JP3142962B2 - Medium for fluorescence detection of coliform bacteria - Google Patents
Medium for fluorescence detection of coliform bacteriaInfo
- Publication number
- JP3142962B2 JP3142962B2 JP19837392A JP19837392A JP3142962B2 JP 3142962 B2 JP3142962 B2 JP 3142962B2 JP 19837392 A JP19837392 A JP 19837392A JP 19837392 A JP19837392 A JP 19837392A JP 3142962 B2 JP3142962 B2 JP 3142962B2
- Authority
- JP
- Japan
- Prior art keywords
- medium
- fluorescence
- present
- fluorescence detection
- coliform bacteria
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【0001】[0001]
【産業上の利用分野】本発明は食品等の検体中に微生物
が存在するか否かを迅速、簡便かつ高感度で判定するた
めの大腸菌群蛍光検出用培地に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a medium for detecting Escherichia coli group fluorescence for rapidly, simply and highly sensitively determining whether or not microorganisms are present in a sample such as food.
【0002】[0002]
【従来の技術】食品、薬品等に代表されるヒトが摂取す
るものの安全性については厳しい管理がなされており、
特に微生物の有無の判定は重要である。このうち、食品
中の大腸菌群の有無の判定は日常検査として特に重要で
ある。2. Description of the Related Art The safety of humans, such as foods and medicines, which are ingested by humans, is strictly controlled.
It is particularly important to determine the presence or absence of microorganisms. Of these, the determination of the presence or absence of coliforms in food is particularly important as a daily test.
【0003】食品中の大腸菌群の検出法としては、従
来、食品サンプルを滅菌生理食塩水等に懸濁してストマ
ッキングして得られた懸濁液を段階希釈し、デゾキシコ
レート培地を用いて24時間培養した後、発育したコロ
ニー数を算出する方法、あるいはブリリアントグリーン
乳糖ブイヨン(BGLB)、乳糖ブイヨン等を用いて2
4〜48時間培養した後、ガスの産生が認められる試験
管の本数を数えて菌数を算定する方法が知られている。
しかしながら、これらの方法では、培養時間が24〜4
8時間程度必要であることから判定に1日以上を要する
こと、コロニーの計測は煩雑であり、その操作には熟練
を要すること、また検体を段階希釈したり、培地を用事
調製することから操作が煩雑である等の問題があった。[0003] As a method for detecting coliforms in foods, a suspension obtained by suspending a food sample in sterile physiological saline or the like and serially diluting the suspension is serially diluted and cultured for 24 hours using a desoxycholate medium. After that, a method of calculating the number of grown colonies, or brilliant green lactose broth (BGLB), lactose broth, etc.
It is known to calculate the number of bacteria by counting the number of test tubes in which gas production is observed after culturing for 4 to 48 hours.
However, in these methods, the culture time is 24 to 4 hours.
Since it takes about 8 hours, it takes more than one day for the determination, and the counting of the colonies is complicated, the operation requires skill, and the operation is necessary because the sample is serially diluted and the medium is prepared. Is complicated.
【0004】一方、最近蛍光基質を利用し、より迅速な
微生物の検出が可能な蛍光法が報告された(特公昭58
−17598号公報及び特開昭57−144995号公
報)。しかしながら、蛍光基質を用いる検出法では、大
腸菌群数が試料原液1ml当たり104 〜105 以上必要
なため、食品等の比較的菌数の少ない大腸菌群を検出す
るには培養操作が必要となり、大腸菌群をより速く増殖
させる培地が要求される。そこで、本発明者らは、大腸
菌群増殖用培地としてジェネレイションタイムが速く、
増殖率がよく、しかもラッグフェイズがない大腸菌群用
増殖培地を開発し、報告した(特開平4−51890号
公報)。On the other hand, recently, a fluorescence method using a fluorescent substrate and capable of detecting microorganisms more rapidly has been reported (Japanese Patent Publication No. 58-58).
-17598 and JP-A-57-144995). However, in the detection method using a fluorescent substrate, the number of coliforms is required to be 10 4 to 10 5 or more per 1 ml of the sample stock solution. There is a need for a medium that allows the coliforms to grow faster. Therefore, the present inventors have a rapid generation time as a medium for growing Escherichia coli,
A growth medium for Escherichia coli with a good growth rate and no lag phase was developed and reported (Japanese Patent Application Laid-Open No. 4-51890).
【0005】[0005]
【発明が解決しようとする課題】しかしながら、この培
地は大腸菌群の増殖には適した培地ではあるが、蛍光基
質をこの培地に組み込み、蛍光検出用の培地として用い
るには濁度が高く、蛍光の消光がみられることから蛍光
検出用の培地としては未だ充分に満足し得るものではな
かった。そこで、より透明度が高く、大腸菌群の蛍光検
出に適した培地の開発が望まれていた。However, although this medium is a medium suitable for the growth of Escherichia coli, it has a high turbidity when a fluorescent substrate is incorporated into this medium and used as a medium for fluorescence detection. Quenching was not yet fully satisfactory as a medium for fluorescence detection. Therefore, development of a medium having higher transparency and suitable for detecting the fluorescence of coliform bacteria has been desired.
【0006】[0006]
【課題を解決するための手段】かかる実情において、本
発明者らは前記特開平4−51890号公報記載の培地
について種々の検討を行ったところ、この培地に含有さ
れている牛胆汁末は大腸菌群等のグラム陰性菌以外の微
生物の生育を抑制し、かつ大腸菌群の生育を促進させる
作用を有するが、その反面、培地の濁度を上昇させ、か
つ蛍光の消光原因となっていることを見出した。そこ
で、当該牛胆汁末に代わり得る成分を探索したところ、
意外にもドデシル硫酸ナトリウムを牛胆汁末に代えて添
加した培地は、濁度が上昇しないだけでなく、これを用
いれば蛍光発現時間が短縮され、より迅速な大腸菌群検
出ができることを見出し、本発明を完成した。Under such circumstances, the present inventors have conducted various studies on the medium described in Japanese Patent Application Laid-Open No. 4-51890, and found that the bovine bile powder contained in this medium is Although it has the effect of suppressing the growth of microorganisms other than Gram-negative bacteria such as groups and promoting the growth of Escherichia coli, it increases the turbidity of the culture medium and causes quenching of fluorescence. I found it. Therefore, when searching for a component that can replace the bovine bile powder,
Surprisingly, the medium to which sodium dodecyl sulfate was added instead of bovine bile powder not only did not increase the turbidity, but also found that the use of this shortened the fluorescence expression time and enabled a more rapid detection of coliform bacteria. Completed the invention.
【0007】すなわち、本発明は、重量で、ペプトン
1.0〜2.5%、塩化ナトリウム0.25〜0.5
%、酵母エキス0.4〜0.6%、リン酸一水素二カリ
ウム0.2〜0.5%、ピルビン酸ナトリウム0.05
〜5.0%、硝酸カリウム0.05〜1.0%、ドデシ
ル硫酸ナトリウム0.01〜1.0%及び4−メチル−
ウンベリフェリル−β−D−ガラクトシド又は4−メチ
ル−ウンベリフェリル−β−D−グルクロニドを含有す
る大腸菌群検出用培地を提供するものである。That is, the present invention relates to a peptone of 1.0 to 2.5% by weight and a sodium chloride of 0.25 to 0.5%.
%, Yeast extract 0.4-0.6%, dipotassium monohydrogen phosphate 0.2-0.5%, sodium pyruvate 0.05
-5.0%, potassium nitrate 0.05-1.0%, sodium dodecyl sulfate 0.01-1.0% and 4-methyl-
It is intended to provide a medium for detecting coliform bacteria containing umbelliferyl-β-D-galactoside or 4-methyl-umbelliferyl-β-D-glucuronide.
【0008】本発明の培地は、前記のようにドデシル硫
酸ナトリウムを含有することを特徴とするが、その配合
量は0.01〜1重量%であり、0.01重量%未満で
は大腸菌群等のグラム陰性菌以外の菌種の抑制が充分で
なく、1重量%を超えると蛍光強度の低下がみられ、好
ましくない。[0008] The medium of the present invention is characterized by containing sodium dodecyl sulfate as described above. The amount of the medium is 0.01 to 1% by weight. Of the bacteria other than Gram-negative bacteria is not sufficient, and if it exceeds 1% by weight, the fluorescence intensity decreases, which is not preferable.
【0009】また、本発明培地の成分である4−メチル
−ウンベリフェリル−β−D−ガラクトシド(4−MU
Gal)及び4−メチル−ウンベリフェリル−β−D−
グルクロニド(4−MUGul)は、蛍光性基質であ
る。4−MUGal及び4−MUGulは通常無色であ
り、このうち4−MUGalは大腸菌群が存在すると、
これが産生するβ−ガラクトシダーゼの作用によって4
−メチル−ウンベリフェロン(4−MU)が生成し、4
−MUGulは大腸菌の中のエシェリシア コリ(Es
cherichia coli)が存在すると、これが
産生するグルクロニダーゼの作用によって4−MUが生
成する。かくして生成した4−MUは、蛍光性物質であ
り、365nmの波長で励起されて、450nmの蛍光を発
するので、これを測定することにより大腸菌群の存在を
確認することができる。Further, 4-methyl-umbelliferyl-β-D-galactoside (4-MU) which is a component of the medium of the present invention is used.
Gal) and 4-methyl-umbelliferyl-β-D-
Glucuronide (4-MUGul) is a fluorescent substrate. 4-MUGal and 4-MUGul are usually colorless, of which 4-MUGal is
By the action of β-galactosidase produced by this, 4
-Methyl-umbelliferone (4-MU) produces 4
-MUGul is Escherichia coli (Es
In the presence of C. cherichia coli, 4-MU is produced by the action of the glucuronidase it produces. The thus generated 4-MU is a fluorescent substance and is excited at a wavelength of 365 nm to emit fluorescence at 450 nm. By measuring this, the presence of the coliform group can be confirmed.
【0010】4−MUGal又は4−MUGulは、本
発明の培地中に10-4〜10-2M程度添加するのが好ま
しい。本発明培地の検出対象である大腸菌群とは、ラク
トース分解酵素を産生する能力を有する一群の微生物
で、エシェリシア属、サイトロバクター属、クレブシエ
ラ属、エンテロバクター属等に属するものをいう。4-MUGal or 4-MUGul is preferably added to the culture medium of the present invention in an amount of about 10 -4 to 10 -2 M. The Escherichia coli group to be detected in the medium of the present invention refers to a group of microorganisms having the ability to produce a lactose-degrading enzyme, belonging to the genus Escherichia, the genus Cytoobacter, the genus Klebsiella, the genus Enterobacter, and the like.
【0011】本発明の培地は、前記成分を混合して液体
培地として用いることもできるが、これに寒天0.1〜
1.5重量%配合して寒天培地として用いることもでき
る。The medium of the present invention can be used as a liquid medium by mixing the above components.
1.5% by weight may be used as an agar medium.
【0012】本発明の大腸菌群検出用培地を用いて、被
検体中の大腸菌群の存在を検出するには、当該培地に被
検体を加えて30〜44.5℃で数時間〜十数時間培養
した後、365nmの光を照射し、450nmの蛍光を測定
すればよい。In order to detect the presence of coliforms in a subject using the medium for detecting coliforms of the present invention, the subject is added to the medium and the mixture is added at 30 to 44.5 ° C. for several hours to several tens of hours. After the culturing, it may be irradiated with light at 365 nm and the fluorescence at 450 nm may be measured.
【0013】[0013]
【実施例】以下、実施例を挙げて更に詳細に説明する
が、本発明はこれらに限定されるものではない。EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
【0014】実施例1 特開平4−51890号公報記載の大腸菌群検出用培地
を基礎培地として、改変培地1〜4を作製し、いずれの
培地が蛍光検出に適するかについて試験を行った。表1
に示す組成の培地を96穴マイクロプレートに0.1ml
ずつ注加し、このプレートに表2記載の菌を接種後、3
5℃で培養を行い、経時的に各プレートに蛍光リーダー
(コロナ社製,MTP−32)を用いて365nmの光を
照射し、450nmの蛍光が発現するまでの時間を測定し
た。この結果を表2に示す。Example 1 Modified mediums 1 to 4 were prepared using a medium for detecting coliform bacteria described in JP-A-4-51890 as a basal medium, and a test was conducted to determine which medium was suitable for fluorescence detection. Table 1
0.1 ml of a medium having the composition shown in
After inoculating the plate with the bacteria described in Table 2, 3
Culture was performed at 5 ° C., and each plate was irradiated with light of 365 nm over time using a fluorescent reader (manufactured by Corona, MTP-32), and the time until 450 nm of fluorescence appeared was measured. Table 2 shows the results.
【0015】[0015]
【表1】 [Table 1]
【0016】[0016]
【表2】 [Table 2]
【0017】この結果から、本発明の培地である改変4
の培地がいずれの菌株においても蛍光検出時間が他の培
地に比べて1〜2時間以上も短縮されることが分かる。From these results, it is found that the medium of the present invention, modified 4
It can be seen that the fluorescence detection time is shortened by 1 to 2 hours or more in any of the strains in any of the strains.
【0018】実施例2 本発明培地を用いて市販食品の大腸菌群検査を行った。
本発明培地を24穴透明プレートに1mlずつ分注してお
き、そこに表3記載の食品10gに90mlの滅菌生理食
塩水を加えて均一になるようにストマッキングした試料
の1mlを接種して35℃で培養した。培養開始後6、
7、8及び24時間目に蛍光リーダー(コロナ社製,M
TP−32)を用いて365nmの光を照射し450nmの
蛍光を測定した(蛍光プレート法)。他方、ストマッキ
ングした試料を10倍段階希釈し、その1mlを滅菌シャ
ーレにとり、ここにあらかじめ加温溶解し45℃程度に
冷却してあるデゾキシコレート培地を注ぎ、培地と試料
をよく混合し、冷却重層後、35℃、20時間培養した
後、菌数を算出した(Deso混釈法)。この結果を表
3に示す。表3より、本発明培地を用いた蛍光プレート
法は従来法であるDeso混釈法とよく一致しているこ
とが分かり、更にDeso混釈法は最低18時間の培養
時間が必要なのに対し蛍光プレート法は6〜8時間の培
養時間で大腸菌群の確認ができた。Example 2 A commercially available food was tested for coliform bacteria using the medium of the present invention.
1 ml of the medium of the present invention was dispensed into a 24-well transparent plate, and 90 ml of sterile physiological saline was added to 10 g of the food shown in Table 3 to inoculate 1 ml of a uniformly stomached sample. Incubated at ℃. After the start of culture 6,
Fluorescence reader (Corona, M
Using TP-32), light of 365 nm was irradiated and fluorescence of 450 nm was measured (fluorescence plate method). On the other hand, the stomached sample is serially diluted 10-fold, and 1 ml of the diluted sample is placed in a sterile petri dish. A desoxycholate medium, which has been heated and dissolved and cooled to about 45 ° C., is poured therein, and the medium and the sample are mixed well. After culturing at 35 ° C. for 20 hours, the number of bacteria was calculated (Deso pour method). Table 3 shows the results. From Table 3, it can be seen that the fluorescent plate method using the medium of the present invention is in good agreement with the conventional Deso pour method, and the Deso pour method requires at least 18 hours of culture time, whereas the fluorescent plate method requires at least 18 hours. The method was able to confirm the coliform bacteria in a culture time of 6 to 8 hours.
【0019】[0019]
【表3】 [Table 3]
【0020】[0020]
【発明の効果】本発明により食品等の検体中の大腸菌群
が存在するか否かを迅速かつ高感度に判定することがで
きる検査が可能となった。According to the present invention, it has become possible to carry out a test capable of quickly and highly sensitively determining whether or not coliforms are present in a sample such as food.
───────────────────────────────────────────────────── フロントページの続き (58)調査した分野(Int.Cl.7,DB名) C12Q 1/10 C12N 1/20 BIOSIS(DIALOG)──────────────────────────────────────────────────の Continued on the front page (58) Field surveyed (Int. Cl. 7 , DB name) C12Q 1/10 C12N 1/20 BIOSIS (DIALOG)
Claims (1)
化ナトリウム0.25〜0.5%、酵母エキス0.4〜
0.6%、リン酸一水素二カリウム0.2〜0.5%、
ピルビン酸ナトリウム0.05〜5.0%、硝酸カリウ
ム0.05〜1.0%、ドデシル硫酸ナトリウム0.0
1〜1.0%及び4−メチル−ウンベリフェリル−β−
D−ガラクトシド又は4−メチル−ウンベリフェリル−
β−D−グルクロニドを含有する大腸菌群蛍光検出用培
地。1. Peptone 1.0-2.5%, sodium chloride 0.25-0.5%, yeast extract 0.4-2.5% by weight
0.6%, dipotassium monohydrogen phosphate 0.2-0.5%,
Sodium pyruvate 0.05-5.0%, potassium nitrate 0.05-1.0%, sodium dodecyl sulfate 0.0
1-1.0% and 4-methyl-umbelliferyl-β-
D-galactoside or 4-methyl-umbelliferyl-
An Escherichia coli group fluorescence detection medium containing β-D-glucuronide.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP19837392A JP3142962B2 (en) | 1992-07-24 | 1992-07-24 | Medium for fluorescence detection of coliform bacteria |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP19837392A JP3142962B2 (en) | 1992-07-24 | 1992-07-24 | Medium for fluorescence detection of coliform bacteria |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH0646891A JPH0646891A (en) | 1994-02-22 |
JP3142962B2 true JP3142962B2 (en) | 2001-03-07 |
Family
ID=16390039
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP19837392A Expired - Lifetime JP3142962B2 (en) | 1992-07-24 | 1992-07-24 | Medium for fluorescence detection of coliform bacteria |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP3142962B2 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996014431A1 (en) * | 1994-11-07 | 1996-05-17 | Studie-En Samenwerkingverband Vlaams Water | Enzymatic method for detecting coliform bacteria or e. coli |
GB2386946A (en) * | 2002-03-27 | 2003-10-01 | Danisco | Detecting microorganisms |
-
1992
- 1992-07-24 JP JP19837392A patent/JP3142962B2/en not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
JPH0646891A (en) | 1994-02-22 |
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