JP6621111B2 - Bacteria inspection method - Google Patents
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- JP6621111B2 JP6621111B2 JP2015233396A JP2015233396A JP6621111B2 JP 6621111 B2 JP6621111 B2 JP 6621111B2 JP 2015233396 A JP2015233396 A JP 2015233396A JP 2015233396 A JP2015233396 A JP 2015233396A JP 6621111 B2 JP6621111 B2 JP 6621111B2
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- 241000894006 Bacteria Species 0.000 title claims description 136
- 238000000034 method Methods 0.000 title claims description 17
- 238000007689 inspection Methods 0.000 title description 3
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 claims description 94
- 229910000037 hydrogen sulfide Inorganic materials 0.000 claims description 94
- 241000607142 Salmonella Species 0.000 claims description 60
- 238000001514 detection method Methods 0.000 claims description 42
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- 241000588724 Escherichia coli Species 0.000 description 13
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- 241000293871 Salmonella enterica subsp. enterica serovar Typhi Species 0.000 description 9
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- 230000001580 bacterial effect Effects 0.000 description 3
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は、菌の検査方法に関する。より詳しくは、硫化水素産生菌又は有機酸産生菌を検出することが可能な技術に関する。 The present invention relates to a method for examining bacteria. More specifically, the present invention relates to a technique capable of detecting hydrogen sulfide-producing bacteria or organic acid-producing bacteria.
細菌の中には、その増殖過程において硫化水素を産生するものがある。中でもサルモネラ属菌の多くは、硫化水素の産生が著しいことが一般的に知られている。 Some bacteria produce hydrogen sulfide during their growth process. Among them, it is generally known that many Salmonella spp. Produce significant hydrogen sulfide.
サルモネラ属菌は、通常、サルモネラ属菌標準試験法NIHSJ-01-ST4（090218）に示された方法で鑑別する。具体的には、ＲＶ培地とＴＴ培地で選択増菌培養後、２種類の分離寒天培地（ＤＨＬ培地等の硫化水素産生性で検出する培地と、ＥＳ培地等の硫化水素産生性に関係なくサルモネラを検出する培地の、それぞれ１種類）に塗抹培養し、集落の産生を検討する。そして、サルモネラと疑われる集落３個をＴＳＩ寒天培地及びＬＩＭ培地に接種し、生化学的性状の確認を行う。更に、抗Ｏ血清による凝集反応により、Ｏ抗原の血清型別を実施してサルモネラ属菌と確定する。 Salmonella is usually differentiated by the method shown in Salmonella standard test method NIHSJ-01-ST4 (090218). Specifically, after selective enrichment culture in RV medium and TT medium, two types of separation agar medium (a medium detected by hydrogen sulfide productivity such as DHL medium, and Salmonella regardless of hydrogen sulfide productivity such as ES medium) Are smeared on one type of medium to detect the colony, and the production of the village is examined. Then, three colonies suspected of being Salmonella are inoculated into TSI agar medium and LIM medium, and biochemical properties are confirmed. Furthermore, the serotype of the O antigen is determined by an agglutination reaction with anti-O serum to establish Salmonella.
また、出願人自身も、発明者として以前に、硫黄源を含有する寒天培地と、アスコルビン酸又はクエン酸水溶液を含有する担体とで構成されることを特徴とする、サルモネラ菌検出用デバイスを提案している（特許文献１参照）。 In addition, the applicant himself has previously proposed a device for detecting Salmonella, comprising an agar medium containing a sulfur source and a carrier containing an aqueous solution of ascorbic acid or citric acid. (See Patent Document 1).
しかしながら、上述した標準試験法は、人の便検体や、食肉、食鳥卵等の食品検体などから検出されたサルモネラ野生株を分離する場合には有効であるが、菌株として保存培地に長期間保存されたものやチフス菌などを用いた場合には、菌の発育状況が悪く、生化学的な性状も十分に発現しないため、増菌培養の必要があることが問題となっていた。また、特に、ＤＨＬ培地等の選択分離培地を用いた場合において、単独の集落をサルモネラ属菌が形成している場合には硫化水素を産生していることが認められるが、菌が密集していると硫化水素を目視することが不可能であるという弱点があった。 However, the standard test method described above is effective when isolating Salmonella wild strains detected from human stool specimens or food specimens such as meat and poultry eggs. In the case of using a preserved bacterium or Salmonella typhi, the growth of the bacterium is poor, and the biochemical properties are not fully expressed. In particular, when a selective separation medium such as a DHL medium is used, when Salmonella is formed in a single colony, it is recognized that hydrogen sulfide is produced. When it was, there was a weak point that it was impossible to see hydrogen sulfide.
更に、特許文献１に記載された方法では、サルモネラ菌検出用デバイスとして用いられるアスコルビン酸又はクエン酸を含ませたろ紙が劣化しやすく、作成にも手間がかかるという問題があった。 Furthermore, the method described in Patent Document 1 has a problem in that filter paper containing ascorbic acid or citric acid used as a device for detecting Salmonella is likely to deteriorate, and it takes time and effort to produce it.
そこで、本発明では、迅速かつ簡便に、硫化水素産生菌又は有機酸産生菌を検出する技術を提供することを主目的とする。 Therefore, the main object of the present invention is to provide a technique for detecting hydrogen sulfide-producing bacteria or organic acid-producing bacteria quickly and easily.
本願発明者らは、上記課題を解決するため硫化水素産生菌を検出する技術について鋭意研究を行い、サルモネラ属菌等の硫化水素産生菌を、腸内細菌であり代謝の際に乳酸等有機酸を産生する、大腸菌、大腸菌群等の有機酸産生菌と接触させることを試みた。その結果、有機酸産生菌は、硫化水素産生菌の硫化水素産生能を高め、硫化水素の産生を顕著に促進する事実を見出した。 In order to solve the above-mentioned problems, the inventors of the present application have conducted intensive research on a technique for detecting hydrogen sulfide-producing bacteria, and have made hydrogen sulfide-producing bacteria such as Salmonella genus bacteria enteric bacteria and organic acids such as lactic acid during metabolism. It tried to contact organic acid-producing bacteria such as Escherichia coli and coliforms that produce. As a result, it has been found that organic acid producing bacteria enhance the hydrogen sulfide producing ability of hydrogen sulfide producing bacteria and significantly promote the production of hydrogen sulfide.
すなわち、本発明では、まず、硫黄源と鉄源とを含有した培地に対象の菌を密集させて接種した後、前記培地に大腸菌群を接種し、所定時間培養する培養工程、
前記培養工程の後、前記対象の菌からの硫化水素の発生を検出する検出工程、
を少なくとも有し、
前記検出工程では、前記培地における前記大腸菌群を接種した箇所に、前記対象の菌からの硫化水素が発生している場合、前記対象の菌をサルモネラ菌と判定する、菌の検出方法を提供する。
また、硫黄源と鉄源とを含有した培地にサルモネラ菌を密集させて接種した後、前記培地に対象の菌を接種し、所定時間培養する培養工程、
前記培養工程の後、前記サルモネラ菌からの硫化水素の発生を検出する検出工程、
を少なくとも有し、
前記検出工程では、前記培地における前記対象の菌を接種した箇所に、前記サルモネラ菌からの硫化水素が発生している場合、前記対象の菌を大腸菌群と判定する、菌の検出方法も提供する。
更に、硫黄源と鉄源とを含有した培地に大腸菌群を密集させて接種した後、前記培地に対象の菌を接種し、所定時間培養する培養工程、
前記培養工程の後、前記対象の菌からの硫化水素の発生を検出する検出工程、
を少なくとも有し、
前記検出工程では、前記培地における前記対象の菌を接種した箇所に、前記対象の菌からの硫化水素が発生している場合、前記対象の菌をサルモネラ菌と判定する、菌の検出方法も提供する。
更にまた、硫黄源と鉄源とを含有した培地に対象の菌を密集させて接種した後、前記培地にサルモネラ菌を接種し、所定時間培養する培養工程、
前記培養工程の後、前記サルモネラ菌からの硫化水素の発生を検出する検出工程、
を少なくとも有し、
前記検出工程では、前記培地における前記サルモネラ菌を接種した箇所に、前記サルモネラ菌からの硫化水素が発生している場合、前記対象の菌を大腸菌群と判定する、菌の検出方法も提供する。
That is, in the present invention, first, after inoculating and inoculating the target fungus in a medium containing a sulfur source and an iron source, a culture step of inoculating the coliform in the medium and culturing for a predetermined time,
After the culturing step, a detection step of detecting the generation of hydrogen sulfide from the target fungus,
Having at least
In the detection step, there is provided a method for detecting a bacterium, in which, when hydrogen sulfide from the target bacterium is generated at a site inoculated with the coliform group in the medium, the bacterium of the target is determined to be Salmonella .
In addition, after inoculating and inoculating Salmonella in a medium containing a sulfur source and an iron source, inoculating the target bacteria in the medium, and culturing for a predetermined time,
After the culturing step, a detection step of detecting generation of hydrogen sulfide from the Salmonella ,
Having at least
In the detection step, there is also provided a method for detecting a bacterium, wherein hydrogen sulfide from the Salmonella bacterium is generated at a location where the bacterium of the object is inoculated in the medium, and the bacterium of the object is determined as an coliform group .
Furthermore, after inoculating and inoculating coliforms in a medium containing a sulfur source and an iron source, inoculating the target bacteria in the medium and culturing for a predetermined time,
After the culturing step, a detection step of detecting the generation of hydrogen sulfide from the target fungus,
Having at least
In the detection step, when hydrogen sulfide is generated from the target bacteria at a location where the target bacteria are inoculated in the medium, a method for detecting the bacteria is also provided in which the target bacteria are determined to be Salmonella. .
Furthermore, after inoculating and inoculating the target fungus in a medium containing a sulfur source and an iron source, inoculating Salmonella in the medium and culturing for a predetermined time,
After the culturing step, a detection step of detecting generation of hydrogen sulfide from the Salmonella ,
Having at least
Wherein in the detection step, the portion inoculated with the Salmonella in the medium, if the hydrogen sulfide from the Salmonella is occurring, it determines bacteria of the target coliforms also provides method for detecting bacteria.
本発明によれば、迅速かつ簡便に、硫化水素産生菌又は有機酸産生菌を検出することが可能となる。なお、ここに記載された効果は、必ずしも限定されるものではなく、本開示中に記載されたいずれかの効果であってもよい。 According to the present invention, hydrogen sulfide-producing bacteria or organic acid-producing bacteria can be detected quickly and easily. Note that the effects described here are not necessarily limited, and may be any of the effects described in the present disclosure.
以下、本発明を実施するための好適な形態について図面を参照しながら説明する。なお、以下に説明する実施形態は、本発明の代表的な実施形態の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。 DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.
図１は、サルモネラ属菌をシャーレ全面に密集させて接種（塗抹）し、培養した場合の培地の様子を撮影した図面代用写真である。通常は、図１の図面代用写真に示すように、サルモネラ属菌を密集させて接種すると、硫化水素の発生は起きない。 FIG. 1 is a drawing-substituting photograph in which the state of a culture medium when Salmonella spp. Usually, as shown in the drawing substitute photograph of FIG. 1, when Salmonella spp.
しかし、本願発明者らは、硫化水素産生菌を有機酸産生菌と接触させることを試みたところ、有機酸産生菌は、硫化水素産生菌の硫化水素産生能を高め、硫化水素の産生を顕著に促進する事実を見出した。 However, the inventors of the present application tried to bring hydrogen sulfide-producing bacteria into contact with organic acid-producing bacteria. The organic acid-producing bacteria increased hydrogen sulfide-producing ability of hydrogen sulfide-producing bacteria and markedly produced hydrogen sulfide. Found the fact that promotes.
より具体的には、サルモネラ属菌等の硫化水素産生菌は、大腸菌、大腸菌群等の有機酸産生菌が産生する有機酸を栄養源として代謝し、硫化水素産生を促進させ、培地上で硫化鉄（FeS）を発現させることが分かった。この硫化鉄の発現は肉眼で確認可能であることから、本発明に係る検出方法を用いれば、硫化水素を産生する菌（硫化水素産生菌）を目視にて検出できる。 More specifically, hydrogen sulfide-producing bacteria such as Salmonella are metabolized by using organic acids produced by organic acid-producing bacteria such as Escherichia coli and coliforms as nutrient sources to promote hydrogen sulfide production and sulfide on the medium. It was found that iron (FeS) was expressed. Since the expression of this iron sulfide can be confirmed with the naked eye, bacteria that produce hydrogen sulfide (hydrogen sulfide producing bacteria) can be visually detected using the detection method according to the present invention.
また、この事実から、有機酸産生菌は、硫化水素産生菌と共生関係にあることが分かり、硫化水素産生菌を用いて、逆に有機酸産生菌を検出できることも判明した。 From this fact, it was also found that the organic acid producing bacteria are in a symbiotic relationship with the hydrogen sulfide producing bacteria, and it was also found that the organic acid producing bacteria can be detected using the hydrogen sulfide producing bacteria.
本発明に係る検出方法を用いれば、迅速かつ簡便に、硫化水素産生菌又は有機酸産生菌を検出することが可能となる。また、培地上に形成された硫化鉄が顕著に目視にて確認できるため、菌の検出の精度を向上することも可能となる。そのため、細菌検査の現場において、硫化水素産生菌又は有機酸産生菌を容易に分離でき、検査効率が格段に向上する。 By using the detection method according to the present invention, hydrogen sulfide-producing bacteria or organic acid-producing bacteria can be detected quickly and easily. Moreover, since the iron sulfide formed on the culture medium can be visually confirmed visually, it is possible to improve the accuracy of detecting bacteria. Therefore, hydrogen sulfide-producing bacteria or organic acid-producing bacteria can be easily separated at the site of bacterial inspection, and the inspection efficiency is greatly improved.
また、前述の通り、従来のサルモネラ菌検出用デバイスであるアスコルビン酸又はクエン酸を含ませたろ紙は、劣化しやすく、作成にも手間がかかる。しかし、本発明に係る検出方法を用いることで、常温で保存等されていた有機酸産生菌の菌株のみを接種すれば、これらが培養の過程で増殖すると共に、アスコルビン酸又はクエン酸と同様に有機酸を産生する。本発明に係る検出方法では、硫化水素産生菌がこの有機酸を栄養源として代謝する原理を用いていることから、本発明に係る検出方法を用いることで、上述したろ紙を作成する工程を行うことなく、サルモネラ属菌等の硫化水素産生菌の検出を行うことができる。 Further, as described above, the filter paper containing ascorbic acid or citric acid, which is a conventional device for detecting Salmonella, is easily deteriorated and takes time and effort. However, by using the detection method according to the present invention, if only the strains of organic acid-producing bacteria that have been stored at room temperature are inoculated, these will proliferate in the course of culturing, as well as ascorbic acid or citric acid. Produces organic acids. In the detection method according to the present invention, since the hydrogen sulfide-producing bacterium uses the principle of metabolizing this organic acid as a nutrient source, the above-described filter paper is prepared by using the detection method according to the present invention. Without detection, hydrogen sulfide-producing bacteria such as Salmonella can be detected.
更に、本発明に係る検出方法を用いれば、硫化水素産生菌又は有機酸産生菌に関する、検出用キット、検出用デバイス等を提供できる。これにより、硫化水素産生菌や有機酸産生菌を新たな生物資源としても活用できる。 Furthermore, if the detection method according to the present invention is used, a detection kit, a detection device, etc. relating to hydrogen sulfide-producing bacteria or organic acid-producing bacteria can be provided. Thereby, hydrogen sulfide-producing bacteria and organic acid-producing bacteria can be used as new biological resources.
以下、本発明に係る検出方法における各工程について、詳細に説明する。 Hereinafter, each step in the detection method according to the present invention will be described in detail.
１．培養工程（Ｉ）
本発明において、培養工程（Ｉ）は、具体的には、硫黄源と鉄源とを含有した培地に対象の菌を密集させて接種した後、前記培地に有機酸産生菌を接種し、所定時間培養する工程（Ｉ−１）、硫黄源と鉄源とを含有した培地に硫化水素産生菌を密集させて接種した後、前記培地に対象の菌を接種し、所定時間培養する工程（Ｉ−２）、硫黄源と鉄源とを含有した培地に有機酸産生菌を密集させて接種した後、前記培地に対象の菌を接種し、所定時間培養する工程（Ｉ−３）、又は、硫黄源と鉄源とを含有した培地に対象の菌を密集させて接種した後、前記培地に硫化水素産生菌を接種し、所定時間培養する工程（Ｉ−４）、である。
1. Culture process (I)
In the present invention, the culturing step (I), specifically, inoculates the target bacteria in a medium containing a sulfur source and an iron source, and then inoculates the medium with organic acid-producing bacteria. Step (I-1) of culturing for a time, Step of inoculating the medium with hydrogen sulfide-producing bacteria in a medium containing a sulfur source and an iron source, and then inoculating the medium with the target bacteria and culturing for a predetermined time (I -2) After inoculating and inoculating organic acid producing bacteria in a medium containing a sulfur source and an iron source, inoculating the medium with the target bacteria and culturing for a predetermined time (I-3), or Step (I-4) is a step (I-4) of inoculating hydrogen sulfide-producing bacteria in the medium after inoculating the medium with the target fungus in a medium containing a sulfur source and an iron source.
（１）培地
前記培地は、硫黄源と鉄源とを含有したものであれば特に限定されず、公知の培地を適宜選択して用いることができるが、寒天培地とすることが好ましい。
(1) Medium The medium is not particularly limited as long as it contains a sulfur source and an iron source, and a known medium can be appropriately selected and used, but an agar medium is preferable.
寒天培地としては、例えば、ＤＨＬ寒天培地（Desoxycholate-hydrogen sulfide-lactose）、ＳＳ寒天培地（Salmonella-Shigella）、ＳＳ−ＳＢ寒天培地（Salmonella-Shigella Sucrose Bromcresolpurple）、ＴＳＩ寒天培地（Triple Sugar Iron）等の寒天培地を挙げることができる。 Examples of the agar medium include DHL agar medium (Desoxycholate-hydrogen sulfide-lactose), SS agar medium (Salmonella-Shigella), SS-SB agar medium (Salmonella-Shigella Sucrose Bromcresolpurple), TSI agar medium (Triple Sugar Iron), etc. Can be mentioned.
前記培地に用いることができる栄養源等も特に限定されず、培養する菌の性質などに応じて適宜選択して用いることができる。
前記栄養源としては、例えば、グルコース、フルクトース、ショ糖、乳糖、澱粉、グリセリン、デキストリン、レシチン等の炭素源：硫酸アンモニウム、硝酸アンモニウム、リン酸‐アンモニウム、リン酸二アンモニウム、塩化アンモニウム等の無機窒素源：アミノ酸、ペプトン等の有機窒素源；ナトリウム、マグネシウム、カリウム、鉄、亜鉛、カルシウム、マンガン等の無機栄養源；その他各種ビタミンなどから選ばれる１種又は２種以上の栄養源などが挙げられる。
Nutrient sources that can be used in the medium are not particularly limited, and can be appropriately selected and used depending on the properties of the bacteria to be cultured.
Examples of the nutrient source include carbon sources such as glucose, fructose, sucrose, lactose, starch, glycerin, dextrin, and lecithin: inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, phosphate-ammonium phosphate, diammonium phosphate, and ammonium chloride. : Organic nitrogen sources such as amino acids and peptones; inorganic nutrient sources such as sodium, magnesium, potassium, iron, zinc, calcium and manganese; and one or more nutrient sources selected from other various vitamins.
（２）対象の菌
前記対象の菌は特に限定されず、本発明に係る検出方法では、細菌、真菌等を含めたあらゆる菌を対象とすることができる。なお、本発明では、多くの菌種を含む、所謂、「菌群」と呼ばれるものも対象とすることができる。
(2) Target bacteria The target bacteria are not particularly limited, and the detection method according to the present invention can target any bacteria including bacteria and fungi. In the present invention, a so-called “bacteria group” including many bacterial species can also be used.
（３）硫化水素産生菌
前記硫化水素産生菌は、硫化水素を産生する菌であれば特に限定されず、例えば、サルモネラ属、シトロバクター属、プロテウス属、エドワージエラ属等に属する菌などが挙げられるが、本発明では、これらの中でも特に、サルモネラ属菌が好ましい。なお、本発明では、前記硫化水素産生菌には、パラチフスＡ菌のように、サルモネラ属菌の中でも硫化水素を産生しない菌は含まれない。
(3) Hydrogen sulfide-producing bacteria The hydrogen sulfide-producing bacteria are not particularly limited as long as they produce hydrogen sulfide, and examples include bacteria belonging to the genus Salmonella, Citrobacter, Proteus, Edwardsiella, etc. However, in the present invention, Salmonella is particularly preferable among these. In the present invention, the hydrogen sulfide-producing bacteria do not include bacteria that do not produce hydrogen sulfide among Salmonella species, such as Paratyphi A bacteria.
また、本発明に係る菌の検出方法を用いれば、硫化水素産生菌の中でも、後述する実施例４で示すように、チフス菌のような硫化水素産生能が弱い細菌であっても検出が可能である。このチフス菌によって引き起こされる感染症（例えば、腸チフスなど）は、未だ衛生環境の悪い地域や発展途上国等で流行しており、非常に問題となっている。本発明により、検査時間の短縮、検出効率の向上を図ることで、硫化水素産生能が弱いチフス菌を含め、硫化水素産生菌による感染症の予防、早期発見等にも役立つ。 In addition, by using the method for detecting a bacterium according to the present invention, even among hydrogen sulfide-producing bacteria, as shown in Example 4 described later, it is possible to detect even a bacterium with weak hydrogen sulfide-producing ability such as Salmonella typhi. It is. Infectious diseases caused by Salmonella typhi (for example, typhoid fever) are still prevalent in regions with poor sanitation and developing countries, and are extremely problematic. By shortening the examination time and improving the detection efficiency according to the present invention, it is useful for the prevention and early detection of infections caused by hydrogen sulfide-producing bacteria, including Salmonella typhi, which has a weak ability to produce hydrogen sulfide.
更に、サルモネラ属菌等の硫化水素産生菌は、長期保存すると、硫化水素産生能が低下することも知られている。しかし、後述する実施例１〜４では、いずれも半年保存したサルモネラ属菌を用いているにもかかわらず、硫化水素の産生を検出することができた。したがって、本発明に係る菌の検出方法を用いることにより、長期保存して硫化水素産生能が低下した硫化水素産生菌も、検出することが可能となる。 Furthermore, it is also known that hydrogen sulfide-producing bacteria such as Salmonella spp. Decrease in hydrogen sulfide-producing ability when stored for a long time. However, in Examples 1 to 4 described later, production of hydrogen sulfide could be detected in spite of using Salmonella spp. Stored for half a year. Therefore, by using the method for detecting a bacterium according to the present invention, it is possible to detect a hydrogen sulfide-producing bacterium that has been stored for a long time and has reduced hydrogen sulfide-producing ability.
（４）有機酸産生菌
前記有機酸産生菌は、乳酸、酪酸、酢酸等の有機酸を産生する菌であれば特に限定されず、例えば、大腸菌、大腸菌群、乳酸菌などが挙げられるが、本発明では、これらの中でも特に、大腸菌、大腸菌群が好ましい。このように、本発明では、有機酸産生菌として、Escherichia coli、Citrobacter、Klebsiella、Enterobacter、Proteus等の多くの菌種を含む、大腸菌群を用いてもよい。
(4) Organic acid-producing bacteria The organic acid-producing bacteria are not particularly limited as long as they produce organic acids such as lactic acid, butyric acid, and acetic acid, and examples thereof include Escherichia coli, coliform bacteria, and lactic acid bacteria. In the invention, among these, Escherichia coli and coliforms are particularly preferable. Thus, in the present invention, Escherichia coli group including many bacterial species such as Escherichia coli, Citrobacter, Klebsiella, Enterobacter, Proteus, etc. may be used as the organic acid producing bacteria.
（５）培養条件
本発明に係る検出方法における培養条件は特に限定されず、培養する菌の性質などに応じて、培養温度、培養時間等を適宜自由に設定することができ、具体的には、例えば、３７℃で６時間、又は３７℃で２４時間培養する条件等が挙げられる。
(5) Culture conditions The culture conditions in the detection method according to the present invention are not particularly limited, and the culture temperature, culture time, etc. can be set as appropriate according to the nature of the bacteria to be cultured, specifically, For example, conditions for culturing at 37 ° C. for 6 hours or 37 ° C. for 24 hours can be mentioned.
２．検出工程（ＩＩ）
本発明において、検出工程（ＩＩ）は、具体的には、培養工程（Ｉ−１）の後、前記対象の菌からの硫化水素の発生を検出する工程（ＩＩ−１）、培養工程（Ｉ−２）の後、前記硫化水素産生菌からの硫化水素の発生を検出する工程（ＩＩ−２）、培養工程（Ｉ−３）の後、前記対象の菌からの硫化水素の発生を検出する工程（ＩＩ−３）、又は、培養工程（Ｉ−４）の後、前記硫化水素産生菌からの硫化水素の発生を検出する工程（ＩＩ−４）、である。
2. Detection step (II)
In the present invention, specifically, the detection step (II) is a step (II-1) of detecting the generation of hydrogen sulfide from the target bacteria after the culture step (I-1), and the culture step (I). -2) After the step (II-2) for detecting the generation of hydrogen sulfide from the hydrogen sulfide-producing bacterium and the culturing step (I-3), the generation of hydrogen sulfide from the target bacterium is detected. It is a step (II-4) of detecting the generation of hydrogen sulfide from the hydrogen sulfide-producing bacterium after the step (II-3) or the culturing step (I-4).
（１）菌の検出方法
検出工程（ＩＩ−１）では、具体的には、前記培地における前記有機酸産生菌を接種した箇所に、前記対象の菌からの硫化水素が発生している場合、前記対象の菌を硫化水素産生菌と判定する。また、検出工程（ＩＩ−２）では、前記培地における前記対象の菌を接種した箇所に、前記硫化水素産生菌からの硫化水素が発生している場合、前記対象の菌を有機酸産生菌と判定する。更に、検出工程（ＩＩ−３）では、前記培地における前記対象の菌を接種した箇所に、前記対象の菌からの硫化水素が発生している場合、前記対象の菌を硫化水素産生菌と判定する。更にまた、検出工程（ＩＩ−４）では、前記培地における前記硫化水素産生菌を接種した箇所に、前記硫化水素産生菌からの硫化水素が発生している場合、前記対象の菌を有機酸産生菌と判定する。
(1) Bacterium detection method In the detection step (II-1), specifically, when hydrogen sulfide from the target fungus is generated at the site where the organic acid-producing bacteria are inoculated in the medium, The target bacterium is determined as a hydrogen sulfide-producing bacterium. In addition, in the detection step (II-2), when hydrogen sulfide from the hydrogen sulfide-producing bacterium is generated at the location inoculated with the target bacterium in the medium, the target bacterium is regarded as an organic acid-producing bacterium. judge. Furthermore, in the detection step (II-3), when hydrogen sulfide from the target bacterium is generated at the location inoculated with the target bacterium in the medium, the target bacterium is determined as a hydrogen sulfide-producing bacterium. To do. Furthermore, in the detection step (II-4), when hydrogen sulfide from the hydrogen sulfide-producing bacterium is generated at the site inoculated with the hydrogen sulfide-producing bacterium in the medium, the target bacterium is produced as an organic acid. Judge as fungus.
本発明において、対象の菌又は硫化水素産生菌からの、硫化水素発生の具体的な検出方法は特に限定されず、例えば、前記培地における、有機酸産生菌、対象の菌、又は硫化水素産生菌を接種した箇所の色調変化を解析することで、硫化水素の発生を検出することが可能である。 In the present invention, a specific method for detecting hydrogen sulfide generation from the target bacterium or hydrogen sulfide-producing bacterium is not particularly limited. For example, the organic acid-producing bacterium, the target bacterium, or the hydrogen sulfide-producing bacterium in the medium is used. It is possible to detect the generation of hydrogen sulfide by analyzing the change in color tone at the location where the seed is inoculated.
より具体的な一例を挙げると、硫黄源と鉄源とを含有した培地に硫化水素産生菌を接種し、該培地上の所定の箇所に有機酸産生菌を接種し、所定時間培養すると、該有機酸産生菌を接種した培地の箇所のみ黒色に変色する。そのため、培地が黒色に変色するか否かを肉眼にて検出することで、硫化水素の発生を検出できる。 To give a more specific example, inoculate a medium containing a sulfur source and an iron source with a hydrogen sulfide-producing bacterium, inoculate a predetermined location on the medium with an organic acid-producing bacterium, and culture for a predetermined time. Only the portion of the medium inoculated with the organic acid producing bacteria turns black. Therefore, the generation of hydrogen sulfide can be detected by detecting with the naked eye whether or not the medium turns black.
以下、実施例に基づいて本発明を更に詳細に説明する。なお、以下に説明する実施例は、本発明の代表的な実施例の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。 Hereinafter, the present invention will be described in more detail based on examples. In addition, the Example demonstrated below shows an example of the typical Example of this invention, and, thereby, the range of this invention is not interpreted narrowly.
＜実施例１＞
本実施例１では、縦８ｃｍ、横２５ｃｍのシャーレにＤＨＬ寒天培地を作製し、該ＤＨＬ寒天培地に非チフスサルモネラ属菌を密集させて全面に接種（塗抹）し、１辺２０ｍｍの正方形各格子に大腸菌群をｎ＝３で１〜１１個点状にそれぞれ接種し、培養前（図２のＡ参照）、３７℃で６時間後（図２のＢ参照）、３７℃で２４時間後（図２のＣ参照）に、目視にて観察した。
<Example 1>
In this Example 1, a DHL agar medium was prepared in a petri dish of 8 cm in length and 25 cm in width, non-typhoidal Salmonella was inoculated on the DHL agar medium and inoculated (smeared) on the entire surface, and each square grid with a side of 20 mm E. coli were inoculated into 1 to 11 dots at n = 3, before culturing (see A in FIG. 2), after 6 hours at 37 ° C. (see B in FIG. 2), and after 24 hours at 37 ° C. (See FIG. 2C).
図２のＡ〜Ｃの図面代用写真に示す通り、大腸菌群を接種した箇所のみ黒色変化を認め、時間の経過とともに黒色が拡大していくのを目視にて確認した。 As shown in FIGS. 2A to 2C, the black color change was observed only at the site where the coliform group was inoculated, and it was visually confirmed that the black color expanded with the passage of time.
すなわち、サルモネラ属菌が培養されたＤＨＬ寒天培地上に接種された大腸菌群の周囲に、硫化鉄(FeS)の黒色反応円が出現する現象が認められた。その一方で、大腸菌群を接種しなかった場合では、この現象が認められなかった。したがって、大腸菌群の周囲では、サルモネラ属菌が嫌気条件下で硫化水素を産生し、増殖していることを目視にて確認できた。これにより、大腸菌群によってサルモネラ属菌の硫化水素産生能が促進されることが推察された。 That is, a phenomenon was observed in which a black reaction circle of iron sulfide (FeS) appeared around the coliform group inoculated on the DHL agar medium in which Salmonella was cultured. On the other hand, this phenomenon was not observed when the coliform group was not inoculated. Therefore, it was confirmed visually that Salmonella spp. Produced hydrogen sulfide under anaerobic conditions and proliferated around the coliform group. Thereby, it was guessed that Escherichia coli group promoted the hydrogen sulfide production ability of Salmonella.
＜実施例２＞
本実施例２では、縦８ｃｍ、横２５ｃｍのシャーレにＤＨＬ寒天培地を作製し、該ＤＨＬ寒天培地に非チフスサルモネラ属菌を密集させて全面に接種し、１辺２０ｍｍの正方形各格子に大腸菌群をｎ＝３で１〜１１個点状にそれぞれ接種し、３７℃で６時間後（図３のＡ参照）、３７℃で２４時間後（図３のＢ参照）に、目視にて観察した。
<Example 2>
In this Example 2, a DHL agar medium is prepared in a petri dish 8 cm long and 25 cm wide, non-typhoidal Salmonella is densely inoculated on the DHL agar medium, inoculated on the entire surface, and E. coli group is placed on each square 20 mm square. N = 3 and inoculated in the form of 1 to 11 dots, which were visually observed after 6 hours at 37 ° C. (see A in FIG. 3) and after 24 hours at 37 ° C. (see B in FIG. 3). .
図３のＡ及びＢの図面代用写真に示す通り、サルモネラ属菌を接種した箇所のみ黒色変化を認め、時間の経過とともに黒色が拡大していくのを観察した。 As shown in the drawing-substituting photographs in FIGS. 3A and 3B, black change was observed only at the site inoculated with Salmonella sp.
次に、黒色反応をしている部分を釣菌し、ＤＨＬ寒天培地に接種したところ、図４の図面代用写真に示すように、黒色コロニー（非チフスサルモネラ属菌）と赤色コロニー（大腸菌群）とが同時に形成された。 Next, when the black reaction part was fished and inoculated into DHL agar medium, black colonies (non-typhoidal Salmonella spp.) And red colonies (E. coli group) as shown in the drawing-substituting photograph in FIG. And formed at the same time.
そのため、大腸菌群はサルモネラ属菌と共生関係にあることが判明し、前述した実施例１の結果も鑑みると、大腸菌群は、サルモネラ属菌による硫化水素産生を促進する機能を有すると結論できた。 Therefore, it was found that the coliform group is in a symbiotic relationship with Salmonella, and in view of the results of Example 1 described above, it could be concluded that the coliform has a function of promoting the production of hydrogen sulfide by Salmonella. .
＜実施例３＞
本実施例３では、大腸菌群のサルモネラ属菌への影響を確認するため、直径９０ｍｍのシャーレ２枚にＤＨＬ寒天培地を作製し、それぞれのシャーレの下半分に非チフスサルモネラ属菌を密集させて接種し、一方のシャーレにおいてサルモネラ属菌を接種しなかった上半分に大腸菌群を接種し（図５のＡ参照）、もう一方はそのままとして、３７℃で２４時間培養後、それぞれ目視にて確認した（図５のＡ及びＢ参照）。
<Example 3>
In this Example 3, in order to confirm the influence of Escherichia coli on Salmonella spp., DHL agar medium was prepared on two petri dishes having a diameter of 90 mm, and non-typhoidal Salmonella spp. Were concentrated in the lower half of each petri dish. Inoculate and inoculate Escherichia coli group on the upper half of the petri dish that was not inoculated with Salmonella (see A in Fig. 5). (See FIGS. 5A and 5B).
図５のＡの図面代用写真に示す通り、大腸菌群が上半分に接種されたシャーレの中央には、硫化鉄（FeS）による明瞭な黒色帯が現れた（シャーレ上方の斜線部分は、赤色であった）。したがって、大腸菌群がサルモネラ属菌による硫化水素産生を促進している事実を確認できた。 As shown in the drawing substitute photo of FIG. 5A, a clear black band made of iron sulfide (FeS) appeared in the center of the petri dish inoculated with coliform bacteria in the upper half (the shaded area above the petri dish is red) there were). Therefore, it was confirmed that the coliform group promotes the production of hydrogen sulfide by Salmonella.
なお、本実施例３において、図６の図面代用写真に示すように、非チフスサルモネラ属菌を下半分に接種し、サルモネラ属菌を接種しなかった上半分に大腸菌群を接種したシャーレにおいて、６時間培養後には大腸菌群を接種した部位は一部黒色を呈したことを確認しており、この結果から、６時間培養後においても大腸菌群がサルモネラ属菌の硫化水素産生能を活発化していることを確認できた。 In Example 3, in the petri dish inoculated with non-typhoidal Salmonella in the lower half and inoculated with coliforms in the upper half not inoculated with Salmonella, as shown in the drawing-substituting photograph of FIG. After incubating for 6 hours, it was confirmed that the inoculated part of the coliform group showed a black color. From this result, the coliform group activated Salmonella's ability to produce hydrogen sulfide even after 6 hours of incubation. I was able to confirm.
＜実施例４＞
直径９０ｍｍのシャーレ２枚にＤＨＬ寒天培地を作製し、それぞれのシャーレにチフス菌を密集させて全面に接種し、一方のシャーレにおいて大腸菌群を点状に４箇所接種し（図７のＢ参照）、３７℃で２４時間培養後、それぞれ目視にて観察した（図７のＡ及びＢ参照）。
<Example 4>
DHL agar medium is prepared in two petri dishes with a diameter of 90 mm, and Salmonella typhi is densely inoculated on each petri dish and inoculated on the entire surface, and four Escherichia coli groups are inoculated on one petri dish (see B in FIG. 7). After culturing at 37 ° C. for 24 hours, each was visually observed (see FIGS. 7A and 7B).
図７のＢの図面代用写真に示す通り、大腸菌群を接種した箇所のみが黒色変化した。したがって、本発明に係る検出方法を用いれば、チフス菌のような硫化水素産生能が弱い硫化水素産生菌であっても、大腸菌群等の有機酸産生菌の存在下で、硫化鉄（FeS）の黒色を明瞭に確認することができ、確実に検出することが可能となることが分かった。 As shown in the drawing-substituting photograph in FIG. 7B, only the portion inoculated with the coliform group turned black. Therefore, when the detection method according to the present invention is used, even if the hydrogen sulfide-producing bacterium such as Salmonella typhi has low ability to produce hydrogen sulfide, iron sulfide (FeS) is used in the presence of organic acid-producing bacteria such as coliforms. It was found that the black color of the film can be clearly confirmed and can be reliably detected.
本発明に係る検出方法を用いれば、迅速かつ簡便に、硫化水素産生菌又は有機酸産生菌を検出することが可能となる。そのため、水道水、飲料、食品、食品に接触する器具、医療機器、これらに用いられる各種部品等における、硫化水素産生菌又は有機酸産生菌の検出を、簡便、迅速かつ高精度に行うことができる。 By using the detection method according to the present invention, hydrogen sulfide-producing bacteria or organic acid-producing bacteria can be detected quickly and easily. Therefore, detection of hydrogen sulfide-producing bacteria or organic acid-producing bacteria in tap water, beverages, foods, instruments that come in contact with foods, medical devices, and various parts used in these can be performed simply, quickly, and with high accuracy. it can.
また、実施例４で示したように、本発明に係る検出方法を用いれば、チフス菌のような硫化水素産生能が弱い硫化水素産生菌であっても、確実に検出することが可能とある。 In addition, as shown in Example 4, when the detection method according to the present invention is used, even a hydrogen sulfide-producing bacterium such as Salmonella typhi having a weak hydrogen sulfide-producing ability can be reliably detected. .
Claims (4)
前記培養工程の後、前記対象の菌からの硫化水素の発生を検出する検出工程、
を少なくとも有し、
前記検出工程では、前記培地における前記大腸菌群を接種した箇所に、前記対象の菌からの硫化水素が発生している場合、前記対象の菌をサルモネラ菌と判定する、菌の検出方法。 After inoculating the target bacteria densely in a medium containing a sulfur source and an iron source, inoculating the coliform group in the medium and culturing for a predetermined time,
After the culturing step, a detection step of detecting the generation of hydrogen sulfide from the target fungus,
Having at least
The method for detecting a bacterium, wherein, in the detection step, when hydrogen sulfide from the target bacterium is generated at a location inoculated with the coliform group in the medium, the bacterium of the target is determined to be a Salmonella bacterium.
前記培養工程の後、前記サルモネラ菌からの硫化水素の発生を検出する検出工程、
を少なくとも有し、
前記検出工程では、前記培地における前記対象の菌を接種した箇所に、前記サルモネラ菌からの硫化水素が発生している場合、前記対象の菌を大腸菌群と判定する、菌の検出方法。 After inoculating and inoculating Salmonella in a medium containing a sulfur source and an iron source, inoculating the target bacteria in the medium and culturing for a predetermined time,
After the culturing step, a detection step of detecting generation of hydrogen sulfide from the Salmonella ,
Having at least
In the detection step, when hydrogen sulfide from the Salmonella is generated at a location where the target fungus is inoculated in the medium, the target fungus is determined as an coliform group .
前記培養工程の後、前記対象の菌からの硫化水素の発生を検出する検出工程、
を少なくとも有し、
前記検出工程では、前記培地における前記対象の菌を接種した箇所に、前記対象の菌からの硫化水素が発生している場合、前記対象の菌をサルモネラ菌と判定する、菌の検出方法。 After inoculating and inoculating coliforms in a medium containing a sulfur source and an iron source, inoculating the target bacteria in the medium and culturing for a predetermined time,
After the culturing step, a detection step of detecting the generation of hydrogen sulfide from the target fungus,
Having at least
In the detection step, when hydrogen sulfide from the target bacterium is generated at a location where the target bacterium is inoculated in the medium, the bacterium detection method determines that the target bacterium is Salmonella .
前記培養工程の後、前記サルモネラ菌からの硫化水素の発生を検出する検出工程、
を少なくとも有し、
前記検出工程では、前記培地における前記サルモネラ菌を接種した箇所に、前記サルモネラ菌からの硫化水素が発生している場合、前記対象の菌を大腸菌群と判定する、菌の検出方法。 After inoculating and inoculating the target fungus in a medium containing a sulfur source and an iron source, inoculating Salmonella in the medium and culturing for a predetermined time,
After the culturing step, a detection step of detecting generation of hydrogen sulfide from the Salmonella ,
Having at least
In the detection step, when hydrogen sulfide from the Salmonella is generated at a location where the Salmonella is inoculated in the medium, the target bacteria is determined as an coliform group .
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