JP2812338B2 - Rapid detection of microanaerobic bacteria - Google Patents
Rapid detection of microanaerobic bacteriaInfo
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- JP2812338B2 JP2812338B2 JP26023689A JP26023689A JP2812338B2 JP 2812338 B2 JP2812338 B2 JP 2812338B2 JP 26023689 A JP26023689 A JP 26023689A JP 26023689 A JP26023689 A JP 26023689A JP 2812338 B2 JP2812338 B2 JP 2812338B2
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- Prior art keywords
- bacteria
- filter
- anaerobic
- anaerobic bacteria
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Description
【発明の詳細な説明】 <産業上の利用分野> 本発明は、例えば長期保存を前提とする食品や飲料な
どの中に、保存中にこれらの製品を腐敗させる腐敗菌や
あるいは食中毒の原因となる菌などのような、いわゆる
嫌気性細菌に属する細菌が存在するか否かを知る場合に
特に好適に用いられる微量嫌気性細菌の検出法に関する
ものである。DETAILED DESCRIPTION OF THE INVENTION <Industrial application field> The present invention relates to the causes of spoilage bacteria or food poisoning that cause spoilage of these products during storage, for example, in foods and beverages premised on long-term storage. The present invention relates to a method for detecting a trace anaerobic bacterium which is particularly preferably used when it is known whether or not a bacterium belonging to a so-called anaerobic bacterium such as a bacterium exists.
<従来の技術> 例えば長期保存を前提とする清酒の場合、火落菌と呼
ばれる清酒中において特異的に成育できる腐敗菌の存在
は、その量がたとえ微量であっても保存中に当該火落菌
が増殖して清酒を腐敗させることとなるので極めて重大
な問題であり、従ってその製造に際しては一般に「火入
れ」と呼ばれる加熱殺菌処理が行われているとともに、
製品出荷に際しては製品中に火落菌が存在するか否かが
厳しくチェックされる。<Conventional technology> For example, in the case of sake that is premised on long-term storage, the presence of spoilage bacteria that can specifically grow in sake called soybean bacteria, This is a very serious problem because it will proliferate and cause the sake to spoil, and therefore, during its production, heat sterilization generally called "burning" is performed,
At the time of product shipment, it is strictly checked whether or not the bacteria are present in the product.
また、近年その普及が著しいいわゆるパック詰め食品
と総称される食品の場合も長期保存を前提としており、
そのためその製造に際しては厳重な殺菌処理が施されて
いるとともに、包装に際しては真空パックなどの特殊な
包装がなされ、外部からの細菌の浸入防止を図ることは
勿論、たとえ製品中に微量の細菌が残留していたとして
も、当該細菌が成育し得ないような条件が与えられてい
る。In addition, in the case of so-called packed foods, which have become very popular in recent years, it is premised on long-term storage,
Therefore, during its manufacture, strict sterilization treatment is applied, and at the time of packaging, special packaging such as vacuum packing is made, not only to prevent the invasion of bacteria from the outside, but also, even if trace amounts of bacteria are contained in the product. Conditions are provided such that the bacteria cannot grow even if they remain.
しかし、細菌の中には例えば食中毒の原因となるボツ
リヌス菌(Clostridium botulinum)やウェルシュ菌(C
lostridium perfringens)などのように、たとえ真空包
装の条件であっても増殖することのできる細菌が存在す
るので、その製造過程においてはこのような細菌の有無
が厳しくチェックされる。However, some bacteria, for example, Clostridium botulinum and C. perfringens (C)
Since there are bacteria such as lostridium perfringens that can grow even under vacuum packaging conditions, the presence of such bacteria is strictly checked during the manufacturing process.
上記火落菌、ボツリヌス菌およびウェルシュ菌はいず
れも嫌気性細菌に属する菌であり、そのためこれらの菌
を検出するにあたっては環境中に通常存在する好気性細
菌の場合の菌体培養と異なり、菌体を以下に述べるよう
な嫌気性雰囲気下において培養し、その後成育したコロ
ニーの数を目視で計数するという方法が採用されてい
る。The fire-killed bacteria, Clostridium botulinum and C. perfringens are all bacteria belonging to an anaerobic bacterium.Therefore, when detecting these bacteria, unlike the cell culture in the case of aerobic bacteria normally present in the environment, the bacterial cells Is cultured in an anaerobic atmosphere as described below, and then the number of grown colonies is visually counted.
すなわち、シャーレ上に拡げた寒天含有培地(以下、
単に寒天培地という)上に一定量の検体液を拡げるとと
もに当該ショーレを密閉式培養器の中に入れ、その後培
養器内の炭酸ガス濃度を5%以上とするか、あるいは当
該培養器内を−500mmHg以下の圧力に減圧するなどとし
て、培養器内の雰囲気を酸化還元電位で0.01V以下の嫌
気性雰囲気とし、この状態で3〜15日間培養して培養後
の培地上に成育したコロニーの数を目視で計数する方法
であり、このような方法を一般に平板寒天培地法、ある
いは略して単にプレート法と呼んでいる。That is, agar-containing medium spread on a Petri dish (hereinafter, referred to as
Spread a certain amount of the sample solution on an agar medium), put the chore in a closed incubator, and then adjust the carbon dioxide concentration in the incubator to 5% or more, or The pressure in the incubator is reduced to a pressure of 500 mmHg or less, and the atmosphere in the incubator is set to an anaerobic atmosphere with an oxidation-reduction potential of 0.01 V or less. This method is generally referred to as a plate agar medium method, or simply a plate method for short.
<発明が解決しようとする問題点> しかしながら、上述のような嫌気性細菌は、菌体培養
を大気圧下で行うことのできる好気性細菌に比べてその
増殖速度が極めて遅く、そのため目視で観察できる程度
の大きさまでに培地上のコロニーが成育するまでには前
述のように3〜15日間というような極めて長い培養期間
が必要である。<Problems to be Solved by the Invention> However, the anaerobic bacteria as described above have a very slow growth rate as compared with the aerobic bacteria which can perform the cell culture under the atmospheric pressure, and thus are visually observed. An extremely long culture period of 3 to 15 days is required as described above until a colony grows on the medium to a size as large as possible.
特に、上記清酒やパック詰め食品のように、製造時に
一応殺菌処理がなされているものの場合は、万一上述の
ような嫌気性細菌が製品中に残留していたとしてもその
数は数細胞以下、あるいは精々10細胞以下というように
極めて微量であり、このように菌体自体の数が微量であ
る場合には菌体の成育が更に悪くなるために、菌体培養
に通常一週間以上を要するのが現実である。In particular, in the case of those that have been sterilized at the time of manufacture, such as the above-mentioned sake and packed food, even if the above-mentioned anaerobic bacteria remain in the product, the number is several cells or less. , Or at most a very small amount of 10 cells or less, and in the case where the number of the cells themselves is very small, the growth of the cells is further deteriorated. That is the reality.
しかし、嫌気性細菌の有無を検査するのに、このよう
な長期間を要したのでは、検査が終了するまで製品を倉
庫内で管理しなければならないという製品管理上の問題
や、あるいは検査の結果嫌気性細菌が検出されたといっ
た場合には、この間に製造された総ての製品を廃棄しな
ければならないといったような問題を生じる可能性もあ
り、その経済的損失は計り知れないものがある。However, if it took such a long time to test for the presence of anaerobic bacteria, there was a problem with product management that the product had to be managed in a warehouse until the test was completed, or there was a problem with testing. If an anaerobic bacterium is detected as a result, problems such as the necessity of discarding all products manufactured during this time may be caused, and the economic loss is enormous. .
本発明はかかる事情に鑑みてなされたものであり、上
記従来方法において必要とされる少なくとも3日以上、
通常一週間以上に及ぶ検査期間を極端に短縮する微量嫌
気性細菌の検出法を提供することを目的とするものであ
る。The present invention has been made in view of such circumstances, and at least three days or more required in the above-described conventional method,
It is an object of the present invention to provide a method for detecting trace anaerobic bacteria, which extremely shortens the examination period usually exceeding one week.
<問題点を解決するための手段> 上記目的を達成するためになされた本発明よりなる微
量嫌気性細菌の検出法は、以下の各工程からなるもので
ある。<Means for Solving the Problems> The method for detecting a trace anaerobic bacterium according to the present invention, which has been made to achieve the above object, comprises the following steps.
すなわち、 (イ)嫌気性細菌を含む検体液を孔径0.45μm以下のフ
ィルターを用いて濾過することにより、検体液中の嫌気
性細菌をフィルター上に捕捉する第一工程 (ロ)嫌気性細菌を捕捉したフィルターを寒天含有培地
上に載せ、この状態にてフィルター上の嫌気性細菌を嫌
気性雰囲気下において12〜48時間培養する第二工程 (ハ)培養終了後に洗浄液を用いてフィルターを洗浄
し、前記第二工程においてフィルターに付着した培地成
分を除去する第三工程 (ニ)洗浄終了後、フィルター上に残留している嫌気性
細菌中のアデノシン−3−リン酸の量を計測する第四工
程 からなる検出法である。(A) The first step of capturing the anaerobic bacteria in the sample solution on the filter by filtering the sample solution containing the anaerobic bacteria using a filter having a pore size of 0.45 μm or less. The captured filter is placed on an agar-containing medium, and in this state, the anaerobic bacteria on the filter are cultured for 12 to 48 hours under an anaerobic atmosphere. (C) After the culture is completed, the filter is washed using a washing solution. A third step of removing the medium component attached to the filter in the second step; (d) after the washing is completed, a fourth step of measuring the amount of adenosine-3-phosphate in the anaerobic bacteria remaining on the filter. This is a detection method consisting of steps.
<作用> 以下に本発明を詳細に説明する。<Operation> Hereinafter, the present invention will be described in detail.
本発明の微量嫌気性細菌の検出法は、液体中に存在す
る嫌気性細菌を孔径0.45μm以下のフィルターを用いて
濾過、捕捉し、次いでこの捕捉した嫌気性細菌を、12〜
48時間という前記従来法に比べて著しく短時間で嫌気性
培養することにより、フィルター上に捕捉された嫌気性
細菌をある程度増殖させ、しかる後に増殖した嫌気性細
菌から、当該細菌中のアデノシン−3−リン酸(以下AT
Pという)を抽出してその量を計測し、この計測値に基
づいて嫌気性細菌を検出することを基本思想とするもの
である。The method for detecting microanaerobic bacteria of the present invention comprises filtering and capturing anaerobic bacteria present in a liquid using a filter having a pore size of 0.45 μm or less, and then filtering the captured anaerobic bacteria from 12 to
By performing anaerobic cultivation in a remarkably short time as compared with the conventional method of 48 hours, the anaerobic bacteria captured on the filter can be proliferated to some extent, and then the adenosine-3 -Phosphoric acid (hereinafter AT
The basic idea is to extract and measure the amount of P, and detect anaerobic bacteria based on the measured value.
液体中に存在する細菌(嫌気細菌に限らない)を検出
するに際して、細菌を孔径0.45μm以下のフィルターを
用いて濾過,捕捉した後、濾過,捕捉した細菌中のATP
量を直接計測することによって細菌の検出、定量を行う
方法に関しては、既に本出願人が出願している(特願昭
63−22075)。当該方法を微量嫌気性細菌の検出に適用
することができれば、培養という煩雑な操作を行うこと
なく嫌気性細菌の検出を行うことが可能である。When detecting bacteria (not limited to anaerobic bacteria) present in a liquid, the bacteria are filtered and captured using a filter with a pore size of 0.45 μm or less, and then ATP in the filtered and captured bacteria is detected.
The applicant has already filed an application for a method for detecting and quantifying bacteria by directly measuring the amount (Japanese Patent Application No.
63-22075). If this method can be applied to the detection of trace anaerobic bacteria, it is possible to detect anaerobic bacteria without performing the complicated operation of culturing.
しかし、本発明者が微量嫌気性細菌の検出にこのよう
な直接検出法の適用を試みたところ、フィルター上に捕
捉される嫌気性細菌の数が例えば100個以上というよう
に多い場合は正確に検出できるが、その数が数個といっ
た場合には菌体中から抽出されるATP量が余りにも少な
過ぎてATPの計測が不可能であることがわかった。勿
論、検体液中の嫌気性細菌の数が少ない場合でも、フィ
ルターによって濾過する検体液の量を多くすれば捕捉さ
れる細菌の数は増加する訳であるが、実際問題としては
孔径0.45μm以下という極めて微細な孔径のフィルター
を用いて多量の検体液を濾過することはフィルターの目
詰まりの問題から困難であり、また仮に可能であったと
しても、検体液が少量しかない場合には適用できない。However, when the present inventor tried to apply such a direct detection method to the detection of trace anaerobic bacteria, when the number of anaerobic bacteria captured on the filter is as large as, for example, 100 or more, it is accurate. Although detection was possible, it was found that when the number was several, the amount of ATP extracted from the cells was too small to measure ATP. Of course, even when the number of anaerobic bacteria in the sample solution is small, the number of captured bacteria increases if the amount of the sample solution filtered by the filter is increased, but as a practical matter, the pore size is 0.45 μm or less. It is difficult to filter a large amount of sample liquid using a filter with an extremely fine pore diameter because of the problem of clogging of the filter, and even if it is possible, it is not applicable if there is only a small amount of sample liquid .
そこで、本発明者はフィルター上に捕捉される細菌の
数が10個以下というような微量であっても当該細菌を確
実に検出することのできる方法について鋭意研究を行
い、その結果、フィルター上に捕捉された微量の嫌気性
細菌を、フィルター上に捕捉したままの状態でATP量の
計測が可能な程度の量に増殖するまで嫌気性培養し、し
かる後に増殖した嫌気性細菌中のATP量を計測するとい
う本発明方法を完成するに至ったのである。Therefore, the present inventors have conducted intensive research on a method that can reliably detect the bacteria even if the number of bacteria captured on the filter is as small as 10 or less, and as a result, on the filter A small amount of the captured anaerobic bacteria is anaerobically cultured in a state where the amount of ATP can be measured while being captured on the filter, and then the amount of ATP in the anaerobic bacteria that has grown is determined. Thus, the method of the present invention for measuring was completed.
本発明を各工程毎に詳述すれば、先ず第一工程におい
ては嫌気性細菌を含む検体液を孔径0.45μm以下のフィ
ルターを用いて濾過することにより、検体液中の嫌気性
細菌をフィルター上に捕捉する。濾過に使用するフィル
ターの孔径は、通常0.40〜0.45μmで十分であるが、例
えば清酒における火落菌の場合は孔径0.20μm以下のも
のを使用しないと捕捉できない。The present invention will be described in detail for each step.First, in the first step, the anaerobic bacteria in the sample solution are filtered on a filter by filtering the sample solution containing the anaerobic bacteria using a filter having a pore size of 0.45 μm or less. To capture. The pore size of the filter used for filtration is usually 0.40 to 0.45 μm. However, for example, in the case of fire-killed bacteria in sake, it cannot be captured unless a pore size of 0.20 μm or less is used.
なお、清酒中の火落菌を検出する場合のように、検査
の対象物がもともと液体である場合は当該液体をそのま
ま検体液として使用すればよいが、パック詰め食品のよ
うに対象物が固形物である場合には、前処理として、細
菌,解離状態のATPおよび発光物質を含まない液体(例
えば後述の第三工程で用いる滅菌生理食塩水や滅菌エタ
ノール含有液)を用いて固形物中の嫌気性細菌を当該液
体中に抽出する操作を行い、得られる抽出液を検体液と
して用いればよい。In addition, when the test target is a liquid, such as in the case of detecting bacterial fleas in sake, the liquid may be used as it is as a sample liquid. In the case of, as a pretreatment, anaerobic treatment in a solid substance is performed by using a liquid containing no bacteria, dissociated ATP and a luminescent substance (for example, a liquid containing sterilized saline or ethanol used in the third step described later). An operation of extracting the bacterium into the liquid may be performed, and the resulting extract may be used as the sample liquid.
次いで第二工程においては、前記第一工程でフィルタ
ー上に捕捉した嫌気性細菌を嫌気性雰囲気下において培
養し、後述の第四工程においてATP量の計測が可能な程
度の量まで増殖させる。Next, in the second step, the anaerobic bacteria captured on the filter in the first step are cultured in an anaerobic atmosphere, and are grown to an amount capable of measuring the amount of ATP in the fourth step described below.
当該嫌気性培養は、嫌気性細菌の捕捉されたフィルタ
ーを、当該フィルターごと細菌捕捉面を上向きにしてシ
ャーレなどの中に拡げた寒天培地上に載せ、しかる後に
当該シャーレを密閉式培養器などの中に入れて所定時間
行う。培養に必要な嫌気性条件は前記従来法の場合と同
じでよく、例えば前記培養器の中に炭酸ガスを吹き込ん
で培養器内の炭酸ガス濃度を5%以上とするか、あるい
は培養器を減圧して−500mmHg以下の圧力とするなどの
手段によって、培養器内の雰囲気を酸化還元電位で0.01
V以下の嫌気性雰囲気とすればよい。In the anaerobic culture, the filter on which the anaerobic bacteria are captured is placed on an agar medium spread in a Petri dish or the like with the filter together with the bacteria capturing surface facing upward, and then the Petri dish is sealed in an incubator or the like. Put it inside for a predetermined time. The anaerobic conditions required for the culture may be the same as those in the conventional method. For example, carbon dioxide gas is blown into the incubator to adjust the concentration of carbon dioxide in the incubator to 5% or more, or the incubator is depressurized. The atmosphere in the incubator is adjusted to an oxidation-reduction potential of 0.01 at a pressure of -500 mmHg or less.
An anaerobic atmosphere of V or less may be used.
本発明者の研究によれば、上記嫌気性培養に要する時
間は培養温度25〜60℃において12〜48時間でよく、培養
時間が12時間より少ない場合はATP量の計測が可能な程
度まで細菌が増殖せず、また48時間を越えて培養するこ
とは細菌の検出を可及的速やかに行うという観点からし
て無駄なことである。According to the study of the present inventors, the time required for the anaerobic culturing may be 12 to 48 hours at a culturing temperature of 25 to 60 ° C. It is wasteful from the viewpoint that bacteria do not proliferate and culture for more than 48 hours is carried out as soon as possible to detect bacteria.
次に第三工程においては、培養終了後のフィルターを
培養器から取り出した後、当該フィルターを後述のよう
な洗浄液を用いて洗浄する。Next, in the third step, the filter after completion of the culture is taken out of the incubator, and the filter is washed with a washing solution as described below.
当該洗浄工程は、前記第二工程においてフィルターに
付着した培地成分を除去することを目的とするものであ
り、更に詳しくは本発明よりなる細菌検出法の誤差の原
因となる、寒天培地中に含まれている遊離のATPをフィ
ルターから除去し、増殖した嫌気性細菌のみをフィルタ
ー上に残留させることを目的として行うものである。従
って、当該洗浄に用いる洗浄液としては、遊離のATPを
含まず、かつ同じく誤差の原因となる細菌および発光物
質の含まれない液体であることが必要であり、例えば12
0℃、15分間オートクレーブにて高圧蒸気滅菌処理した
限外濾過処理純水に、エタノールを10%の濃度となるよ
うに溶解した滅菌エタノール含有液や前記限外濾過処理
純水に食塩を0.9%の濃度となるように溶解した滅菌食
塩水(あるいは滅菌生理食塩水)を用いる。The washing step is intended to remove the medium components attached to the filter in the second step, and more specifically, causes an error in the bacteria detection method according to the present invention, and is contained in the agar medium. The purpose is to remove free ATP from the filter and leave only the grown anaerobic bacteria on the filter. Therefore, it is necessary that the washing liquid used for the washing be a liquid that does not contain free ATP and does not contain bacteria and luminescent substances that also cause errors.
0.9% sodium chloride is added to a sterilized ethanol-containing solution obtained by dissolving ethanol to a concentration of 10% in ultrafiltration-treated pure water subjected to high-pressure steam sterilization in an autoclave at 0 ° C for 15 minutes or the ultrafiltration-treated pure water. Use a sterile saline solution (or a sterile physiological saline solution) dissolved to a concentration of.
更に第四工程においてはフィルターに残留している嫌
気性細菌の細胞膜を、酵素、アニオン系界面活性剤など
の溶菌剤を用いて溶解して細胞中のATPを抽出し、更に
抽出したATPの量を生物発光現象を利用して定量し、当
該ATP量に基づいて検体液中に存在する嫌気性微生物の
検出を行う。In the fourth step, the cell membrane of the anaerobic bacteria remaining in the filter is dissolved using a lytic agent such as an enzyme or an anionic surfactant to extract ATP in the cells, and the amount of the extracted ATP Is quantified using the bioluminescence phenomenon, and anaerobic microorganisms present in the sample liquid are detected based on the ATP amount.
上記ATP量の定量方法は、発光素であるルシフェリン
の存在下、発光酵素であるルシフェラーゼを作用させて
発光させ、その発光量を螢光検出器(光電子検出管)に
て電気的に検出し、これをATP量に換算することによっ
て行う。The method for quantifying the amount of ATP is as follows: in the presence of luciferin, which is a luminescent element, luciferase, which is a luminescent enzyme, is caused to emit light, and the amount of luminescence is electrically detected by a fluorescence detector (photoelectron detection tube); This is performed by converting it to an ATP amount.
なお、上述の説明で明らかなごとく、本発明の嫌気性
細菌の検出法は、検体液中に含まれる嫌気性細菌をフィ
ルター上に濾過,捕捉し、次いで当該捕捉した嫌気性細
菌を嫌気性培養によって一旦増殖させ、この増殖した嫌
気性細菌中のATP量を計測して検体液中に存在する嫌気
性微生物の検出を行う方法であるから、最終的に計測さ
れるATP量から検体液中に当初存在した嫌気性細菌の個
数(細胞数)を求めることまでは困難であり、本発明法
の意義は検体液中に嫌気性細菌が存在していたか否かを
従来法より短時間内に知ることができるという点にあ
る。そして、食品や飲料中における嫌気性細菌の存在
は、前述のごとくその量にかかわらず存在していること
自体が重大な問題なのであり、従って実用的には嫌気性
細菌が存在するか否かを迅速に検出することができれば
十分なのである。As is clear from the above description, the method for detecting anaerobic bacteria of the present invention involves filtering and capturing anaerobic bacteria contained in a sample solution on a filter, and then culturing the captured anaerobic bacteria in an anaerobic culture. This is a method for measuring the amount of ATP in the grown anaerobic bacteria and detecting the anaerobic microorganisms present in the sample liquid. It is difficult to determine the number of anaerobic bacteria (cell number) that initially existed, and the significance of the method of the present invention is to know whether or not anaerobic bacteria were present in a sample solution within a shorter time than the conventional method. The point is that you can do it. The presence of anaerobic bacteria in foods and beverages is a serious problem itself, as described above, irrespective of its amount, and therefore, in practice, it is important to determine whether anaerobic bacteria are present. It is enough if it can be detected quickly.
<実施例> 以下に本発明の実施例を説明する。<Example> An example of the present invention will be described below.
実施例 開栓直後の日本酒(日本酒Aとする)、および当該日
本酒に適量の火落菌を添加した菌添加日本酒2酒(それ
ぞれ日本酒Bおよび日本酒Cとする)を検体液とし、そ
れぞれその10mlを0.20μm孔径のメンブレンフィルター
にて濾過し、菌体を捕捉した。Example Sake immediately after opening (referred to as sake A), and two sakes with fungi added to each of the sakes (referred to as sake B and sake C, respectively) obtained by adding an appropriate amount of Hakufungi to the sample liquid, and 10 ml of each were used as sample liquids. The cells were filtered through a membrane filter having a pore size of μm to capture the cells.
次いで、上記各フィルターを、それぞれ日本醸造協会
の定めるSI寒天培地(エタノール含有率5%、寒天含有
率1%組成の寒天培地)を拡げたシャーレの上に、菌体
捕捉面を上向きにして載せ、更にこれらのシャーレを密
閉式培養器の中に入れた。Next, each of the above filters was placed on a petri dish in which a SI agar medium (an agar medium having an ethanol content of 5% and an agar content of 1% composition) specified by the Japan Brewing Association was spread, with the bacterial cell capturing surface facing upward. And these petri dishes were placed in a closed incubator.
当該培養器内を−500mmHg以下に減圧した上で更に培
養器内に炭酸ガスを封入し、当該培養器内が酸化還元電
位で0.01V以下の嫌気性雰囲気となっていることを確認
(メチレンブルー検出薬を用いて常法により確認)した
後、この状態にて温度30℃で24時間培養を行った。After reducing the pressure in the incubator to -500 mmHg or less, further inject carbon dioxide gas in the incubator, and confirm that the inside of the incubator has an anaerobic atmosphere with an oxidation-reduction potential of 0.01 V or less (methylene blue detection After confirmation by a conventional method using a drug), the cells were cultured in this state at a temperature of 30 ° C. for 24 hours.
培養終了後各フィルターを取り出し、それぞれを前述
したような滅菌エタノール含有液にて十分に洗浄し、そ
の後各フィルターをATP計測に供した。なお、ATP計測に
はIMSCO社製のMicrosure−100(螢光検出器)を用い
た。測定は同一検体液について3回行い、それら繰り返
し3点の平均値を第1表に示した。After the completion of the culture, each filter was taken out, and each was sufficiently washed with a sterilized ethanol-containing solution as described above, and then each filter was subjected to ATP measurement. The ATP measurement was performed using a Microsure-100 (fluorescence detector) manufactured by IMSCO. The measurement was performed three times for the same sample liquid, and the average value of the three repeated points is shown in Table 1.
比較例(従来法)として、上記各日本酒A、B、Cの
それぞれについて、その1mlを上述したと同じSI寒天培
地上に採り、日本醸造協会の定める平板寒天培地法に準
じて、上記実施例の場合と同じ嫌気性条件下にて温度30
℃で培養したところ、培地上にコロニーが形成されたこ
とを目視にて観察できるまでに10日間を要した。As a comparative example (conventional method), 1 ml of each of the above sakes A, B, and C was taken on the same SI agar medium as described above, and the above-mentioned example was prepared according to the plate agar medium method defined by the Japan Brewing Association. Temperature 30 under the same anaerobic conditions as
After culturing at a temperature of 10 ° C., it took 10 days before the formation of colonies on the medium could be visually observed.
培養後の火落菌コロニー数を目視にて計数し、その結
果を第1表に併記した。なお、第1表にはこのような測
定の繰り返し3点の平均値を示してある。The number of fire-killed colonies after culturing was visually counted, and the results are shown in Table 1. Table 1 shows the average value of three repetitions of such measurement.
第1表から明らかなように、従来法における火落菌計
測値がゼロである日本酒Aについては、本発明法による
ATP計測値もゼロであり、一方日本酒Bのように従来法
における火落菌の計測値が約1個と極めて微量であって
も本発明法にてATPを確実に検出、還元すれば火落菌が
存在することを確実に検出することができる。 As is clear from Table 1, sake A in which the measured value of fire-killed bacteria in the conventional method is zero is determined by the method of the present invention.
The ATP measurement value is also zero. On the other hand, even if the measurement value of the fire-killing bacteria in the conventional method is as small as about 1 as in sake B, if the ATP is reliably detected and reduced by the method of the present invention, the fire-killing bacteria can be reduced. The presence can be reliably detected.
<効果> 本発明法によれば、従来のプレート法(平板寒天培地
法)における菌コロニー数の目視による計数といった煩
雑かつ専門的な操作や、通常一週間以上にも渡る長期間
の培養を行わないと結果が判明しないといった問題点が
総て解決され、たとえ微量しか存在しない嫌気性細菌で
あっても、これを12〜48時間という従来より著しく短い
期間で検出することができる。<Effect> According to the method of the present invention, a complicated and specialized operation such as visual counting of the number of bacterial colonies in a conventional plate method (plate agar medium method), or a long-term culture usually over one week or more is performed. All the problems that the result is not clear without it are solved, and even an anaerobic bacterium having a small amount can be detected in a significantly shorter time of 12 to 48 hours than before.
その結果、食品,飲料などの安全性や品質の向上に大
いに貢献し、また製品管理の面でも従来より効率的な管
理が行えるようになって経済的にも極めて大きな利益を
もたらすことは必定である。As a result, it is inevitable that it will greatly contribute to the improvement of the safety and quality of foods and beverages, etc., and will be able to manage products more efficiently than before, resulting in extremely great economic benefits. is there.
Claims (1)
検出法。 (イ)嫌気性細菌を含む検体液を孔径0.45μm以下のフ
ィルターを用いて濾過することにより、検体液中の嫌気
性細菌をフィルター上に捕捉する第一工程 (ロ)嫌気性細菌を捕捉したフィルターを寒天含有培地
上に載せ、この状態にてフィルター上の嫌気性細菌を嫌
気性雰囲気下において12〜48時間培養する第二工程 (ハ)培養終了後に洗浄液を用いてフィルターを洗浄
し、前記第二工程においてフィルターに付着した培地成
分を除去する第三工程 (ニ)洗浄終了後、フィルター上に残留している嫌気性
細菌中のアデノシン−3−リン酸の量を計測する第四工
程1. A method for rapid detection of trace anaerobic bacteria comprising the following steps: (A) The first step of capturing the anaerobic bacteria in the sample liquid on the filter by filtering the sample liquid containing the anaerobic bacteria using a filter having a pore size of 0.45 μm or less. (B) The anaerobic bacteria were captured. The filter is placed on an agar-containing medium, and in this state, the anaerobic bacteria on the filter are cultured under an anaerobic atmosphere for 12 to 48 hours. (C) After the culture, the filter is washed with a washing solution, Third step of removing medium components attached to the filter in the second step (d) After completion of washing, fourth step of measuring the amount of adenosine-3-phosphate in the anaerobic bacteria remaining on the filter
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP26023689A JP2812338B2 (en) | 1989-10-06 | 1989-10-06 | Rapid detection of microanaerobic bacteria |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP26023689A JP2812338B2 (en) | 1989-10-06 | 1989-10-06 | Rapid detection of microanaerobic bacteria |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH03123497A JPH03123497A (en) | 1991-05-27 |
JP2812338B2 true JP2812338B2 (en) | 1998-10-22 |
Family
ID=17345245
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JP26023689A Expired - Fee Related JP2812338B2 (en) | 1989-10-06 | 1989-10-06 | Rapid detection of microanaerobic bacteria |
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JP (1) | JP2812338B2 (en) |
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1989
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JPH03123497A (en) | 1991-05-27 |
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