JP2005143329A - Method for asepticizing zoo- and phytoplankton and method for culturing rotifer using the same method for asepticization - Google Patents
Method for asepticizing zoo- and phytoplankton and method for culturing rotifer using the same method for asepticization Download PDFInfo
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Abstract
Description
本発明は、動植物プランクトンの無菌化方法，および当該無菌化方法を用いたワムシの培養方法に関し、特に、特定抗生物質を用いた動植物プランクトンの無菌化方法，および当該無菌化方法を用いたワムシの培養方法に関する。 The present invention relates to a method for sterilizing animal and plant plankton, and a method for cultivating rotifer using the sterilization method, and in particular, a method for sterilizing animal and plant plankton using a specific antibiotic, and a method for rotifer using the sterilization method. The present invention relates to a culture method.
動植物プランクトンの無菌化に関しては、種々の方法が報告されている。例えば，無菌化処理として、次亜塩素酸ナトリウムやグルタールアルデヒドの単独処理、次亜塩素酸ナトリウムと抗生物質混合液を用いた処理方法が知られている。 Various methods have been reported for sterilization of animal and phytoplankton. For example, as a sterilization treatment, a single treatment with sodium hypochlorite or glutaraldehyde, or a treatment method using sodium hypochlorite and an antibiotic mixture is known.
これらの方法で調製した培養液の無菌検査には、マリーン寒天培地やその類似の培地1種類のみで行なっている。 The sterility test of the culture solution prepared by these methods is carried out using only one type of marine agar medium or similar medium.
また、AM9と呼ばれる抗生物質混合液（ストレプトマイシン、ペニシリンG、硫酸ポリミキシンB、テトラサイクリン、クロラムフェニコール、ネオマイシン）を用いて、動物プランクトンであるTigriopusを無菌化する方法が知られている（著者：Provasoli et al、題名：Axenic Cultivation of the Brine Shrimp Artemia salina.雑誌名：Annals of the New York Academy of Sciences 77(2), p250-261 (1959)）。 Also known is a method of sterilizing the zooplankton Tigriopus using an antibiotic mixture called AM9 (streptomycin, penicillin G, polymyxin B sulfate, tetracycline, chloramphenicol, neomycin) (author: Provasoli et al, title: Axenic Cultivation of the Brine Shrimp Artemia salina. Journal name: Annals of the New York Academy of Sciences 77 (2), p250-261 (1959)).
また、抗生物質混合液（AM9）を用いて無菌ワムシを調製する方法が知られている。これは、無菌ワムシを用いてを用いて８種の藻類のワムシ増殖に対する影響を調べるものである(著者：Kazutsugu HIRAYAMA et al.題名：Nutritional Effect of Eight Species of Marine Phytoplankton on Population Growth of the Rotifer, Brachionus plicatilis.雑誌名：Bulletin of the Japanese Society of Scientific Fisheries 45(1), p11-16 (1979)) In addition, a method for preparing a sterile rotifer using an antibiotic mixture (AM9) is known. This is to examine the effect of 8 kinds of algae on rotifer growth using a sterile rotifer (Author: Kazutsugu HIRAYAMA et al. Title: Nutritional Effect of Eight Species of Marine Phytoplankton on Population Growth of the Rotifer, Brachionus plicatilis. Journal name: Bulletin of the Japanese Society of Scientific Fisheries 45 (1), p11-16 (1979))
また、ワムシの耐久卵を、次亜塩素酸ナトリウム処理により無菌化するために
その濃度と処理時間を最適化し、耐久卵の孵化率が良く、かつ、無菌化する
ための条件を規定した無菌培養が知られている（著者：Philippe Douillet
題名：Disinfection of rotifer cysts leading to bacteria-free populations.
雑誌名：Journal of Experimental Marine Biology and Ecology
224, p183-192 (1998)）。
In addition, in order to sterilize rotifer durable eggs by sodium hypochlorite treatment, the concentration and processing time are optimized, the hatchability of durable eggs is good, and the sterilized culture stipulates the conditions for sterilization (Author: Philippe Douillet
Title: Disinfection of rotifer cysts leading to bacteria-free populations.
Journal Name: Journal of Experimental Marine Biology and Ecology
224, p183-192 (1998)).
また、ワムシの耐久卵を、メルチオレート、グルタールアルデヒド処理により無菌化するためその濃度と処理時間を検討し、ワムシを無菌化した後、種々の細菌を添加し、ワムシの培養に対する影響を調べた文献が知られている（著者：Rombaut G., et al.題名：Selection of bacteria enhancing the growth rate of axenically hatched rotifers (Brachionus plicatilis).雑誌名：Aquaculture
176, p195-207 (1999)）。
176, p195-207 (1999)).
しかしながら、上記従来の無菌化方法によれば、実験室で培養時に混入する可能性がある細菌や菌類は、海洋性の細菌だけではないため、海洋性検査培地のみの無菌化レベルでしかない。 However, according to the conventional sterilization method, the bacteria and fungi that may be mixed during culturing in the laboratory are not only marine bacteria, but are only sterilized only by the marine test medium.
また、上記従来の無菌化方法によれば、用いる抗生物質にも問題があった。すなわち、抗生物質混合液中にクロラムフェニコールが含まれており、当該抗生物質が、ワムシなどの仔魚用プランクトンの産卵とふ化に悪影響を及ぼすという問題がある。 Moreover, according to the conventional sterilization method, there is a problem with the antibiotic used. That is, there is a problem that chloramphenicol is contained in the antibiotic mixture, and the antibiotic has an adverse effect on egg production and hatching of plankton for larvae such as rotifers.
また、上述の抗生物質混合液（AM9）を用いて無菌ワムシを調製する方法では、ワムシに悪影響を及ぼす抗生物質（クロラムフェニコール）を使用しており、かつ、細菌検査に１種類の検査培地しか用いていないので、無菌化の確認が不十分であるという問題を有する。 In addition, the method for preparing sterile rotifers using the above-mentioned antibiotic mixture (AM9) uses an antibiotic (chloramphenicol) that has an adverse effect on rotifers, and one type of test for bacterial testing Since only the medium is used, there is a problem that confirmation of sterilization is insufficient.
また、上述の無菌化条件を検討した例、処理濃度、処理時間を検討した例によれば、細菌検査に１種類の検査培地しか用いていないことから、やはり無菌化の確認が不十分であるという問題を有する。 Moreover, according to the example which examined the sterilization conditions mentioned above, the example which examined processing density | concentration, and processing time, since only one type of test | inspection culture medium is used for a bacteria test, the confirmation of sterilization is still insufficient. Have the problem.
したがって、海洋性のみならず、土壌、水系、空気中等に存在する細菌類をも死滅させより完全な無菌化状態の動植物プランクトンの無菌化方法が要望されるが、当該方法はこれまで知られていない。 Therefore, there is a demand for a method for sterilizing animals and phytoplankton in a more complete sterilized state by killing not only marine but also bacteria existing in soil, water system, air etc., but this method has been known so far. Absent.
そこで、本発明は、海洋性のみならず、土壌、水系、空気中等に存在し、飼育段階において発生し得る細菌を効率的に除去し高い無菌状態を付与する無菌化方法、及びこれを用いたワムシの培養方法を提供することにある。 Therefore, the present invention is a sterilization method that efficiently removes bacteria that are present not only in marine nature but also in soil, water systems, air, and the like and can be generated in the breeding stage, and imparts a high sterility state, and the same. It is to provide a method for cultivating rotifer.
上記目的を達成するために、発明者らは、無菌レベルを向上させるべく、海洋上の細菌のみならず、実験室等陸上で混入可能な細菌についても検討を行った結果、本発明の動植物プランクトンの無菌化方法、及び当該無菌化方法を用いたワムシの培養方法を見出すに至った。 In order to achieve the above object, the inventors have studied not only marine bacteria but also bacteria that can be mixed on land such as laboratories in order to improve the sterility level. And a method for cultivating a rotifer using the sterilization method.
本発明の動植物プランクトンの無菌化方法は、次亜塩素酸ナトリウム、メルチオレート、グルタールアルデヒド、過酸化水素、塩化ベンザルコニウム（逆性石鹸）からなる群から選択される少なくとも1種により処理した後、作用機序の異なる複数の抗生物質により処理することを特徴とする。 The method for sterilizing animal and phytoplankton according to the present invention comprises treating with at least one selected from the group consisting of sodium hypochlorite, merthiolate, glutaraldehyde, hydrogen peroxide, and benzalkonium chloride (reverse soap). It is characterized by treatment with a plurality of antibiotics having different mechanisms of action.
また、本発明の動植物プランクトンの無菌化方法の好ましい実施態様において、前記作用機序の異なる複数の抗生物質が、アンピシリン、カナマイシン、ナリジクス酸、ストレプトマイシンからなる群から選択される少なくとも1種であることを特徴とする。 Further, in a preferred embodiment of the method for sterilizing an animal or phytoplankton of the present invention, the plurality of antibiotics having different action mechanisms are at least one selected from the group consisting of ampicillin, kanamycin, nalidixic acid and streptomycin. It is characterized by.
また、本発明の動植物プランクトンの無菌化方法の好ましい実施態様において、前記抗生物質の各抗生物質の濃度が、それぞれ10〜150μg/mlであることを特徴とする。 Further, in a preferred embodiment of the method for sterilizing animals and phytoplankton of the present invention, the concentration of each antibiotic of the antibiotic is 10 to 150 μg / ml.
また、本発明のワムシの培養方法は、上記本発明の無菌化方法を使用して、無菌ワムシを培養するワムシの培養方法であって、ワムシ耐久卵を次亜塩素酸ナトリウム、メルチオレート、グルタールアルデヒド、過酸化水素、塩化ベンザルコニウム（逆性石鹸）からなる群から選択される少なくとも1種の処理剤により洗浄し、当該次亜塩素酸ナトリウムなどを除去し、次いで、耐久卵をふ化させ、前記作用機序の異なる複数の抗生物質の存在下、培養することを特徴とする。 The rotifer culture method of the present invention is a rotifer culture method for cultivating a sterile rotifer using the sterilization method of the present invention, wherein the rotifer durable egg is treated with sodium hypochlorite, merthiolate, glutar. Wash with at least one treatment agent selected from the group consisting of aldehyde, hydrogen peroxide, and benzalkonium chloride (reverse soap) to remove the sodium hypochlorite, and then to hatch durable eggs And culturing in the presence of a plurality of antibiotics having different mechanisms of action.
また、本発明のワムシの培養方法の好ましい実施態様において、前記処理剤の濃度が、０．１〜１％であることを特徴とする。 In a preferred embodiment of the method for cultivating rotifers of the present invention, the concentration of the treatment agent is 0.1 to 1%.
また、本発明のワムシの培養方法の好ましい実施態様において、前記処理剤での洗浄時間が、１分〜１０分であることを特徴とする。 Further, in a preferred embodiment of the method for cultivating rotifers of the present invention, the washing time with the treatment agent is 1 minute to 10 minutes.
また、本発明のワムシの培養方法の好ましい実施態様において、前記処理剤で洗浄後、滅菌人口海水によりさらに洗浄し、当該次亜塩素酸ナトリウムを除去することを特徴とする。 In a preferred embodiment of the method for cultivating rotifers of the present invention, the sodium hypochlorite is removed by further washing with sterilized artificial seawater after washing with the treatment agent.
また、本発明のワムシの培養方法の好ましい実施態様において、前記処理剤を除去後、耐久卵をふ化させることを特徴とする。 In a preferred embodiment of the method for cultivating rotifers of the present invention, after the treatment agent is removed, durable eggs are hatched.
また、本発明のワムシの培養方法の好ましい実施態様において、前記作用機序の異なる複数の抗生物質が、アンピシリン、カナマイシン、ナリジクス酸、ストレプトマイシンからなる群から選択される少なくとも1種であることを特徴とする。 Also, in a preferred embodiment of the method for cultivating rotifers of the present invention, the plurality of antibiotics having different action mechanisms is at least one selected from the group consisting of ampicillin, kanamycin, nalidixic acid, and streptomycin. And
本発明の無菌化方法によれば、仔魚用プランクトンの培養に最適な無菌化状態を提供することが可能であるという有利な効果を奏する。 According to the sterilization method of the present invention, there is an advantageous effect that it is possible to provide an optimum sterilization state for culturing larval plankton.
また、本発明の無菌化方法を用いたワムシの培養方法によれば、混在する菌相の影響を受けないため、ワムシ用の資料評価、ワムシの栄養要求性試験、ワムシ遺伝子解析等において高い再現性を有する基礎的知見を得ることが可能であるというという有利な効果を奏する。 In addition, according to the rotifer culture method using the sterilization method of the present invention, since it is not affected by the mixed microflora, it is highly reproducible in rotifer data evaluation, rotifer auxotrophy test, rotifer gene analysis, etc. There is an advantageous effect that it is possible to obtain basic knowledge having sex.
また、本発明の無菌化方法を用いたワムシの培養法によれば、ワムシ培養において、外的要因を排除したデータが得られ，ワムシ培養に悪影響を与える因子などの解析により、安定したワムシ培養に必要な条件を見出すことができるという有利な効果を奏する。 In addition, according to the rotifer culture method using the sterilization method of the present invention, data that excludes external factors can be obtained in rotifer culture, and stable rotifer culture can be performed by analyzing factors that adversely affect rotifer culture. There is an advantageous effect that the necessary conditions can be found.
本発明の動植物プランクトンの無菌化方法では、まず、次亜塩素酸ナトリウム、メルチオレート、グルタールアルデヒド、過酸化水素、塩化ベンザルコニウム（逆性石鹸）からなる群から選択される少なくとも1種により処理する。これらの処理は、耐久卵を含め動植物プランクトンに付着、もしくは混在している菌類、細菌類に対して効果的に当該動植物プランクトンの無菌化を達成する観点から採用するものである。係る処理によって、かび類、酵母類を死滅されることが可能である。ここでは、上記処理を特に例示しているが、かび類、酵母類を死滅させることが十分であり、かつ、耐久卵を含め動植物プランクトンが死滅しないような化学薬品であれば、他に使用可能であり、特に限定されるものではない。 In the sterilization method of animal and phytoplankton of the present invention, first, treatment is performed with at least one selected from the group consisting of sodium hypochlorite, merthiolate, glutaraldehyde, hydrogen peroxide, and benzalkonium chloride (reverse soap). To do. These treatments are employed from the viewpoint of effectively sterilizing the animal and plant plankton with respect to fungi and bacteria adhering to or mixed with the animal and plant plankton including the durable egg. By such treatment, fungi and yeasts can be killed. Here, the above treatment is specifically exemplified, but other chemicals can be used as long as it is sufficient to kill fungi and yeasts and does not kill animal and phytoplankton including durable eggs. There is no particular limitation.
さらに、本発明においては、上記処理後、作用機序の異なる複数の抗生物質により処理する。これは、前記処理剤では細菌類を完全に死滅させることができず、さらに、その後に混入してくると予想される土壌、水系、空気中等に存在する細菌類について対処しようと試みた結果、作用機序の異なる複数の抗生物質を採用するに至ったものである。 Furthermore, in the present invention, after the treatment, treatment is performed with a plurality of antibiotics having different action mechanisms. This is because the treatment agent cannot completely kill the bacteria, and further, as a result of trying to deal with bacteria present in the soil, water system, air, etc. that are expected to be mixed afterwards, This has led to the adoption of multiple antibiotics with different mechanisms of action.
このような作用機序の異なる複数の抗生物質としては、アンピシリン、カナマイシン、ナリジクス酸、ストレプトマイシンからなる群から選択される少なくとも1種を挙げることができる。本発明において使用可能な抗生物質としては、アンピシリン、カナマイシン、ナリジクス酸、ストレプトマイシンの類似体(アナログ)も含む。これらの類似体も、構造、作用機序が同様であれば、同様の効果を達成し得るからである。たとえば、アンピシリンは、ペニシリン類、セフェム類に置換可能であり、カナマイシン、ストレプトマイシンは、アミノグリコシド系物質に置換可能であり、ナリジクス酸は、DNA合成阻害剤(ピロミド酸、ピペミド酸、ノフロキサシン、オフロキサシン、エノキサシン)に置換可能であり、これらを、上記アンピシリン等に代えて使用することができる。ここで、少なくとも1種としたが、海洋性以外の多数の細菌を死滅させるという観点から、アンピシリン、カナマイシン、ナリジクス酸、ストレプトマイシンの1種以上乃至全部を使用してもよい。 Examples of the plurality of antibiotics having different action mechanisms include at least one selected from the group consisting of ampicillin, kanamycin, nalidixic acid, and streptomycin. Antibiotics that can be used in the present invention also include analogs (analogues) of ampicillin, kanamycin, nalidixic acid and streptomycin. This is because these analogs can achieve the same effect as long as they have the same structure and mechanism of action. For example, ampicillin can be replaced with penicillins and cephems, kanamycin and streptomycin can be replaced with aminoglycosides, and nalidixic acid can be a DNA synthesis inhibitor (pyromido acid, pipemidic acid, nofloxacin, ofloxacin, enoxacin) ), Which can be used in place of the above ampicillin and the like. Here, although at least one species is used, one or more or all of ampicillin, kanamycin, nalidixic acid, and streptomycin may be used from the viewpoint of killing many bacteria other than marine bacteria.
また、本発明の動植物プランクトンの無菌化方法において、好適には、前記抗生物質の各抗生物質の濃度が、それぞれ10〜150μg/mlである。特に、細菌を死滅させワムシなど動植物プランクトンに悪影響を及ぼさない薬剤終濃度というという観点から、アンピシリン及びその類似体においては、２０〜１５０μg/ml、好ましくは100μg/ml前後である。また、カナマイシン及びその類似体においては、細菌を死滅させワムシなど動植物プランクトンに悪影響を及ぼさない薬剤終濃度という観点から、１０〜１００μg/ml、好ましくは60μg/ml前後である。ナリジクス酸及びその類似体においては、細菌を死滅させワムシなど動植物プランクトンに悪影響を及ぼさない薬剤終濃度という観点から、１５〜５０μg/ml、好ましくは60μg/ml前後である。ストレプトマイシン及びその類似体においては、細菌を死滅させワムシなど動植物プランクトンに悪影響を及ぼさない薬剤終濃度という観点から、１０〜１００μg/ml、好ましくは60μg/ml前後である。 In the method for sterilizing animals and phytoplankton of the present invention, preferably, the concentration of each antibiotic in the antibiotic is 10 to 150 μg / ml. In particular, from the viewpoint of a final drug concentration that kills bacteria and does not adversely affect animal and phytoplankton such as rotifers, it is 20 to 150 μg / ml, preferably around 100 μg / ml in ampicillin and its analogs. In addition, in the case of kanamycin and its analogs, the concentration is 10 to 100 μg / ml, preferably around 60 μg / ml, from the viewpoint of the final drug concentration that kills bacteria and does not adversely affect animal and phytoplankton such as rotifers. In the case of nalidixic acid and analogs thereof, the concentration is 15 to 50 μg / ml, preferably around 60 μg / ml from the viewpoint of the final concentration of the drug that kills bacteria and does not adversely affect animal and phytoplankton such as rotifers. In the case of streptomycin and analogs thereof, the concentration is 10 to 100 μg / ml, preferably around 60 μg / ml, from the viewpoint of the final drug concentration that kills bacteria and does not adversely affect animal and phytoplankton such as rotifers.
次に、本発明のワムシの培養方法について説明する。本発明のワムシの培養方法は、上記本発明の無菌化方法を使用して、無菌ワムシを培養するワムシの培養方法であって、ワムシ耐久卵を次亜塩素酸ナトリウム、メルチオレート、グルタールアルデヒド、過酸化水素、塩化ベンザルコニウム（逆性石鹸）からなる群から選択される少なくとも1種による処理剤で洗浄し、当該処理剤を除去し、次いで、耐久卵をふ化させ、前記作用機序の異なる複数の抗生物質の存在下、培養する。処理剤の種類、濃度、抗生物質の説明については、上述の本発明の動植物プランクトンの無菌化方法をそのまま参照することができる。 Next, the rotifer culture method of the present invention will be described. The method for cultivating a rotifer of the present invention is a rotifer culture method for cultivating a sterile rotifer using the sterilization method of the present invention, wherein the rotifer is a sodium hypochlorite, merthiolate, glutaraldehyde, Wash with a treatment agent selected from the group consisting of hydrogen peroxide and benzalkonium chloride (reverse soap), remove the treatment agent, and then hatch a durable egg, Incubate in the presence of different antibiotics. For the description of the type, concentration, and antibiotics of the treatment agent, the above-described method for sterilizing animals and plants plankton of the present invention can be directly referred to.
また、本発明のワムシの培養方法の好ましい実施態様において、前記処理剤の濃度が、０．１〜１％である。当該濃度と、後述する処理時間との関係は、概ね反比例の関係にある。 In a preferred embodiment of the method for cultivating rotifers of the present invention, the concentration of the treatment agent is 0.1 to 1%. The relationship between the concentration and the processing time described later is generally inversely proportional.
好ましい実施態様において、前記処理剤での洗浄時間は、細菌を死滅させ動植物プランクトンに悪影響を及ぼさないという観点から、１〜１０分である。また、本発明のワムシの培養方法の好ましい実施態様において、前記処理剤による洗浄後、滅菌人口海水によりさらに洗浄し、当該処理剤を除去する。そして、前記処理剤を除去後、耐久卵をふ化させることができる。 In a preferred embodiment, the washing time with the treatment agent is 1 to 10 minutes from the viewpoint of killing bacteria and not adversely affecting the flora and fauna plankton. In a preferred embodiment of the method for cultivating a rotifer of the present invention, after washing with the treatment agent, the treatment agent is further washed with sterilized artificial seawater to remove the treatment agent. And after removing the said processing agent, a durable egg can be hatched.
また、本発明では、前記作用機序の異なる複数の抗生物質として、アンピシリン、カナマイシン、ナリジクス酸、ストレプトマイシンからなる群から選択される少なくとも1種を使用することができる。これらによって、上述の薬剤によって死滅しなかった細菌及びその後に混入してくると予想される土壌、水系、空気中等に存在する細菌類をも死滅させることが可能となる。
海洋性以外の細菌類をも滅菌することが可能となる。
In the present invention, at least one selected from the group consisting of ampicillin, kanamycin, nalidixic acid, and streptomycin can be used as the plurality of antibiotics having different action mechanisms. By these, it is possible to kill bacteria that have not been killed by the above-mentioned drugs and bacteria that are expected to be mixed in the soil, water system, and air.
Bacteria other than marine can be sterilized.
以下、本発明を実施例により更に具体的に説明するが、本発明は、下記実施例に限定して解釈される意図ではない。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not intended to be interpreted as being limited to the following examples.
実施例１
本実施例では、処理剤として、次亜塩素酸ナトリウムを使用した。ワムシ耐久卵の培養開始のスターターとして用い、次亜塩素酸ナトリウム処理後、抗生物質を使用することでワムシの無菌培養が可能となった。使用した抗生物質は、作用機序の異なるアンピシリン、カナマイシン、ナリジクス酸、ストレプトマイシンなどである。これらの抗生物質を混合して、混合液として用いた。
Example 1
In this example, sodium hypochlorite was used as the treating agent. As a starter for cultivating rotifer durable eggs, aseptic culture of rotifer was made possible by using antibiotics after sodium hypochlorite treatment. Antibiotics used include ampicillin, kanamycin, nalidixic acid, streptomycin and the like having different mechanisms of action. These antibiotics were mixed and used as a mixed solution.
無菌処理後のワムシ培養液の細菌検査は、真菌検査にポテトデキストロース寒天培地(菌類、酵母用)、細菌検査にマリーン寒天培地(海洋性細菌用)とトリプトソイ寒天培地(一般細菌用)を使用した。 Bacterial examination of rotifer culture after sterilization was performed using potato dextrose agar (for fungi and yeast) for fungal testing, and marine agar (for marine bacteria) and tryptosoy agar (for general bacteria) for bacterial testing. .
抗生物質の選択にあっては、１）短期間でワムシに悪影響を示さないもの、２）多種にわたる細菌を死滅させるため作用機序の異なる数種類を混交、３）抗生物質感受性菌が確実に死滅する濃度で抗生物質を使用、４）抗生物質処理後は、ワムシへの影響を最小限に抑えるため抗生物質を培地より除去した。以上の点から、使用種、濃度及び使用期間を定めた。 In selecting antibiotics, 1) those that do not adversely affect rotifers in a short period of time, 2) a mixture of several types with different mechanisms of action to kill a wide variety of bacteria, and 3) surely killing antibiotic-sensitive bacteria 4) After antibiotic treatment, the antibiotic was removed from the medium to minimize the effect on rotifer. From the above points, the use species, concentration, and use period were determined.
具体的に以下のように行なった。操作は、すべてクリーンベンチ内で行い,使用する試薬、機器類はすべて滅菌済みのものを使用した。 Specifically, it was performed as follows. All operations were performed in a clean bench, and all reagents and equipment used were sterilized.
１．耐久卵を終濃度0.5％の次亜塩素酸ナトリウムを含む滅菌人工海水で３分間洗浄した。
２．耐久卵を滅菌人工海水で３回洗浄し、次亜塩素酸ナトリウムを除去した。
３．滅菌人工海水に耐久卵を移し、光を照射しふ化させた。
４．ふ化後、無菌培養を行なったクロレラ濃縮液を給餌し培養した。
５．アンピシリン(100μg/ml)、カナマイシン(60μg/ml)、ナリジクス酸(30μg/ml)、ストレプトマイシン(60μg/ml)を培養液に加え培養した(2週間程度)。(括弧内に各抗生物質の終濃度を示す。)
６．ワムシをプランクトンネットで洗浄し抗生物質を除去し、培養した。
７．3種類の培地にて無菌検査を行い、無菌化を確認した。
1. Durable eggs were washed with sterilized artificial seawater containing sodium hypochlorite with a final concentration of 0.5% for 3 minutes.
2. Durable eggs were washed three times with sterile artificial seawater to remove sodium hypochlorite.
3. Durable eggs were transferred to sterilized artificial seawater and irradiated with light to hatch.
4). After hatching, the chlorella concentrate which had been subjected to aseptic culture was fed and cultured.
5). Ampicillin (100 μg / ml), kanamycin (60 μg / ml), nalidixic acid (30 μg / ml) and streptomycin (60 μg / ml) were added to the culture and cultured (about 2 weeks). (The final concentration of each antibiotic is shown in parentheses.)
6). The rotifer was washed with plankton net to remove antibiotics and cultured.
7. Sterilization was confirmed with three types of media to confirm sterilization.
無菌試験については以下のとおりである。まず、それぞれ３種類の寒天培地を別々に蒸気滅菌後、シャーレ（ペトリ皿）に分注後固める。これらの寒天培地にワムシ培養液を１００μｌ程度無菌的に塗り広げる。これらのシャーレを２５℃にて１〜２週間程度培養し、コロニーの有無を確認する。細菌が培養液中に混在している場合、どれか１つもしくは全ての寒天培地上に細菌のコロニーが生じる。無菌であればコロニーは確認できないこととなる。 The sterility test is as follows. First, each of the three types of agar medium is separately steam sterilized, then dispensed into a petri dish (petri dish) and then solidified. Aseptically spread about 100 μl of the rotifer broth on these agar media. These petri dishes are cultured at 25 ° C. for about 1 to 2 weeks, and the presence or absence of colonies is confirmed. When bacteria are mixed in the culture medium, bacterial colonies are formed on any one or all of the agar medium. If it is sterile, no colonies can be confirmed.
上述の無菌化の結果、マリーン寒天培地とトリプトソイ寒天培地をもちいた無菌検査では生じた細菌のコロニー形態が異なる物が確認され、マリーン寒天培地（その類似培地も含む）のみでは検査しきれない（培養不可）細菌が混在するが、本発明の無菌化方法によれば、これらの細菌類も殆ど観察されず、より無菌化状態を達成することが判明した。 As a result of the sterilization described above, sterility tests using marine agar medium and tryptic soy agar medium confirmed that the colony morphology of the produced bacteria was different, and it was not possible to test only with marine agar medium (including its similar medium) ( Although culture is not possible, bacteria are mixed, but according to the sterilization method of the present invention, these bacteria are hardly observed, and it has been found that a more sterilized state is achieved.
本発明は、水産業における増養殖分野において、確実に無菌化処理を行なうことが可能であり、魚類種苗生産を実施する上で、貢献度が極めて高い。 The present invention can reliably perform sterilization in the field of aquaculture in the fishery industry, and has a very high contribution in implementing fish seedling production.
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| CN105660466A (en) * | 2016-01-15 | 2016-06-15 | 中山大学 | Method for breeding grouper fry by controlling illumination |
| CN114621874A (en) * | 2021-12-28 | 2022-06-14 | 宁波浮田生物技术有限公司 | Microalgae culture medium and application |
| CN114946720A (en) * | 2022-05-25 | 2022-08-30 | 项尚飞 | Method for determining acute and chronic toxicity of cephalexin to brachionus calyciflorus |
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