JP2013116053A - Medium for detecting enterohemorrhagic escherichia coli - Google Patents
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Abstract
Description
本発明は、食中毒の原因となる腸管出血性大腸菌を他の大腸菌と区別して検出し得る培地及び腸管出血性大腸菌の検出方法に関する。 The present invention relates to a medium capable of detecting enterohemorrhagic E. coli causing food poisoning separately from other E. coli and a method for detecting enterohemorrhagic E. coli.
大腸菌O157をはじめとする腸管出血性大腸菌はベロ毒素を産生する大腸菌の血清型の一種であり、一般的に食中毒の原因となる細菌として知られており、また本食中毒は重篤化することがあることから、本細菌の制御は食品衛生及び安全の点からも重要である(非特許文献1)。 Enterohemorrhagic Escherichia coli such as Escherichia coli O157 is a kind of serotype of Escherichia coli that produces verotoxin, and is generally known as a bacterium that causes food poisoning, and food poisoning may become serious. Therefore, control of this bacterium is important from the viewpoint of food hygiene and safety (Non-patent Document 1).
腸管出血性大腸菌の選択分離培地としてはセフィキシム及び亜テルル酸カリウムを添加したマッコンキー基礎寒天培地に特定の糖を加えた培地、すなわちO157の場合にはソルビトールを利用できないのでソルビトールを添加し、発育してきたコロニーのうち糖利用反応を認めない無色のコロニーを腸管出血性大腸菌O157として分離する、同様にO111及びO26の場合にはソルボース、ラムノースをそれぞれ添加し同様に糖利用反応を認めないコロニーを分離するといったものが古くから用いられてきた。この場合には目的とする細菌のコロニーに検出可能な反応が見られず、目的以外のコロニーに検出可能な反応が発生するため目的菌のコロニーが見難くなる。 As a selective separation medium for enterohemorrhagic Escherichia coli, a sorbitol cannot be used in the case of a medium with a specific sugar added to a MacConkey basic agar medium supplemented with cefixime and potassium tellurite, that is, sorbitol cannot be used in the case of O157, and so Colorless colonies that do not recognize sugar utilization reaction are isolated as enterohemorrhagic Escherichia coli O157. Similarly, in the case of O111 and O26, sorbose and rhamnose are added to isolate colonies that do not recognize sugar utilization reaction. Things to do have been used for a long time. In this case, no detectable reaction is seen in the target bacterial colonies, and a detectable reaction occurs in the non-target colonies, making it difficult to see the target bacterial colonies.
一方で近年では検出可能な色原体化合物を有する酵素基質により腸管出血性大腸菌に特異的な酵素を指標とし、腸管出血性大腸菌を検出できる培地が種々開発されている(非特許文献2、3及び特許文献1、2)。 On the other hand, in recent years, various media have been developed that can detect enterohemorrhagic Escherichia coli using enzymes specific to enterohemorrhagic Escherichia coli as an index using an enzyme substrate having a detectable chromogenic compound (Non-patent Documents 2 and 3). And Patent Documents 1 and 2).
これらの検出可能な色原体化合物を有する酵素基質の添加による腸管出血性大腸菌の培地ではO157、O111、O26といった主要な腸管出血性大腸菌の血清型を推定的に鑑別することが出来る培地であるが、複数の色原体化合物を有する酵素基質を使用する必要があり、非常に高価である。更にこれらの培地において3種の菌の性状のうち2種は同系色のコロニーを形成するため一概にして3種を鑑別できるとは言い難い。また、食品を検体とする検出培地としては、血清型の鑑別は重要ではなく、検体食品中に食中毒を生じる可能性のある腸管出血性大腸菌が存在するか否かが最重要である。
従って、本発明の課題は、血清型によらず、腸管出血性大腸菌全般を選択的に検出できる培地を提供することにある。
In the medium of enterohemorrhagic E. coli by the addition of an enzyme substrate having these detectable chromogenic compounds, it is a medium that can preferentially differentiate the major enterohemorrhagic E. coli serotypes such as O157, O111, and O26. However, it is necessary to use an enzyme substrate having a plurality of chromogenic compounds, which is very expensive. Further, in these media, two of the three types of fungi form colonies of similar colors, so it is difficult to say that the three types can be distinguished collectively. Further, as a detection medium using food as a specimen, discrimination of serotype is not important, and it is most important whether or not enteric hemorrhagic Escherichia coli that may cause food poisoning exists in the specimen food.
Accordingly, an object of the present invention is to provide a medium capable of selectively detecting all enterohemorrhagic Escherichia coli regardless of serotype.
そこで本発明者は、高価な色原体化合物を有する酵素基質を1種のみ使用して、腸管出血性大腸菌全般を他の大腸菌群と区別して検出可能な培地を開発すべく種々検討した結果、セフィキシム及び亜テルル酸塩を添加した栄養培地に、胆汁酸又はその塩、青色の色原体化合物を遊離し得るβ−ガラクトシダーゼ基質、低濃度の乳糖、及び特定濃度のセロビオースを添加した培地を用いれば、血清型にかかわらず腸管出血性大腸菌を明瞭な青色のコロニーとして検出できることを見出し、本発明を完成した。 Therefore, the present inventor has conducted various studies to develop a medium that can detect enterohemorrhagic Escherichia coli in general from other coliforms using only one enzyme substrate having an expensive chromogenic compound, Use a nutrient medium supplemented with cefixime and tellurite and a medium supplemented with bile acid or its salt, a β-galactosidase substrate that can release blue chromogenic compounds, a low concentration of lactose, and a specific concentration of cellobiose. For example, it was found that enterohemorrhagic Escherichia coli can be detected as a clear blue colony regardless of the serotype, and the present invention has been completed.
すなわち、本発明は、(a)セフィキシム、(b)亜テルル酸塩、(c)胆汁酸又はその塩、(d)青色の色原体化合物を遊離し得るβ−ガラクトシダーゼ基質、(e)乳糖0.01〜2g/L(検出時の濃度)、及び(f)セロビオース5〜20g/L(検出時の濃度)を含有する腸管出血性大腸菌検出用培地を提供するものである。
また、本発明は、上記の培地に検体を接種し、培養後青色のコロニーを検出することを特徴とする腸管出血性大腸菌の検出方法を提供するものである。
That is, the present invention includes (a) cefixime, (b) tellurite, (c) bile acid or a salt thereof, (d) a β-galactosidase substrate capable of releasing a blue chromogenic compound, (e) lactose An intestinal hemorrhagic Escherichia coli detection medium containing 0.01-2 g / L (concentration at detection) and (f) cellobiose 5-20 g / L (concentration at detection) is provided.
The present invention also provides a method for detecting enterohemorrhagic Escherichia coli, which comprises inoculating a specimen into the above medium and detecting a blue colony after culture.
本発明の培地を用いれば、食中毒の原因となる腸管出血性大腸菌が、血清型にかかわらず、他の大腸菌群と区別して、青色のコロニーとして明確に検出できる。従って、広範囲な飲食品中の腸管出血性大腸菌が簡便かつ速やかに検出可能となる。 Using the medium of the present invention, enterohemorrhagic Escherichia coli causing food poisoning can be clearly detected as a blue colony, regardless of the serotype, as distinguished from other coliforms. Therefore, enterohemorrhagic Escherichia coli in a wide range of foods and drinks can be detected easily and quickly.
本発明の腸管出血性大腸菌検出用培地は、(a)セフィキシム、(b)亜テルル酸塩、(c)胆汁酸又はその塩、(d)青色の色原体化合物を遊離し得るβ−ガラクトシダーゼ基質、(e)乳糖0.01〜2g/L(検出時の濃度)、及び(f)セロビオース5〜20g/L(検出時の濃度)を含有する。 The medium for detecting enterohemorrhagic Escherichia coli according to the present invention comprises (a) cefixime, (b) tellurite, (c) bile acid or a salt thereof, and (d) β-galactosidase capable of releasing a blue chromogenic compound. Contains substrate, (e) lactose 0.01-2 g / L (concentration upon detection), and (f) cellobiose 5-20 g / L (concentration upon detection).
(a)セフィキシムはプロテウスやモルガネラ等の発育を抑制し、(b)亜テルル酸塩は腸管出血性大腸菌以外の大腸菌やアエロモナス等の発育を抑制するため、腸管出血性大腸菌の分離に重要である。亜テルル酸塩としては、亜テルル酸ナトリウム、亜テルル酸カリウム等の亜テルル酸アルカリ金属塩が好ましい。セフィキシムは本発明培地中の検出時の濃度として0.01〜0.5mg/L含有するのが好ましく、0.01〜0.1mg/L含有するのがより好ましい。亜テルル酸塩は本発明培地中の検出時の濃度として0.1〜10mg/L含有するのが好ましく、1〜8mg/L含有するのがより好ましい。 (A) Cefixime suppresses the growth of Proteus, Morganella, etc. (b) Tellurite inhibits the growth of Escherichia coli and Aeromonas other than enterohemorrhagic Escherichia coli, so it is important for the isolation of enterohemorrhagic Escherichia coli . As the tellurite, alkali metal tellurite such as sodium tellurite and potassium tellurite is preferable. Cefixime is preferably contained in an amount of 0.01 to 0.5 mg / L, more preferably 0.01 to 0.1 mg / L, as a concentration in the culture medium of the present invention. It is preferable to contain 0.1-10 mg / L of tellurite as a density | concentration at the time of the detection in this invention culture medium, and it is more preferable to contain 1-8 mg / L.
(c)胆汁酸又はその塩は、グラム陽性菌を抑制し、また形成したコロニーを赤色系にすることにより、腸管出血性大腸菌以外の細菌のコロニーを排除しやすくする作用を有する。胆汁酸としては、コール酸、デオキシコール酸が挙げられる。胆汁酸の塩としては、ナトリウム塩、カリウム塩等のアルカリ金属塩が挙げられる。(c)胆汁酸又はその塩は、グラム陽性菌の抑制及び腸管出血性大腸菌以外の細菌のコロニーの呈色性の点から、本発明培地の検出時の濃度として0.5〜10g/L含有するのが好ましく、1〜5g/L含有するのがより好ましい。 (C) The bile acid or a salt thereof has an effect of facilitating elimination of colonies of bacteria other than enterohemorrhagic Escherichia coli by suppressing gram-positive bacteria and making the formed colonies red. Examples of bile acids include cholic acid and deoxycholic acid. Examples of bile acid salts include alkali metal salts such as sodium salts and potassium salts. (C) Bile acid or a salt thereof contains 0.5 to 10 g / L as a concentration at the time of detection of the culture medium of the present invention from the viewpoint of suppression of Gram-positive bacteria and coloration of bacterial colonies other than enterohemorrhagic Escherichia coli It is preferable to contain 1 to 5 g / L.
(d)青色の色原体化合物を遊離し得るβ−ガラクトシダーゼ基質は、腸管出血性大腸菌を色原体化合物に依存する青色のコロニーを形成させるために用いる。腸管出血性大腸菌を含む大腸菌群はβ−ガラクトシダーゼを産生するので、検体中に腸管出血性大腸菌を含む大腸菌群が含まれれば基質が分解され、青色の色原体化合物が遊離する。これらの基質に含まれる青色の色原体化合物としては、5−ブロモ−4−クロロ−3−インドール、5−ブロモ−3−インドール、6−ブロモ−2−ナフトール、2−メチル−3−インドール、6−クロロ−3−インドール等が挙げられる。また、β−ガラクトシダーゼ基質としては、β−ガラクトピラノシドが好ましい。(d)青色の色原体化合物を遊離し得るβ−ガラクトシダーゼ基質の具体例として5−ブロモ−4−クロロ−3−インドキシル−β−D−ガラクトピラノシド、5−ブロモ−3−インドキシル−β−D−ガラクトピラノシド、5−ブロモ−4−クロロ−3−インドキシル−β−D−ガラクトピラノシド、5−ブロモ−3−インドキシル−β−D−ガラクトピラノシド、6−ブロモ−2−ナフチル−β−D−ガラクトピラノシド、1−メチル−3−インドキシル−β−D−ガラクトピラノシド、6−クロロ−3−インドキシル−β−D−ガラクトピラノシドなどが挙げられる。
(d)青色の色原体化合物を遊離し得るβ−ガラクトシダーゼ基質は腸管出血性大腸菌のコロニーの呈色性の点から、本発明培地の検出時の濃度として0.01〜1g/L含有するのが好ましく、0.05〜0.5g/L含有するのがより好ましい。
(D) A β-galactosidase substrate capable of releasing a blue chromogenic compound is used to cause enterohemorrhagic E. coli to form a blue colony dependent on the chromogenic compound. Since the coliform group including enterohemorrhagic Escherichia coli produces β-galactosidase, if the sample contains an coliform group including enterohemorrhagic Escherichia coli, the substrate is decomposed and the blue chromogenic compound is released. The blue chromogenic compounds contained in these substrates include 5-bromo-4-chloro-3-indole, 5-bromo-3-indole, 6-bromo-2-naphthol, 2-methyl-3-indole , 6-chloro-3-indole and the like. The β-galactosidase substrate is preferably β-galactopyranoside. (D) Specific examples of β-galactosidase substrate capable of releasing blue chromogenic compound include 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside, 5-bromo-3-india Xyl-β-D-galactopyranoside, 5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside, 5-bromo-3-indoxyl-β-D-galactopyranoside 6-bromo-2-naphthyl-β-D-galactopyranoside, 1-methyl-3-indoxyl-β-D-galactopyranoside, 6-chloro-3-indoxyl-β-D-galacto Examples include pyranoside.
(D) The β-galactosidase substrate capable of releasing the blue chromogenic compound contains 0.01 to 1 g / L as a concentration at the time of detection of the medium of the present invention from the viewpoint of coloration of colony of enterohemorrhagic Escherichia coli. Is more preferable, and 0.05 to 0.5 g / L is more preferable.
(e)乳糖は、腸管出血性大腸菌を含む大腸菌群が資化しうる糖であるが、腸管出血性大腸菌のみを青色の色原体化合物に由来した呈色コロニーを形成させ、更にその発色を増強させる作用を有する。本発明培地の検出時の濃度として0.01〜2g/L含有するのが好ましく、0.5〜2g/L含有するのがより好ましい。 (E) Lactose is a sugar that can be assimilated by coliforms including enterohemorrhagic Escherichia coli, but only enterohemorrhagic Escherichia coli forms a colored colony derived from a blue chromogenic compound and further enhances its color development Have the effect of It is preferable to contain 0.01-2 g / L as a density | concentration at the time of detection of this invention culture medium, and it is more preferable to contain 0.5-2 g / L.
(f)セロビオースは大腸菌以外の菌により資化しうる糖である。腸管出血性大腸菌以外の菌のコロニーの呈色性の点から、本発明培地の検出時の濃度として5〜20g/L含有するのが好ましく、7.5〜15g/L含有するのがより好ましい。 (F) Cellobiose is a sugar that can be assimilated by bacteria other than E. coli. From the viewpoint of coloration of colonies of bacteria other than enterohemorrhagic Escherichia coli, the concentration of the present invention medium is preferably 5 to 20 g / L, more preferably 7.5 to 15 g / L. .
本発明培地において、十分量の(f)セロビオースと低濃度の(e)乳糖は、腸管出血性大腸菌を青色のコロニーとして検出する点で重要である。セフィキシム及び亜テルル酸カリウムを添加した栄養培地に腸管出血性大腸菌を含む大腸菌群に特異的であるβ−ガラクトシダーゼに対する青色の色原体化合物を有する酵素基質を加えるだけでは、セフィキシム及び亜テルル酸カリウムに耐性である腸管出血性大腸菌以外の一部の大腸菌群についても青色のコロニーを形成する。これを回避するために大腸菌が利用できずその他の大腸菌群が利用できる(f)セロビオースを十分量加えることにより、セフィキシム及び亜テルル酸カリウムに耐性の腸管出血性大腸菌以外の菌は、青色以外の集落を形成するが、その結果目的菌である腸管出血性大腸菌の青色の発色が極端に弱くなり明瞭ではなくなることを認めた。これに低濃度の(e)乳糖を加えると、通常全ての大腸菌群は乳糖を利用するため、β−ガラクトシダーゼに対する色原体化合物を加えてあっても発育した全ての大腸菌群はピンク色を帯びることが想定されるが、実際には腸管出血性大腸菌は血清型に拘らずピンク色の発色を認めないだけでなく、更に青色の発色が増強され明瞭な青色コロニーを形成することを認めた。また培地上に発育した腸管出血性大腸菌以外のコロニーは青色以外のコロニーを形成する、あるいは発育しないため、腸管出血性大腸菌を青色のコロニーとして容易に鑑別出来る。 In the medium of the present invention, a sufficient amount of (f) cellobiose and a low concentration of (e) lactose are important in terms of detecting enterohemorrhagic E. coli as blue colonies. Simply adding an enzyme substrate with a blue chromogenic compound for β-galactosidase that is specific for coliforms including enterohemorrhagic E. coli to a nutrient medium supplemented with cefixime and potassium tellurite, cefixime and potassium tellurite Blue colonies are also formed in some coliforms other than enterohemorrhagic Escherichia coli resistant to. In order to avoid this, E. coli cannot be used and other coliform groups can be used. (F) By adding a sufficient amount of cellobiose, bacteria other than enterohemorrhagic E. coli resistant to cefixime and potassium tellurite are not blue. A colony was formed, but as a result, it was recognized that the blue coloration of the intestinal hemorrhagic Escherichia coli, which is the target bacterium, became extremely weak and unclear. When a low concentration of (e) lactose is added to this, all coliforms usually use lactose, so even if the chromogenic compound for β-galactosidase is added, all the grown coliforms are pink. In fact, enterohemorrhagic Escherichia coli not only showed pink coloration regardless of the serotype, but also showed that blue coloration was enhanced to form a clear blue colony. In addition, colonies other than enterohemorrhagic Escherichia coli grown on the medium form colonies other than blue or do not grow, so that enterohemorrhagic Escherichia coli can be easily identified as a blue colony.
本発明培地には、更にニュートラルレッドを加えても良い。ニュートラルレッドは胆汁酸又はその塩とともに形成したコロニーの色を赤色系にすることにより、腸管出血性大腸菌以外の細菌のコロニーを排除しやすくする。ニュートラルレッドを加えない場合でも腸管出血性大腸菌以外の菌はほぼ無色のコロニーあるいは黄緑色のコロニーを形成し青色の腸管出血性大腸菌のコロニーと鑑別可能であるが、検出時の濃度として0.001〜0.008g/L含有させたときに培地が無色透明で腸管出血性大腸菌以外のコロニーが明瞭なピンク色を呈するためよりコロニーの鑑別が容易になる。ニュートラルレッドは腸管出血性大腸菌以外の細菌のコロニーの呈色性の点から、本発明培地の検出時の濃度として0.001〜0.008g/L含有するのが好ましく、0.002〜0.006g/L含有するのがより好ましい。また一般的に培地に使用されている含有量である0.02〜0.03g/Lの場合には培地全体が赤色系になってしまうため、コロニーの検出性が低下することがある。 Neutral red may be further added to the medium of the present invention. Neutral red makes it easy to eliminate colonies of bacteria other than enterohemorrhagic Escherichia coli by making the color of colonies formed with bile acids or salts thereof red. Even when neutral red is not added, bacteria other than enterohemorrhagic Escherichia coli can be distinguished from blue intestinal hemorrhagic Escherichia coli colonies by forming almost colorless colonies or yellow-green colonies, but the concentration at the time of detection is 0.001. When containing 0.008 g / L, the culture medium is colorless and transparent, and colonies other than enterohemorrhagic Escherichia coli exhibit a clear pink color, making colony identification easier. Neutral red is preferably contained in an amount of 0.001 to 0.008 g / L as a concentration at the time of detection of the medium of the present invention, from the viewpoint of coloration of colonies of bacteria other than enterohemorrhagic Escherichia coli. It is more preferable to contain 006 g / L. In addition, in the case of 0.02 to 0.03 g / L, which is a content generally used in a culture medium, the whole culture medium becomes red, and colony detectability may be reduced.
本発明培地には上記成分の他、栄養成分、無機塩、pH調整剤等を配合することが出来る。 In addition to the above components, nutrient media, inorganic salts, pH adjusters, and the like can be added to the culture medium of the present invention.
栄養成分としては、ペプトン、獣肉エキス、酵母エキス、魚肉エキス等が好ましい。無機塩類としては塩化ナトリウム等の無機酸金属塩、クエン酸ナトリウムなどの有機酸金属塩などが挙げられる。 As a nutritional component, peptone, animal meat extract, yeast extract, fish meat extract and the like are preferable. Inorganic salts include inorganic acid metal salts such as sodium chloride and organic acid metal salts such as sodium citrate.
また、本発明培地は、形成した腸管出血性大腸菌以外の細菌のコロニーの呈色性及び排除のしやすさの点から、検出時のpHが7.0以上であるのが好ましく、pH7.0〜8.0であるのがより好ましい。 In addition, the culture medium of the present invention preferably has a pH of 7.0 or more at the time of detection from the viewpoint of coloration and ease of elimination of colonies of bacteria other than the enterohemorrhagic Escherichia coli formed, and pH 7.0 More preferably, it is -8.0.
また、本発明培地には、検出可能なβ−グルクロニダーゼ基質を加える事により、腸管出血性大腸菌の中で最も検出頻度が高いO157を推定的に検出することも可能である。 Further, by adding a detectable β-glucuronidase substrate to the culture medium of the present invention, it is possible to detect preferentially O157 having the highest detection frequency among enterohemorrhagic Escherichia coli.
本発明培地を用いて、被検体中の腸管出血性大腸菌を検出するには、当該培地に検体を接種して25〜40℃、24〜48時間培養した後に、コロニーの呈色を観察すればよい。 In order to detect enterohemorrhagic Escherichia coli in a subject using the medium of the present invention, the sample is inoculated and cultured at 25 to 40 ° C. for 24 to 48 hours, and then the color of the colony is observed. Good.
本発明培地に適用される検体としては、肉類、魚介類などの生鮮食料品、便などの臨床検体、海水、調理場、病院などのふき取り検体等が挙げられるが、これらの検体を予めトリプトソーヤブイヨン培地で培養した培養液や更にこれを増菌用培地で培養した培養液も用いることができる。 Samples applied to the culture medium of the present invention include fresh foods such as meat and seafood, clinical samples such as stool, wiping samples from seawater, kitchens, hospitals, etc. A culture solution cultured in a soya bouillon medium or a culture solution obtained by culturing this in a medium for enrichment can also be used.
次に実施例を挙げて本発明を詳細に説明するが、本発明はこれら実施例に限定されるものではない。
実施例1
(1)培地の作製
各培地組成を表1に示した。
本発明培地は1リットル使用量を1リットルの精製水に加え、100℃、20分間加温溶解し、約50℃になるまで冷却後、セフィキシム及び亜テルル酸カリウムを加えよく混合した後プラスチックシャーレ(90φmm)に20mlずつ分注して培地が固まるまで静置し本発明の腸管出血性大腸菌検出用培地を作製した。また対照品として本発明培地より乳糖を抜いたもの及びセロビオースを抜いたものをそれぞれ同様に作製した。
陽性コントロールとしてトリプトソイ寒天培地(TSA)は1リットル使用量を1リットルの精製水に加え、121℃、15分間高圧蒸気滅菌し良く撹拌後、プラスチックシャーレ(90φmm)に20mlずつ分注して培地が固まるまで静置した。
EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.
Example 1
(1) Production of medium Table 1 shows the composition of each medium.
In the culture medium of the present invention, add 1 liter of use amount to 1 liter of purified water, dissolve by heating at 100 ° C. for 20 minutes, cool to about 50 ° C., add cefixime and potassium tellurite, mix well, and then add plastic petri dish. 20 ml each was dispensed into (90 mm) and allowed to stand until the medium solidified to prepare the enterohemorrhagic Escherichia coli detection medium of the present invention. Further, as a control product, a product obtained by removing lactose from the medium of the present invention and a product obtained by removing cellobiose were prepared in the same manner.
As a positive control, trypsoy soy agar medium (TSA) was added to 1 liter of purified water in 1 liter amount, and autoclaved at 121 ° C for 15 minutes under high pressure steam sterilization. Let stand until it hardens.
(2)菌株の供試
供試菌株はトリプトソイ寒天培地で24時間前培養したものを供試菌として用い、これを白金線を用いて画線塗抹により接種し、35℃、24時間培養後のコロニー色調を確認した。
結果を表2〜表4に示す。表中、TSAはトリプケース・ソイブイヨン培地を示す。また、乳糖抜き、セロビオース抜き等は、本発明培地(表1)から、それらの成分を抜いた培地であることを示す。
(2) test test strain of strain used as test bacteria those 24 hours precultured in tryptic soy agar medium, which was inoculated with streaked with a platinum wire, 35 ° C., after 24 hours of culture The colony color was confirmed.
The results are shown in Tables 2-4. In the table, TSA indicates a trycase soy bouillon medium. Moreover, lactose removal, cellobiose removal, etc. show that it is a culture medium which removed those components from this invention culture medium (Table 1).
表1の培地を用いて、腸管出血性大腸菌を含む各供試菌株の発育を確認したところ、本発明培地は腸管出血性大腸菌のみを青色の有色コロニーとして検出でき、それ以外の発育した菌についてはピンク色のコロニーとして排除できることを認めた。一方、乳糖を含まない培地では腸管出血性大腸菌以外のピンク色の発色は十分であったが、腸管出血性大腸菌の発色が弱く鑑別が良好でないことを認めた。またセロビオースを含まない培地では腸管出血性大腸菌ではないE.hermanniiが腸管出血性大腸菌と鑑別できないことを認めた。
ここで乳糖は全ての大腸菌及びE.hermanniiの両方が利用できる糖であり、乳糖を加えた場合には全ての菌がピンク色の呈色をもたらす事が容易に推測できるにもかかわらず、低濃度の乳糖を加えた場合、腸管出血性大腸菌には全くピンク色の発色を認めず、青色の色原体化合物由来の発色のみが増強されたという事実は全く予測外であった。
When the growth of each test strain containing enterohemorrhagic Escherichia coli was confirmed using the medium of Table 1, the medium of the present invention was able to detect only enterohemorrhagic Escherichia coli as a blue colored colony, and other grown bacteria Found that it could be eliminated as a pink colony. On the other hand, in the medium containing no lactose, pink coloration other than enterohemorrhagic Escherichia coli was sufficient, but the coloration of enterohemorrhagic Escherichia coli was weak and the differentiation was not good. In addition, E. coli is not enterohemorrhagic E. coli in a medium without cellobiose. It was found that hermannii could not be differentiated from enterohemorrhagic E. coli.
Here, lactose is all E. coli and E. coli. Both Hermannii are available sugars, and it is easy to guess that all bacteria will produce a pink color when lactose is added, but intestinal hemorrhage when low concentrations of lactose are added The fact that no pink coloration was observed in the genus Escherichia coli and only the color development derived from the blue chromogenic compound was enhanced was completely unexpected.
本発明培地(表1)における乳糖の濃度を確認したところ、本発明培地における乳糖濃度は0gであればX−GALによる発色が非常に悪く、0.01〜2g/L、特に0.5〜2g/Lでは腸管出血性大腸菌の青色の発色が明瞭になり、3g以上になると乳糖を利用する全ての菌のコロニーがピンク色を帯びるため腸管出血性大腸菌とそれ以外の菌の鑑別能が低下することを認めた。従って腸管出血性大腸菌とそれ以外の菌の鑑別能を良好にするために低濃度の乳糖として0.01〜2g/L、特に0.5〜2g/L含有することが好ましいことを認めた。 When the concentration of lactose in the medium of the present invention (Table 1) was confirmed, if the lactose concentration in the medium of the present invention was 0 g, the color development by X-GAL was very poor, 0.01-2 g / L, especially 0.5- At 2g / L, the blue color of enterohemorrhagic Escherichia coli becomes clear, and when it exceeds 3g, colonies of all the fungi that use lactose are pink, and the ability to distinguish enterohemorrhagic Escherichia coli from other bacteria decreases. Admitted to do. Therefore, in order to improve the discrimination ability between enterohemorrhagic Escherichia coli and other bacteria, it was found that it is preferable to contain 0.01 to 2 g / L, particularly 0.5 to 2 g / L as a low concentration of lactose.
本発明培地(表1)のニュートラルレッド濃度を検討したところ、ニュートラルレッドを加えない場合でも十分腸管出血性大腸菌とそれ以外の菌の鑑別は可能であるが、腸管出血性大腸菌以外の菌のコロニー色調が薄黄緑色で青色のコロニーと誤判定する可能性がある。また通常一般的に使用される濃度である0.02g/Lでは各発育菌株のコロニー性状は変わらないが、培地色調が赤色であるため、発育した菌のコロニー色調が見づらくなる。一方、その約10分の1量である0.0025g/Lまで低下させると培地色が透明であるにも拘らず、腸管出血性大腸菌以外の明瞭な集落の着色が認められた。0.001g/Lまで濃度を低下させると腸管出血性大腸菌以外のコロニーの着色が薄くなりすぎることが認められた。従って、本発明培地にニュートラルレッドを加える場合には、ニュートラルレッド濃度を0.001〜0.008g/L、特に0.002〜0.006g/Lにすることで培地が無色透明となり同時に着色集落が明瞭に確認できることを見出した。またニュートラルレッドは酸性pHでは赤色を呈する性質があり、本培地における現象は培地pHが7.0以上であることが重要であり、これを下回ると培地色が赤色を呈するようになってしまう。 Examination of the neutral red concentration of the culture medium of the present invention (Table 1) revealed that enterohemorrhagic Escherichia coli and other bacteria could be sufficiently differentiated even when neutral red was not added, but colonies of bacteria other than enterohemorrhagic Escherichia coli There is a possibility that the color tone is light yellow-green and erroneously determined as a blue colony. Moreover, although the colony property of each growth strain does not change at the commonly used concentration of 0.02 g / L, since the color of the culture medium is red, it becomes difficult to see the colony color of the grown bacteria. On the other hand, when the amount was reduced to about 1/10 of 0.0025 g / L, clear colony coloration other than enterohemorrhagic Escherichia coli was observed even though the medium color was transparent. When the concentration was reduced to 0.001 g / L, it was found that the coloration of colonies other than enterohemorrhagic Escherichia coli became too light. Therefore, when neutral red is added to the culture medium of the present invention, the neutral red concentration is 0.001 to 0.008 g / L, especially 0.002 to 0.006 g / L. It was found that can be clearly confirmed. Neutral red has the property of exhibiting a red color at an acidic pH, and it is important that the pH of the medium is 7.0 or more, and below this, the medium color becomes red.
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