eighth embodiment.
When the adsorbent layer is yellow in the whole egg and it is difficult to distinguish it from the redox dye or bile acid bond-deposited dye, even if only egg white is used as the adsorbent, the embodiment of this method invention is used. included.
Further, a method of injecting animal serum as an adsorbent and injecting it in the same manner as when using an egg and heating to form an adsorbent layer is also an embodiment of the invention according to claim 9.
What was manufactured by these methods is the culture medium of the invention according to claim 4.
[0028]
Embodiment 10
As an adsorbent, as an organic compound-based adsorbent, one of a hydrophobic chromatographic carrier and an ion exchange resin is laid in a culture vessel to form an adsorbent layer.
Since the ion-exchange resin is usually granular, when the bottom of the culture vessel is flat, the granule becomes 2 to 3 particle layers. When the culture vessel is a test tube, the bottom hemispherical portion is The amount is enough to fill.
Then, the same composition as that obtained by removing the adsorbent from the composition of the second embodiment is injected onto the adsorbent.
The culture medium produced by this method is an embodiment of the invention described in claim 4.
[0029]
【The invention's effect】
Since the culture medium of the invention according to claim 1 has the above composition, even if a redox dye is mixed, the inhibitory effect on the growth of the bacteria is extremely reduced as compared with the conventional culture medium, and its growth supporting ability is It is equivalent to a conventional liquid culture medium for microorganism culture that does not contain redox dyes. The redox dye is incorporated into the cells as the bacteria grow, reacts with the reductase in the cells, and the cells are reliably colored. However, fine growth also develops in a color that is clearly different from that of the adsorbent that settles on the bottom of the medium container, and has an effect that can be confirmed early.
In the culture medium of the invention of claim 2, the redox dye is at least selected from STC, β-STC, OTC, β-OTC, α-PTC, β-PTC, γ-PTC, MDT and TTC. Since one kind of pigment is mixed, as the bacteria grow, these pigments are taken into the cells having the reductase and have the effect of reliably producing red color.
[0030]
When neutral red is mixed in the culture medium of the invention described in claim 3, the fungus produces an effect of developing a red color as it grows, as in the invention of claim 2.
Among the culture media according to the third aspect of the present invention, those in which methylene blue is mixed have the effect that the fungus develops a blue color as it grows. Also in this case, the growth support ability is not reduced at all.
In particular, as an additive, a bile acid or a bile salt is mixed, so that when the microbial cells proliferate and are taken into the microbial cells together with the bile acid-binding pigment, the bile is contained in the microbial cells. The acid-bonded pigment is fixed and does not elute again outside the cells, but the cells are surely colored, and when the cells propagate and form colonies, the propagation can be easily recognized.
[0031]
In addition to the effects of the invention of claim 1, 2 or 3, the medium of the invention of claim 4 is silica gel, alumina, and admixture of the adsorbent, which precipitates at the bottom of the container, Alternatively, white or milky white filter paper deposited or pasted on the bottom of the culture medium container will surely grow the bacteria that grow on it, and the color is white, so the pigment is clearly red or blue, There is an effect that can be observed from the initial stage of the proliferation.
When the adsorbent is activated carbon, the bottom surface of the culture vessel is black, which is clearly different from the color due to the growth of the bacteria, and has an effect that can be easily discriminated. When the activated carbon is used and the two adsorbents are covered with filter paper and the white adsorbent is used, the contrast with the colonies of bacteria is good and easy to distinguish.
[0032]
As an invention of a manufacturing method according to a fifth aspect, the constituent elements of the liquid culture medium for cultivating microorganisms with the colored indicator according to the first aspect or the first aspect may be mixed sequentially or at once in any order. The liquid culture medium for cultivating microorganisms with the described colored indicator can be produced, and the production at the inspection site can be simplified.
As an invention of a manufacturing method according to a sixth aspect, the composition elements of the liquid medium for cultivating microorganisms with the colored indicator according to the third or fourth aspect may be sequentially or temporarily fitted in any order. Item 3. A liquid culture medium for culturing microorganisms with a colored indicator according to item 3 or 4 can be produced, and the production at the inspection site can be simplified.
[0033]
The method invention according to claim 7 or 8 is a method in which a filter paper as an adsorbent is previously attached to the bottom surface in the culture vessel, and it is simple and can produce a product in which the adsorbent is reliably positioned on the entire bottom surface. When this is used to cultivate the fungus, the effect of inhibiting the growth of the fungus acts in the vicinity of the fungus body, making the effect more reliable, and by contrast with the stained fungus, the initial growth of the fungus A liquid medium for cultivating microorganisms with a colored indicator that can be clearly recognized from the stage can be produced.
In the method according to claim 9, the adsorbent is solidified and stabilized at the bottom of the culture vessel, and the reaction image does not bleed and is stable.
[0034]
Experimental Example As a liquid culture medium, Middle Brook 7H9 is added to 4 ml as a liquid culture medium for acid-fast bacilli, and 20 microgram / milliliter of STC is added as a redox dye serving as an indicator. Further, 0.10 gram of silica gel is used as this example. As a control medium, Middle Brook 7H9 is used as a normal liquid medium, and as a comparative example, the above silica gel, which is one of the components of the present invention, is removed. In addition, the bacterial solution concentration was 10 −6 milligram / milliliter, and each was cultured for 2 weeks. The results are as follows.
[0035]
[Table 1]
In the Examples of the present invention and the control Middlebrook 7H9 alone, the growth of similar bacteria was observed in the same number of days (2 weeks) until the medium inoculated with 10 −6 milligram / milliliter. In the case where no was added, no bacterial growth was observed.
In addition, in the product of the present invention, the growth of the bacteria showed a clear red color, clearly distinguished from the precipitated white silica gel, and it was confirmed that it exhibited a sufficient indicator function.
[0037]
In addition, when the redox dye and bile acid bond-deposited dye are less than the above ranges, the coloring is insufficient, and as the range is exceeded, the effect of inhibiting the growth of bacteria occurs.
The adsorbent is preferably in an amount within the above range, and if it is less than this range, sufficient ability to support bacterial growth cannot be obtained. Amount.
If the amount of bile acid or bile salt is also below the above range, the coloring effect is not sufficient, and if it exceeds the range, the growth of the fungus is suppressed, which is not preferable.
Claims (9)
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| JP2000152552A JP3813410B2 (en) | 1999-05-26 | 2000-05-24 | Liquid culture medium for microbial culture containing coloring indicator and method for producing the same |
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| JP14679899 | 1999-05-26 | ||
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| JP2000152552A JP3813410B2 (en) | 1999-05-26 | 2000-05-24 | Liquid culture medium for microbial culture containing coloring indicator and method for producing the same |
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| US9637770B2 (en) | 2011-03-07 | 2017-05-02 | Mekorot Water Company, Ltd. | Methods and devices for handling biofouling |
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