JP2022076001A - Agar medium for microorganism culture - Google Patents
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Abstract
Description
本発明は、微生物を培養する寒天培地、すなわち固化成分として寒天を含有する固体培地およびその製造方法に関するものである。 The present invention relates to an agar medium for culturing microorganisms, that is, a solid medium containing agar as a solidifying component and a method for producing the same.
細菌、真菌などの微生物の検査は、臨床分野や獣医学分野における感染症診断の他、食品分野、環境分野における食中毒菌、衛生指標菌の検出などを目的として幅広い分野で実施されている。近年は、遺伝子法や免疫学的方法による迅速検査や自動分析機器が普及してきているものの、培養法により微生物の分離・同定を行うことは現在もなお微生物検査の基本である。 Inspection of microorganisms such as bacteria and fungi is carried out in a wide range of fields for the purpose of diagnosing infectious diseases in the clinical field and veterinary field, as well as detecting food poisoning bacteria and hygiene indicator bacteria in the food field and the environmental field. In recent years, although rapid diagnostic tests and automated analytical instruments using genetic and immunological methods have become widespread, the separation and identification of microorganisms by culture methods is still the basis of microbiological tests.
固化成分として寒天を使用する固体培地は、微生物の単離、鑑別用培地として使用され、また、薬剤感受性試験のディスク拡散法においても使用される。さらに、遺伝子法や直接ＥＩＡ法などのための試料調製を目的に、固体培地を使用する増菌培養が実施されることも多い。 A solid medium using agar as a solidifying component is used as a medium for isolating and differentiating microorganisms, and is also used in a disk diffusion method for drug susceptibility testing. Further, enrichment culture using a solid medium is often carried out for the purpose of sample preparation for the genetic method, the direct EIA method, or the like.
寒天は、ガラクトースを基本骨格とする直鎖の多糖類であり、紅藻のうちテングサ属やオゴノリ属の海藻を原料として得られる。熱可塑性であり、蒸留水を加えて加熱溶解すると１００℃前後で液化（ゾル化）し、それを冷却すると３５～４５℃付近で固化（ゲル化）する。 Agar is a linear polysaccharide having galactose as its basic skeleton, and is obtained from seaweeds of the genus Gelidiaceae and Gracilaria among red algae. It is thermoplastic, and when it is heated and dissolved by adding distilled water, it liquefies (soles) at around 100 ° C, and when it is cooled, it solidifies (gels) at around 35 to 45 ° C.
固体培地に含有されている寒天は、三次元の網目構造を形成してゲル状態で存在しているが、その網目構造中に多量の水分を蓄えていると考えられている。蓄えられた水は時間の経過によって、また、保存温度の変化に晒されることによって寒天から遊離する。遊離して出てきた水を一般に離水と呼び、容器内の固体培地の寒天ゲルの表面の離水が容器内で蒸発し、その水蒸気が凝結して容器の内壁や寒天ゲル表面に付着した水を凝水と呼ぶ。本明細書においては以下、特に断りの無い限り、離水と凝水を区別せずに凝水という。 The agar contained in the solid medium forms a three-dimensional network structure and exists in a gel state, but it is considered that a large amount of water is stored in the network structure. The stored water is released from the agar over time and by being exposed to changes in storage temperature. The water that comes out free is generally called water separation, and the water separation on the surface of the agar gel of the solid medium in the container evaporates in the container, and the water vapor condenses and adheres to the inner wall of the container and the surface of the agar gel. It is called coagulation. In the present specification, unless otherwise specified, the term "water coagulation" is used without distinguishing between water separation and water coagulation.
固体培地の寒天ゲルの表面の凝水が多いと、培地に生育した菌が流れてコロニーが形成されないことや、コロニーを形成したとしてもコロニーの拡散やコロニー同士の融合により菌の分離が困難になること、その他、雑菌の混入（コンタミネーション）などの問題が生じ得る。 If there is a lot of water on the surface of the agar gel in the solid medium, the bacteria that have grown on the medium will flow and colonies will not be formed, and even if colonies are formed, it will be difficult to separate the bacteria due to the diffusion of the colonies and the fusion of the colonies. In addition to this, problems such as contamination by various germs may occur.
寒天培地の凝水の問題を回避する方法として、検体を接種する前に、平板培地の表面に付着している水滴を無菌的に乾燥させる方法がある。その場合、通常、３５～３７℃のインキュベーター中で、シャーレを倒置し、蓋の上に少し開くように本体の方を傾けて置いて培地表面を乾燥させる（非特許文献１）。乾燥時間は３０～６０分程度であるが、インキュベーターの棚の置き場所によって乾燥に要する時間が異なるため、複数の平板培地の表面を適度な乾燥状態にするためには、乾燥中にインキュベーターの棚の上のシャーレの位置を換える作業が必要になる。また、培地の種類によってはインキュベーター中に置いても乾燥しにくいものもある。そのため、凝水が出にくい寒天培地が望まれている。 As a method of avoiding the problem of water coagulation of the agar medium, there is a method of aseptically drying the water droplets adhering to the surface of the plate medium before inoculating the sample. In that case, usually, in an incubator at 35 to 37 ° C., the petri dish is inverted and the main body is tilted so as to open slightly on the lid to dry the surface of the medium (Non-Patent Document 1). The drying time is about 30 to 60 minutes, but the time required for drying differs depending on the location of the incubator shelf. Therefore, in order to make the surfaces of multiple plate media in an appropriate dry state, the incubator shelf is being dried. It is necessary to change the position of the petri dish on the top. Also, depending on the type of medium, it may be difficult to dry even if it is placed in an incubator. Therefore, an agar medium that does not easily condense water is desired.
血液寒天培地の離水を抑制するために、血液寒天培地に高濃度（２０～４０％）のトレハロースを添加する方法が提案されている（特許文献１）。しかしながら、トレハロースは価格が高価なため、トレハロースを高濃度で含有する培地は高コストであるという問題がある。 In order to suppress the separation of water from the blood agar medium, a method of adding a high concentration (20 to 40%) of trehalose to the blood agar medium has been proposed (Patent Document 1). However, since trehalose is expensive, there is a problem that a medium containing a high concentration of trehalose is expensive.
本発明は、上記の現状に鑑みてなされたものであり、凝水が抑制され、調製が容易な微生物培養用の寒天培地およびその製造方法を提供することを目的としている。 The present invention has been made in view of the above-mentioned current situation, and an object of the present invention is to provide an agar medium for culturing microorganisms in which water coagulation is suppressed and easy to prepare, and a method for producing the same.
本発明者は、上記課題を解決するため鋭意検討した結果、従来、微生物培養用の寒天培地で使用されている重量平均分子量が３０万～４５万である寒天（以下、従来の微生物培養用の寒天という。）を固化成分とする寒天培地に、重量平均分子量が１５万以下、特に２万～１５万である低分子量寒天を固化成分として添加することにより、４℃～２５℃の保存温度の変化に晒した場合に培地表面に付着する凝水が抑制されることを新たに見出した。さらに、本発明者は、重量平均分子量が２万～１５万である低分子量寒天の添加量を、重量平均分子量が３０万～４５万である従来の微生物培養用の寒天の含有量に対して５％～５０％の割合とすることにより、凝水を抑制しつつ培地表面をコロニー形成に適切な状態に保ち、コロニーの微小化を抑えることができることを見出し、本発明を完成させるに至った。 As a result of diligent studies to solve the above problems, the present inventor has found that agar having a weight average molecular weight of 300,000 to 450,000, which has been conventionally used in an agar medium for culturing microorganisms (hereinafter, for conventional microbial culture). By adding low molecular weight agar having a weight average molecular weight of 150,000 or less, particularly 20,000 to 150,000, as a solidifying component to an agar medium containing agar as a solidifying component, a storage temperature of 4 ° C to 25 ° C can be obtained. It was newly found that the coagulation adhering to the surface of the medium was suppressed when exposed to the change. Furthermore, the present inventor has added an amount of low molecular weight agar having a weight average molecular weight of 20,000 to 150,000 with respect to the content of conventional agar for microbial culture having a weight average molecular weight of 300,000 to 450,000. We have found that by setting the ratio to 5% to 50%, it is possible to keep the surface of the medium in an appropriate state for colony formation while suppressing water coagulation, and to suppress the miniaturization of colonies, which has led to the completion of the present invention. ..
すなわち、本発明は以下のような構成からなるものである。
（１）少なくとも（ａ）および（ｂ）を固化成分として含有することを特徴とする、微生物の培養に用いる固体培地。
（ａ）重量平均分子量が３０万～４５万である寒天、
（ｂ）重量平均分子量が１５万以下である低分子量寒天。
（２）前記低分子量寒天の重量平均分子量が２万～１５万であることを特徴とする（１）に記載の固体培地。
（３）前記重量平均分子量が３０万～４５万である寒天の含有量（Ａ）に対する前記低分子量寒天の含有量（Ｂ）の比が０．０５以上０．５以下であることを特徴とする（１）または（２）に記載の固体培地。
（４）微生物の培養に用いる固体培地の製造方法であって、固化成分として（ａ）および（ｂ）を使用することを特徴とする固体培地の製造方法。
（ａ）重量平均分子量が３０万～４５万である寒天、
（ｂ）重量平均分子量が１５万以下である低分子量寒天。
（５）前記低分子量寒天の重量平均分子量が２万～１５万であることを特徴とする（４）に記載の固体培地の製造方法。
（６）前記重量平均分子量が３０万～４５万である寒天の含有量（Ａ）に対する前記低分子量寒天の含有量（Ｂ）の比が０．０５以上０．５以下であることを特徴とする（４）または（５）に記載の固体培地の製造方法。
That is, the present invention has the following configuration.
(1) A solid medium used for culturing microorganisms, which comprises at least (a) and (b) as solidifying components.
(A) Agar having a weight average molecular weight of 300,000 to 450,000,
(B) Low molecular weight agar having a weight average molecular weight of 150,000 or less.
(2) The solid medium according to (1), wherein the low molecular weight agar has a weight average molecular weight of 20,000 to 150,000.
(3) The ratio of the content (B) of the low molecular weight agar to the content (A) of the agar having a weight average molecular weight of 300,000 to 450,000 is 0.05 or more and 0.5 or less. The solid medium according to (1) or (2).
(4) A method for producing a solid medium used for culturing microorganisms, which comprises using (a) and (b) as solidifying components.
(A) Agar having a weight average molecular weight of 300,000 to 450,000,
(B) Low molecular weight agar having a weight average molecular weight of 150,000 or less.
(5) The method for producing a solid medium according to (4), wherein the low molecular weight agar has a weight average molecular weight of 20,000 to 150,000.
(6) The ratio of the content (B) of the low molecular weight agar to the content (A) of the agar having a weight average molecular weight of 300,000 to 450,000 is 0.05 or more and 0.5 or less. The method for producing a solid medium according to (4) or (5).
本発明によれば、固化成分として従来の微生物培養用の寒天を使用する固体培地に、重量平均分子量が１５万以下、特に２万～１５万である低分子量寒天を固化成分として添加することによって、ゲル状態の寒天の三次元網目構造の中に低分子量の寒天が入り込んで網目構造を補強し、離水が抑制され、結果として凝水を抑制可能な寒天培地とすることができる。 According to the present invention, by adding low molecular weight agar having a weight average molecular weight of 150,000 or less, particularly 20,000 to 150,000, as a solidifying component to a solid medium using conventional agar for culturing microorganisms as a solidifying component. , Low molecular weight agar enters into the three-dimensional network structure of gel-like agar to reinforce the network structure, water separation is suppressed, and as a result, agar medium capable of suppressing water coagulation can be obtained.
さらに、重量平均分子量が２万～１５万である低分子量寒天の添加量を重量平均分子量が３０万～４５万である従来の微生物培養用の寒天の含有量に対して５％～５０％の割合とすることにより、凝水を抑制しつつ培地表面をコロニー形成に適した状態とし、コロニーの微小化を抑えることができる。 Further, the amount of low molecular weight agar added having a weight average molecular weight of 20,000 to 150,000 is 5% to 50% of the content of conventional agar for microbial culture having a weight average molecular weight of 300,000 to 450,000. By setting the ratio, it is possible to suppress water coagulation, make the surface of the medium suitable for colony formation, and suppress the miniaturization of colonies.
また、本発明によれば、従来の微生物培養用の寒天を固化成分として使用する固体培地に、重量平均分子量が２万～１５万である低分子量寒天を固化成分として添加することにより固体培地を製造することができるため、特別な操作を要することなく、容易に培地を製造することができる。 Further, according to the present invention, a solid medium is added by adding low molecular weight agar having a weight average molecular weight of 20,000 to 150,000 as a solidifying component to a conventional solid medium using agar for culturing microorganisms as a solidifying component. Since it can be produced, the medium can be easily produced without requiring any special operation.
本発明の固体培地は、従来の微生物培養用の寒天に加えて、重量平均分子量が１５万以下、特に２万～１５万である低分子量寒天を固化成分として含有することを特徴とする。低分子量寒天を添加すると、ゲル状態において従来の微生物培養用の寒天が形成する三次元の網目構造の中に低分子量の寒天が入り込み、網目構造を複雑化して補強する。それにより、網目構造が保持できる水分量が増え、かつ、温度変化による網目構造の変化が起きにくくなることで離水が抑制され、結果として凝水を抑制可能になる。後に実施例で示す様に、重量平均分子量２万～１５万の低分子量寒天を用いることで前記の効果が得られる。
実際に微生物を培養する際には、従来の微生物培養用の寒天を含有する基礎培地の成分に加えて重量平均分子量が１５万以下、特に２万～１５万である低分子量寒天を添加したものを本発明の培地として用いることができる。なお、寒天の重量平均分子量の測定方法は特に限定されないが、高速液体クロマトグラフィー（ＨＰＬＣ）によるゲル浸透クロマトグラフィー法（ＧＰＣ法）により測定することができる。具体的には、国際公開第２０１６／１６７３７３号公報に記載されているように、寒天を０．１５％濃度となるよう精製水に懸濁し、９５℃～９７℃で加温溶解後、５０℃に冷却し、カラム（ＴＯＳＯＨ ＴＳＫ－ＧＥＬ ｆｏｒ ＨＰＬＣ，ＴＳＫ－ＧＥＬ ＧＭＰＷＸＬ等）を使用し、分子量既知のプルランを標準物質として測定することができる。
The solid medium of the present invention is characterized by containing, in addition to the conventional agar for culturing microorganisms, a low molecular weight agar having a weight average molecular weight of 150,000 or less, particularly 20,000 to 150,000, as a solidifying component. When low molecular weight agar is added, the low molecular weight agar enters into the three-dimensional network structure formed by the conventional agar for culturing microorganisms in the gel state, which complicates and reinforces the network structure. As a result, the amount of water that can be retained by the network structure increases, and changes in the network structure due to temperature changes are less likely to occur, so that water separation is suppressed, and as a result, water coagulation can be suppressed. As will be shown later in Examples, the above effect can be obtained by using a low molecular weight agar having a weight average molecular weight of 20,000 to 150,000.
When actually culturing microorganisms, in addition to the components of the conventional basal medium containing agar for culturing microorganisms, low molecular weight agar having a weight average molecular weight of 150,000 or less, particularly 20,000 to 150,000, is added. Can be used as the medium of the present invention. The method for measuring the weight average molecular weight of agar is not particularly limited, but it can be measured by gel permeation chromatography (GPC method) by high performance liquid chromatography (HPLC). Specifically, as described in International Publication No. 2016/163733, agar is suspended in purified water so as to have a concentration of 0.15%, heated and dissolved at 95 ° C to 97 ° C, and then 50 ° C. It can be measured by using a column (TOSOH TSK-GEL for HPLC, TSK-GEL GMPWXL, etc.) and purulan having a known molecular weight as a standard substance.
本発明で使用する低分子量寒天の重量平均分子量は２万～１５万であり、ゼリー強度（１．５％濃度のゲル。以下、濃度の記載は省略する。）は３０～２２０ｇ／ｃｍ２程度である。凝水抑制効果を高めるために、添加する低分子量寒天の重量平均分子量を２万～１２万とすることが好ましく、７万～１２万とすることがより好ましく、８万～１１万とすることがさらに好ましく、９万～１０．５万とすることがよりさらに好ましく、９．５万～１０．２万とすることが特に好ましい。また、低分子量寒天の重量平均分子量が２万～１２万である場合は、ゼリー強度が３０～１８０ｇ／ｃｍ２であることが好ましく、低分子量寒天の重量平均分子量が７万～１２万である場合は、ゼリー強度が１００～１８０ｇ／ｃｍ２であることが好ましく、低分子量寒天の重量平均分子量が８万～１１万である場合は、ゼリー強度は１２０～１７０ｇ／ｃｍ２であることが好ましく、低分子量寒天の重量平均分子量が９万～１０．５万である場合は、ゼリー強度が１３０～１６０ｇ／ｃｍ２であることが好ましく、低分子量寒天の重量平均分子量が９．５万～１０．２万である場合は、ゼリー強度が１４０～１５０ｇ／ｃｍ２であることが好ましい。 The weight average molecular weight of the low molecular weight agar used in the present invention is 20,000 to 150,000, and the jelly strength (1.5% concentration gel; hereinafter, the description of the concentration is omitted) is about 30 to 220 g / cm 2 . Is. In order to enhance the effect of suppressing water coagulation, the weight average molecular weight of the low molecular weight agar to be added is preferably 20,000 to 120,000, more preferably 70,000 to 120,000, and more preferably 80,000 to 110,000. Is even more preferable, and 90,000 to 105,000 is even more preferable, and 95,000 to 102,000 is particularly preferable. When the weight average molecular weight of the low molecular weight agar is 20,000 to 120,000, the jelly strength is preferably 30 to 180 g / cm 2 , and the weight average molecular weight of the low molecular weight agar is 70,000 to 120,000. In this case, the jelly strength is preferably 100 to 180 g / cm 2 , and when the weight average molecular weight of the low molecular weight agar is 80,000 to 110,000, the jelly strength is preferably 120 to 170 g / cm 2 . When the weight average molecular weight of low molecular weight agar is 90,000 to 105,000, the jelly strength is preferably 130 to 160 g / cm 2 , and the weight average molecular weight of low molecular weight agar is 95,000 to 10 When it is 20,000, the jelly strength is preferably 140 to 150 g / cm 2 .
本発明で使用する低分子量寒天は、重量平均分子量２万～１５万である。製造方法は特に限定されないが、特開平５－３１７００８号公報や特開平１０－１４６１７４号公報に記載されているように、重量平均分子量が約２０万～約４０万である市販の一般的な寒天の製造工程に酸処理や熱処理、酵素処理などにより寒天分子を切断する工程を追加することにより製造されたものであっても良い。使用する酸濃度、加熱温度などを調節することにより分子量やゼリー強度を適宜調節する技術も公知である（梅村澄夫ら，低分子量寒天の開発と利用に関する研究（第１報），岐阜県産業技術センター研究報告，２００９）。
なお、本発明で使用できる市販の低分子量寒天としては、例えば、ウルトラ寒天ＡＸ－３０（伊那食品工業株式会社、ゼリー強度：３０～６０ｇ／ｃｍ２）、ウルトラ寒天ＡＸ－１００（伊那食品工業株式会社製、ゼリー強度：１００～１５０ｇ／ｃｍ２）、ウルトラ寒天ＢＸ－３０（伊那食品工業株式会社製、ゼリー強度：３０～６０ｇ／ｃｍ２）、ウルトラ寒天ＢＸ－１００（伊那食品工業株式会社製、ゼリー強度：１００～１５０ｇ／ｃｍ２）、ウルトラ寒天ＵＸ－３０（伊那食品工業株式会社製、ゼリー強度：３０～６０ｇ／ｃｍ２）、ウルトラ寒天ＵＸ－１００（伊那食品工業株式会社、ゼリー強度：１００～１５０ｇ／ｃｍ２）などが挙げられる。
The low molecular weight agar used in the present invention has a weight average molecular weight of 20,000 to 150,000. The production method is not particularly limited, but as described in JP-A-5-317008 and JP-A-10-146174, a general commercially available agar having a weight average molecular weight of about 200,000 to about 400,000. It may be manufactured by adding a step of cleaving agar molecules by acid treatment, heat treatment, enzyme treatment or the like to the manufacturing step of. A technique for appropriately adjusting the molecular weight and jelly strength by adjusting the acid concentration and heating temperature to be used is also known (Sumio Umemura et al., Research on Development and Utilization of Low Molecular Weight Agar (1st Report), Gifu Industrial Technology Center Research Report, 2009).
Examples of commercially available low molecular weight agar that can be used in the present invention include Ultra Agar AX-30 (Ina Food Industry Co., Ltd., Jelly strength: 30-60 g / cm 2 ) and Ultra Agar AX-100 (Ina Food Industry Co., Ltd.). Company-made, jelly strength: 100-150 g / cm 2 ), Ultra agar BX-30 (made by Ina Food Industry Co., Ltd., jelly strength: 30-60 g / cm 2 ), ultra-agar BX-100 (made by Ina Food Industry Co., Ltd.) , Jelly strength: 100-150 g / cm 2 ), Ultra agar UX-30 (manufactured by Ina Food Industry Co., Ltd., jelly strength: 30-60 g / cm 2 ), Ultra agar UX-100 (Ina Food Industry Co., Ltd., jelly strength : 100-150 g / cm 2 ) and the like.
従来の微生物培養用の寒天の重量平均分子量は約４０万、より詳細には３０万～４５万程度であり、ゼリー強度は５００～８００ｇ／ｃｍ２程度である。本発明において寒天培地に固化成分として使用する従来の微生物培養用の寒天の量は、それぞれの培地に応じて適宜選択すれば良いが、例えば培地１Ｌ当たり８ｇ～２０ｇの範囲から適宜含有量を選択できる。 The weight average molecular weight of conventional agar for culturing microorganisms is about 400,000, more specifically, about 300,000 to 450,000, and the jelly strength is about 500 to 800 g / cm 2 . In the present invention, the amount of conventional agar for culturing microorganisms used as a solidifying component in the agar medium may be appropriately selected according to each medium, and for example, the content is appropriately selected from the range of 8 g to 20 g per 1 L of the medium. can.
後に実施例に示す様に、従来の微生物培養用の寒天の含有量を一定にし、重量平均分子量２万～１５万の低分子量寒天の添加量を変化させると、低分子量寒天の添加量が増加するのに伴い、凝水量が減少する傾向がある。凝水量が減少すれば、発育したコロニーが流れてコロニーが形成されない問題が改善される。一方、低分子量寒天の添加によりゼリー強度が大きく上昇する（低分子量寒天を添加していない培地と比較したゼリー強度の上昇値が約１００ｇ／ｃｍ２を超える）と、発育した微生物のコロニー形成状態（拡がり状態）に影響し、コロニーが微小化する傾向がある。
そのため、本発明の固体培地で使用する低分子量寒天の添加量は、凝水量およびゼリー強度の両面において培地表面をコロニー形成に適切な状態に保ち、コロニーの微小化を抑えつつ凝水を抑制できる範囲とすることが好ましい。当業者であれば本発明の実施例を参考にして低分子量寒天の添加量を設定することができる。例えば、培地１Ｌ当たりの低分子量寒天の添加量を０．８ｇ以上とすることが好ましく、１ｇ以上とすることがより好ましく、２ｇ以上とすることがさらに好ましく、２．５ｇ以上とすることがよりさらに好ましい。また、培地１Ｌ当たりの低分子量寒天の添加量を７．５ｇ以下とすることが好ましく、６ｇ以下とすることがより好ましく、５ｇ以下とすることがさらに好ましいが、低分子量寒天の添加によるゼリー強度の上昇に起因するコロニーの微小化の状況に応じて、適宜、低分子量寒天の添加量を４ｇ以下、３．５ｇ以下などとすることができる。
As shown later in Examples, when the content of conventional agar for microbial culture is kept constant and the amount of low molecular weight agar added having a weight average molecular weight of 20,000 to 150,000 is changed, the amount of low molecular weight agar added increases. As a result, the amount of coagulation tends to decrease. If the amount of water coagulation is reduced, the problem that the grown colonies flow and the colonies are not formed is improved. On the other hand, when the jelly strength is greatly increased by the addition of the low molecular weight agar (the increase value of the jelly strength is more than about 100 g / cm 2 as compared with the medium to which the low molecular weight agar is not added), the colonization state of the developed microorganisms. It affects (spreading state) and tends to make colonies smaller.
Therefore, the amount of low molecular weight agar added in the solid medium of the present invention keeps the surface of the medium in an appropriate state for colonization in terms of both the amount of water coagulation and the strength of jelly, and can suppress water coagulation while suppressing the miniaturization of colonies. It is preferably in the range. A person skilled in the art can set the amount of low molecular weight agar added with reference to the examples of the present invention. For example, the amount of low molecular weight agar added per 1 L of medium is preferably 0.8 g or more, more preferably 1 g or more, further preferably 2 g or more, and more preferably 2.5 g or more. More preferred. Further, the amount of low molecular weight agar added per 1 L of medium is preferably 7.5 g or less, more preferably 6 g or less, still more preferably 5 g or less, but the jelly strength due to the addition of low molecular weight agar. The amount of low molecular weight agar added can be appropriately set to 4 g or less, 3.5 g or less, or the like, depending on the state of micronization of the colony due to the increase in the amount of the agar.
本発明の固体培地において使用する従来の微生物培養用の寒天の含有量（Ａ）に対する低分子量寒天の添加量（Ｂ）の比（Ｂ／Ａ）は、培地表面をコロニー形成に適切な状態に保ち、コロニーの微小化を抑えつつ凝水を抑制できる範囲とすることが好ましい。例えば、本発明の固体培地において使用する従来の微生物培養用の寒天の含有量（Ａ）に対する低分子量寒天の添加量（Ｂ）の比（Ｂ／Ａ）を、０．０５以上（例えば０．８ｇ／１５ｇ）とすることが好ましいが、０．１以上（例えば１．５ｇ／１５ｇ）とすることがより好ましく、０．１２以上（例えば１．８ｇ／１５ｇ）とすることがさらに好ましく、０．１４以上（例えば２．１ｇ／１５ｇ）とすることがよりさらに好ましく、０．１６以上（例えば２．５ｇ／１５ｇ）とすることが特に好ましい。また、従来の微生物培養用の寒天の含有量（Ａ）に対する低分子量寒天の添加量（Ｂ）の比（Ｂ／Ａ）は、０．５以下（例えば７．５ｇ／１５ｇ）とすることが好ましく、０．４以下（例えば６ｇ／１５ｇ）とすることがより好ましく、０．３４以下（例えば５ｇ／１５ｇ）とすることがさらに好ましいが、低分子量寒天の添加によるゼリー強度の上昇に起因するコロニーの微小化の状況に応じて、適宜、０．３以下（例えば４．５ｇ／１５ｇ）、０．２７以下（例えば４ｇ／１５ｇ）、０．２５以下（例えば３．７ｇ／１５ｇ）、０．２３以下（例えば３．４ｇ／１５ｇ）、０．２以下（例えば３．０ｇ／１５ｇ）などとすることができる。 The ratio (B / A) of the addition amount (B / A) of the low molecular weight agar to the content (A) of the conventional agar for culturing microorganisms used in the solid medium of the present invention makes the medium surface suitable for colonization. It is preferable to keep it in a range where water coagulation can be suppressed while suppressing the miniaturization of colonies. For example, the ratio (B / A) of the amount of low molecular weight agar added (B) to the content (A) of conventional agar for culturing microorganisms used in the solid medium of the present invention is 0.05 or more (for example, 0. 8 g / 15 g) is preferable, but 0.1 or more (for example, 1.5 g / 15 g) is more preferable, and 0.12 or more (for example, 1.8 g / 15 g) is more preferable, and 0. It is more preferably .14 or more (for example, 2.1 g / 15 g), and particularly preferably 0.16 or more (for example, 2.5 g / 15 g). Further, the ratio (B / A) of the addition amount (B / A) of the low molecular weight agar to the content (A) of the conventional agar for culturing microorganisms may be 0.5 or less (for example, 7.5 g / 15 g). It is preferably 0.4 or less (for example, 6 g / 15 g), more preferably 0.34 or less (for example, 5 g / 15 g), but it is caused by an increase in jelly strength due to the addition of low molecular weight agar. 0.3 or less (for example, 4.5 g / 15 g), 0.27 or less (for example, 4 g / 15 g), 0.25 or less (for example, 3.7 g / 15 g), 0, depending on the state of colony miniaturization. It can be .23 or less (for example, 3.4 g / 15 g), 0.2 or less (for example, 3.0 g / 15 g), and the like.
本発明の固体培地において培養の対象となる微生物は、寒天培地に生育し得る細菌または真菌である。細菌の具体例として、Staphylococcus属菌、Streptococcus属菌、Enterococcus属菌、Bacillus属菌、Corynebacterium属菌、Listeria属菌、Peptococcus属菌、Clostridium属菌、Eubacterium属菌、Propionibacterium属菌、Lactobacillus属菌、Actinomyces属菌、Nocardia属菌、Mycobacterium属菌などのグラム陽性菌、Neisseria属菌、Branhamella属菌、Haemophilus属菌、Escherichia属菌、Salmonella属菌、Shigella属菌、Klebsiella属菌、Bordetella属菌、Enterobacter属菌、Serratia属菌、Campylobacter属菌、Vibrio属菌、Pseudomonas属菌、Aeromonas属菌、Acinetobacter属菌、Brucella属菌、Legionella属菌、Citrobacter属菌、Hafnia属菌、Proteus属菌、Morganella属菌、Providencia属菌、Yersinia属菌、Xanthomonas属菌、Flavobacterium属菌、Helicobacter属菌、Veillonella属菌、Bacteroides属菌、Fusobacterium属菌、Treponema属菌、Leptospira属菌、Mycoplasma属菌、Ureaplasma属菌などのグラム陰性菌が挙げられ、真菌の具体例として、Aspergillus属菌、Candida属菌、Cryptococcus属菌、Mucor属菌、Mortierella属菌、Trichophyton属菌、Histoplasma属菌などが挙げられるが、これらに限定されない。 The microorganism to be cultured in the solid medium of the present invention is a bacterium or fungus that can grow on an agar medium. Specific examples of the bacteria include Staphylococcus spp., Streptococcus spp., Enterococcus spp., Bacillus spp., Corynebacterium spp., Listeria spp., Peptococcus spp., Clostridium spp., Eubacterium spp., Propionibacterium spp., Lactobacillus spp. Gram-positive bacteria such as Actinomyces , Nocardia , Mycobacterium , Neisseria , Branhamella , Haemophilus , Escherichia , Salmonella , Shigella , Klebsiella , Bordetella , Enterobacter Genus, Serratia , Campylobacter , Vibrio , Pseudomonas , Aeromonas , Acinetobacter , Brucella , Legionella , Citrobacter , Hafnia , Proteus , Morganella , Providencia , Yersinia , Xanthomonas , Flavobacterium , Helicobacter , Veillonella , Bacteroides , Fusobacterium , Treponema , Leptospira , Mycoplasma , Ureaplasma , etc. Gram-negative bacteria are mentioned, and specific examples of fungi include, but are not limited to, Aspergillus spp., Candida spp., Cryptococcus spp., Mucor spp., Mortierella spp., Trichophyton spp., Histoplasma spp. ..
本発明の固体培地の基礎培地となる培地は、細菌、真菌が発育可能な寒天培地である。具体例として普通寒天培地、標準寒天培地、トリプトソイ寒天培地（SCD寒天培地）、血液寒天培地、チョコレート寒天培地、マンニット食塩培地、エッグヨーク食塩寒天培地、スタフィロコッカスＮｏ．１１０培地、マッコンキー寒天培地、CT-SMAC寒天培地、ドリガルスキー寒天培地（DRIG培地）、ドリガルスキー改良培地（BTB乳糖寒天培地）、デスオキシコーレイト寒天培地、DHL寒天培地、ハートインフュジョン寒天培地、ブレインハートインフュジョン寒天培地、SSB寒天培地、サルモネラ・シゲラ寒天培地(SS寒天培地)、TCBS寒天培地、ビブリオ寒天培地、TSI寒天培地、クリグラー寒天培地、アルギニン・グリセロール・塩類寒天培地（AGS寒天培地）、ベアードパーカー寒天培地、NAC寒天培地、スキロー培地、NGKG寒天培地、ミュラーヒントン寒天培地、サブロー寒天培地、ポテトデキストロース寒天培地、カンジダ寒天培地などが挙げられるが、これらに限定されない。 The medium serving as the basal medium of the solid medium of the present invention is an agar medium on which bacteria and fungi can grow. Specific examples include ordinary agar medium, standard agar medium, trypto-soy agar medium (SCD agar medium), blood agar medium, chocolate agar medium, mannit salt medium, egg yoke salt agar medium, and Staphylococcus No. 110 medium, McConkey agar medium, CT-SMAC agar medium, Drigalski agar medium (DRIG medium), Drigalsky improved medium (BTB lactose agar medium), Desoxycholate agar medium, DHL agar medium, Heart Infusion agar medium, Brainhart Infusion Agar, SSB Agar, Salmonella Shigera Agar (SS Agar), TCBS Agar, Vibrio Agar, TSI Agar, Krigler Agar, Arginine glycerol / Salt Agar (AGS Agar) , Baird Parker Agar, NAC Agar, Skillo Medium, NGKG Agar, Muller Hinton Agar, Sabouraud Agar, Potato Dextrose Agar, Candida Agar, etc., but not limited to these.
本発明の固体培地は、固化成分として従来の微生物培養用の寒天に加えて重量平均分子量２万～１５万の低分子量寒天を使用することにより製造することができる。後に実施例に示す様に、固化成分として従来の微生物培養用の寒天に加えて重量平均分子量２万～１５万の低分子量寒天を添加する以外は、基礎培地となる培地の製造方法と同様の工程を経て製造され得る。すなわち、固化成分として（ａ）重量平均分子量が３０万から４５万である寒天、および（ｂ）重量平均分子量２万～１５万の低分子量寒天、を使用して調製した培地成分溶液に滅菌処理を施した後、シャーレなどの容器に分注して固化することにより製造することができる。 The solid medium of the present invention can be produced by using a low molecular weight agar having a weight average molecular weight of 20,000 to 150,000 in addition to the conventional agar for culturing microorganisms as a solidifying component. As shown later in Examples, the same method as the method for producing a medium as a basal medium is used except that a low molecular weight agar having a weight average molecular weight of 20,000 to 150,000 is added in addition to the conventional agar for culturing microorganisms as a solidifying component. It can be manufactured through a process. That is, the medium component solution prepared using (a) agar having a weight average molecular weight of 300,000 to 450,000 and (b) low molecular weight agar having a weight average molecular weight of 20,000 to 150,000 as the solidifying component is sterilized. It can be manufactured by dispensing it into a container such as a petri dish and solidifying it.
本発明に使用される試料は、細菌、真菌を含む可能性のある試料であれば特に限定されない。ヒト、他の動物の生体由来、食品、環境由来の検体、それらの培養液、さらには、医薬品、化粧品の成分などが試料として挙げられる。これらの試料は、抽出、濃縮などの前処理を行っても良い。 The sample used in the present invention is not particularly limited as long as it is a sample that may contain bacteria and fungi. Specimens derived from living organisms of humans and other animals, foods, and the environment, their culture solutions, and components of pharmaceuticals and cosmetics can be mentioned as samples. These samples may be subjected to pretreatment such as extraction and concentration.
本発明の固体培地は、必要に応じて、分離対象とする微生物に適した発色酵素基質、分離対象とする微生物以外の発育を抑制するための選択剤、その他の成分をさらに添加することもできる。通常、選択分離培地で使用され得る成分の種類、濃度における添加であれば、本発明の効果に影響することはない。 If necessary, the solid medium of the present invention may be further added with a color-developing enzyme substrate suitable for the microorganism to be isolated, a selection agent for suppressing the growth of microorganisms other than the microorganism to be isolated, and other components. .. Usually, if it is added at the type and concentration of the component that can be used in the selective separation medium, the effect of the present invention is not affected.
本発明の固体培地は、微生物の単離、鑑別用培地としての使用の他に、薬剤感受性試験のディスク拡散法においても使用することができ、さらには、遺伝子法や直接ＥＩＡ法などのための試料調製を目的にした、増菌培養用の培地としても使用することができる。 The solid medium of the present invention can be used not only as a medium for isolation and differentiation of microorganisms, but also in a disk diffusion method for drug susceptibility testing, and further for a genetic method, a direct EIA method, or the like. It can also be used as a medium for enrichment culture for the purpose of sample preparation.
本発明の固体培地の形態は特に限定されないが、優れた凝水抑制効果という観点から、平板固形培地の形態が最も好ましい。 The form of the solid medium of the present invention is not particularly limited, but the form of a flat plate solid medium is most preferable from the viewpoint of excellent water coagulation suppressing effect.
以下に本発明の実施例を示すが、本発明はこれら実施例により限定されるものではない。 Examples of the present invention are shown below, but the present invention is not limited to these examples.
（血液寒天培地における凝水試験）
羊血液寒天培地を基礎培地とし、低分子量寒天ＳＷ（ＳＳＫセールス株式会社製、重量平均分子量：約１０万、ゼリー強度：１４０～１５０ｇ／ｃｍ２）を添加して凝水抑制効果を調べた。
(Water coagulation test in blood agar medium)
Using sheep blood agar medium as the basal medium, low molecular weight agar SW (manufactured by SSK Sales Co., Ltd., weight average molecular weight: about 100,000, jelly strength: 140 to 150 g / cm 2 ) was added to examine the effect of suppressing water coagulation.
（１）培地の準備
以下の表１に示した培地成分１を秤量し、以下の（２）で羊血液を添加した後の培地量が２Ｌとなるように精製水に懸濁後、１２１℃で１５分間高圧滅菌した。従来の微生物培養用の寒天と低分子量寒天を区別するため、表１において、従来の微生物培養用の寒天を寒天Ａ、低分子量寒天ＳＷを寒天Ｂと記載する。
(1) Preparation of medium Weigh the medium component 1 shown in Table 1 below, suspend it in purified water so that the amount of medium after adding sheep blood in (2) below is 2 L, and then record at 121 ° C. Was sterilized under high pressure for 15 minutes. In order to distinguish between the conventional agar for culturing microorganisms and the low molecular weight agar, the agar for conventional culturing is referred to as agar A and the low molecular weight agar SW is referred to as agar B in Table 1.
（２）羊血液の添加
高圧滅菌後、培地を５０℃に冷却した後、予め無菌的に採取した羊脱繊維血液を、培地１Ｌ当たり５０ｍＬとなるように添加した。その後、培地を１８ｍＬずつシャーレに分注して固化した。
(2) Addition of sheep blood After high-pressure sterilization, the medium was cooled to 50 ° C., and then aseptically collected sheep defibered blood was added so as to be 50 mL per 1 L of the medium. Then, 18 mL of the medium was dispensed into a petri dish and solidified.
（３）凝水量の測定
（１）、（２）で調製した培地入りシャーレを４℃、倒置にて２０時間保存した後、２５℃、倒置にてシャーレを３０分間放置し、その後、培地入りシャーレの重量を電子天秤にて測定した。続いてシャーレを縦置きにして３０分間放置して培地表面に滲み出た水を濾紙によって拭き取った後、再び培地入りシャーレの重量を測定し、（滲み出た水の拭き取り前のシャーレの重量）－（滲み出た水を拭き取った後のシャーレの重量）を凝水量として算出した。
なお、低分子量寒天ＳＷの各添加量につき１０枚のシャーレを凝水試験対象とし、重量の測定はシャーレ１枚毎に行った。
(3) Measurement of the amount of coagulated water The petri dish containing the medium prepared in (1) and (2) was stored at 4 ° C. in an inverted position for 20 hours, then left at 25 ° C. in an inverted position for 30 minutes, and then contained in a medium. The weight of the petri dish was measured with an electronic balance. Subsequently, the petri dish was placed vertically and left for 30 minutes to wipe off the water exuded on the surface of the medium with a filter paper, and then the weight of the petri dish containing the medium was measured again (the weight of the petri dish before wiping off the exuded water). -(Weight of petri dish after wiping off the exuded water) was calculated as the amount of water coagulation.
In addition, 10 petri dishes were subject to the water coagulation test for each addition amount of the low molecular weight agar SW, and the weight was measured for each petri dish.
（４）培地のゼリー強度の測定
（１）、（２）で調製した培地を上記（３）の凝水量測定用のものとは別に用意し、２０℃の恒温槽にて２０時間保存した後、レオメーター（ＦＵＤＯＨ ＲＨＥＯ ＭＥＴＥＲ ＲＴＣ－３００２Ｄ）（株式会社レイテック製）の測定部が培地に１ｍｍ侵入する強度をゼリー強度として測定した。なお、低分子量寒天ＳＷの各添加量につきシャーレ１枚をゼリー強度測定の対象とし、１枚のシャーレの３箇所を測定して平均値をゼリー強度として算出した。
(4) Measurement of jelly strength of medium The medium prepared in (1) and (2) is prepared separately from the one for measuring the amount of coagulation in (3) above, and stored in a constant temperature bath at 20 ° C. for 20 hours. , The strength at which the measuring unit of the Leometer (FUDOH RHEO MTER RTC-3002D) (manufactured by Raytec Co., Ltd.) penetrated the medium by 1 mm was measured as the jelly strength. For each amount of low molecular weight agar SW added, one petri dish was targeted for jelly strength measurement, three points of one petri dish were measured, and the average value was calculated as the jelly strength.
（５）結果
培地の凝水量およびゼリー強度の測定結果を表２に示す。凝水量の測定結果は、低分子量寒天ＳＷの各添加量における平均凝水量を、低分子量寒天ＳＷを添加していない培地（Ｎｏ．１）の平均凝水量と比較した凝水削減量および凝水削減率とともに表示し、また、ゼリー強度は、低分子量寒天ＳＷを添加していない培地（Ｎｏ．１）のゼリー強度からの上昇値とともに表示している。
(5) Results Table 2 shows the measurement results of the amount of coagulated water and the jelly strength of the medium. The measurement result of the amount of coagulation was that the average amount of coagulation at each addition amount of the low molecular weight agar SW was compared with the average amount of coagulation of the medium (No. 1) to which the low molecular weight agar SW was not added. It is displayed together with the reduction rate, and the jelly intensity is displayed together with the increase value from the jelly intensity of the medium (No. 1) to which the low molecular weight agar SW is not added.
表２に示した様に、培地１Ｌ当たりの低分子量寒天ＳＷの添加量が１ｇの培地（Ｎｏ．２）においても、低分子量寒天ＳＷを添加していない培地（Ｎｏ．１）の凝水量と比較した削減率は６４．６％という良好な値であった。また、凝水削減率は低分子量寒天ＳＷの添加量の増加に伴い上昇し、培地１Ｌ当たりの低分子量寒天ＳＷの添加量が３ｇ以上の培地では７６．０％以上という非常に良好な凝水削減率を示した。一方、ゼリー強度は、低分子量寒天ＳＷの添加量の増加に伴い上昇し、培地１Ｌ当たりの低分子量寒天ＳＷの添加量が４ｇの培地（Ｎｏ．５）では、低分子量寒天ＳＷを添加していない培地（Ｎｏ．１）のゼリー強度よりも１１０ｇ／ｃｍ２高い値を示した。また、培地１Ｌ当たりの低分子量寒天ＳＷの添加量が５ｇの培地（Ｎｏ．６）のゼリー強度は７００ｇ／ｃｍ２を超えていた。 As shown in Table 2, even in the medium (No. 2) in which the amount of low molecular weight agar SW added per 1 L of the medium is 1 g, the amount of coagulated water in the medium (No. 1) to which the low molecular weight agar SW is not added The compared reduction rate was a good value of 64.6%. In addition, the water coagulation reduction rate increases as the amount of low molecular weight agar SW added increases, and the amount of low molecular weight agar SW added per 1 L of medium is 76.0% or more in a medium of 3 g or more, which is very good. The reduction rate is shown. On the other hand, the jelly strength increases with an increase in the amount of low molecular weight agar SW added, and in the medium (No. 5) in which the amount of low molecular weight agar SW added per 1 L of the medium is 4 g, the low molecular weight agar SW is added. It showed a value 110 g / cm 2 higher than the jelly intensity of the non-medium (No. 1). Further, the jelly strength of the medium (No. 6) in which the amount of low molecular weight agar SW added per 1 L of the medium was 5 g exceeded 700 g / cm 2 .
（血液寒天培地における培養試験）
羊血液寒天培地を基礎培地として低分子量寒天ＳＷ（ＳＳＫセールス株式会社製、重量平均分子量：約１０万、ゼリー強度：１４０～１５０ｇ／ｃｍ２）を添加した培地にStreptococcus pyogenes、Streptococcus agalactiae、Streptococcus intermedius、Streptococcus pneumoniae、Streptococcus Group G、Enterobacter cloacae、Moraxella catarrhalis、Staphylococcus aureusを接種し、菌の発育状況を調べた。
(Culture test in blood agar medium)
Streptococcus pyogenes , Streptococcus agalactiae , Streptococcus intermedius to a medium containing sheep blood agar medium as a basal medium and low molecular weight agar SW (manufactured by SSK Sales Co., Ltd., weight average molecular weight: about 100,000, jelly strength: 140-150 g / cm 2 ). , Streptococcus pneumoniae , Streptococcus Group G, Enterobacter cloacae , Moraxella catarrhalis , Staphylococcus aureus were inoculated and the growth status of the fungus was investigated.
（１）培地の準備
以下の表３に示した培地成分２を秤量し、以下の（２）で羊血液を添加した後の培地量が３Ｌとなるように精製水に懸濁後、１２１℃で１５分間高圧滅菌した。表３において、従来の微生物培養用の寒天を寒天Ａ、低分子量寒天ＳＷを寒天Ｂと記載する。
(1) Preparation of medium Weigh the medium component 2 shown in Table 3 below, suspend it in purified water so that the amount of medium after adding sheep blood in (2) below is 3 L, and then record at 121 ° C. Was sterilized under high pressure for 15 minutes. In Table 3, the conventional agar for culturing microorganisms is referred to as agar A, and the low molecular weight agar SW is referred to as agar B.
（２）羊血液の添加
高圧滅菌後、培地を５０℃に冷却した後、予め無菌的に採取した羊脱繊維血液を、培地１Ｌ当たり５０ｍＬとなるように添加した。その後、培地を１８ｍＬずつシャーレに分注して固化した。
(2) Addition of sheep blood After high-pressure sterilization, the medium was cooled to 50 ° C., and then aseptically collected sheep defibered blood was added so as to be 50 mL per 1 L of the medium. Then, 18 mL of the medium was dispensed into a petri dish and solidified.
（３）細菌の接種と培養
前培養した各菌株を滅菌生理食塩水に懸濁し、McFarland No. 1の菌液を作製した。その後、前記菌液を（１）、（２）で調製した培地に画線し、３５℃で１８時間、５％炭酸ガス環境下での培養または好気培養を行い、発育した細菌のコロニーサイズを測定した。なお、シャーレ１枚につき１菌株を接種して培養試験を行い、優勢に発育した１コロニーを代表としてサイズの測定を行った。
(3) Bacterial inoculation and culture Each pre-cultured strain was suspended in sterile physiological saline to prepare McFarland No. 1 bacterial solution. Then, the bacterial solution was plotted on the medium prepared in (1) and (2), and cultured at 35 ° C. for 18 hours in a 5% carbon dioxide gas environment or aerobic culture to grow the colony size of the bacteria. Was measured. In addition, one strain was inoculated into one petri dish and a culture test was conducted, and the size was measured with one colony that grew predominantly as a representative.
（４）結果
５％炭酸ガス環境下での培養により発育したコロニーのサイズを表４に、また、好気培養により発育したコロニーのサイズを表５に示す。
(4) Results Table 4 shows the sizes of colonies grown by culturing in a 5% carbon dioxide gas environment, and Table 5 shows the sizes of colonies grown by aerobic culture.
表４および５に示した様に、５％炭酸ガス環境下での培養、好気培養のいずれにおいても、培地１Ｌ当たりの低分子量寒天ＳＷの添加量が３ｇの培地では低分子量寒天ＳＷを添加していない培地と同等のコロニーサイズであったが、培地１Ｌ当たりの低分子量寒天ＳＷの添加量が４ｇの培地ではコロニーサイズが小さくなる傾向が見られ、培地１Ｌ当たりの低分子量寒天ＳＷの添加量が５ｇの培地では、コロニーサイズがさらに小さくなっていた。 As shown in Tables 4 and 5, low molecular weight agar SW was added to the medium in which the amount of low molecular weight agar SW added per 1 L of the medium was 3 g in both the culture under the 5% carbon dioxide gas environment and the aerobic culture. The colony size was the same as that of the medium without the medium, but the colony size tended to be smaller in the medium in which the amount of low molecular weight agar SW added per 1 L of the medium was 4 g, and the addition of the low molecular weight agar SW per 1 L of the medium was observed. In the medium with an amount of 5 g, the colony size was further reduced.
実施例１の結果（表２）によると、培地１Ｌ当たりの低分子量寒天ＳＷの添加量が４ｇ、５ｇの培地では、低分子量寒天ＳＷを添加していない培地と比較したゼリー強度の上昇値がそれぞれ１１０ｇ／ｃｍ２、１５０ｇ／ｃｍ２であることから、低分子量寒天を添加していない培地と比較してゼリー強度が約１００ｇ／ｃｍ２以上上昇している培地（低分子量寒天ＳＷの場合には、培地１Ｌ当たりの添加量が４ｇ以上の培地）ではゼリー強度が上昇する程、コロニーが小さくなることが示唆された。 According to the results of Example 1 (Table 2), in the medium in which the amount of low molecular weight agar SW added per 1 L of the medium was 4 g and 5 g, the increase value of the jelly strength was higher than that in the medium to which the low molecular weight agar SW was not added. Since the contents are 110 g / cm 2 and 150 g / cm 2 , respectively, the jelly strength is increased by about 100 g / cm 2 or more as compared with the medium to which low molecular weight agar is not added (in the case of low molecular weight agar SW). It was suggested that the colonies became smaller as the jelly strength increased in the medium in which the amount added per 1 L of the medium was 4 g or more.
（低分子量寒天ＳＷのゼリー強度測定）
低分子量寒天ＳＷ（ＳＳＫセールス株式会社製、重量平均分子量：約１０万）の３種類のロットについて、ゼリー強度を調べた。
(Measurement of jelly strength of low molecular weight agar SW)
The jelly strength was examined for three types of lots of low molecular weight agar SW (manufactured by SSK Sales Co., Ltd., weight average molecular weight: about 100,000).
（１）寒天ゲルの準備
各ロットの低分子量寒天ＳＷを１．５％濃度となるように精製水に懸濁し、ｐＨを７．０に調整した。寒天懸濁液を１００℃で４０分間加温溶解した。５０℃に冷却した後、１８ｍＬずつシャーレに分注して固化した。
(1) Preparation of agar gel The low molecular weight agar SW of each lot was suspended in purified water so as to have a concentration of 1.5%, and the pH was adjusted to 7.0. The agar suspension was heated and dissolved at 100 ° C. for 40 minutes. After cooling to 50 ° C., 18 mL each was dispensed into a petri dish and solidified.
（２）ゼリー強度の測定
（１）で調製した寒天ゲルを２０℃の恒温槽にて２０時間保存した後、レオメーター（ＦＵＤＯＨ ＲＨＥＯ ＭＥＴＥＲ ＲＴＣ－３００２Ｄ）（株式会社レイテック製）の測定部が培地に１ｍｍ侵入する強度をゼリー強度として測定した。なお、低分子量寒天ＳＷの各添加量につきシャーレ１枚をゼリー強度測定の対象とし、１枚のシャーレの３箇所を測定して平均値をゼリー強度として算出した。
(2) Measurement of jelly intensity After storing the agar gel prepared in (1) in a constant temperature bath at 20 ° C. for 20 hours, the measuring unit of the reometer (FUDOH RHEO MTER RTC-3002D) (manufactured by Raytec Co., Ltd.) is the medium. The strength of penetrating 1 mm into the jelly was measured as the jelly strength. For each amount of low molecular weight agar SW added, one petri dish was targeted for jelly strength measurement, three points of one petri dish were measured, and the average value was calculated as the jelly strength.
（３）結果
寒天ゲルのゼリー強度の測定結果を溶液のｐＨ実測値とともに表６に示す。
(3) Results The measurement results of the jelly strength of the agar gel are shown in Table 6 together with the measured pH values of the solution.
低分子量寒天ＳＷのゼリー強度は、表６に示したとおり、１４０／ｃｍ２または１５０ｇ／ｃｍ２であった。 The jelly intensity of the low molecular weight agar SW was 140 / cm 2 or 150 g / cm 2 as shown in Table 6.
（低分子量寒天添加培地の経時変化試験１）
羊血液寒天培地を基礎培地とし、低分子量寒天ＳＷ（ＳＳＫセールス株式会社製、重量平均分子量：約１０万、ゼリー強度：１４０～１５０ｇ／ｃｍ２）を添加して調製後、１０℃、倒置にて３．５ヶ月保存した培地における凝水試験を行った。
(Time-dependent change test of low molecular weight agar-added medium 1)
Using sheep blood agar medium as the basal medium, add low molecular weight agar SW (manufactured by SSK Sales Co., Ltd., weight average molecular weight: about 100,000, jelly strength: 140-150 g / cm 2 ) to prepare, and then invert at 10 ° C. A water coagulation test was performed on a medium stored for 3.5 months.
（１）培地の準備
以下の表７に示した培地成分３を秤量し、以下の（２）で羊血液を添加した後の培地量が１Ｌとなるように精製水に懸濁後、１２１℃で１５分間高圧滅菌した。従来の微生物培養用の寒天と低分子量寒天を区別するため、表７において、従来の微生物培養用の寒天を寒天Ａ、低分子量寒天ＳＷを寒天Ｂと記載する。
(1) Preparation of medium Weigh the medium component 3 shown in Table 7 below, suspend it in purified water so that the amount of medium after adding sheep blood in (2) below is 1 L, and then record at 121 ° C. Was sterilized under high pressure for 15 minutes. In order to distinguish between the conventional agar for culturing microorganisms and the low molecular weight agar, in Table 7, the agar for conventional culturing is referred to as agar A and the low molecular weight agar SW is referred to as agar B.
（２）羊血液の添加
高圧滅菌後、培地を５０℃に冷却した後、予め無菌的に採取した羊脱繊維血液を、培地１Ｌ当たり５０ｍＬとなるように添加した。その後、培地を１８ｍＬずつシャーレに分注して固化した。
(2) Addition of sheep blood After high-pressure sterilization, the medium was cooled to 50 ° C., and then aseptically collected sheep defibered blood was added so as to be 50 mL per 1 L of the medium. Then, 18 mL of the medium was dispensed into a petri dish and solidified.
（３）凝水量の測定
（１）、（２）で調製した培地入りシャーレを１０℃、倒置にて３．５ヶ月保存した（培地調製日、保存開始日とも２０１６年１２月８日）。その後、４℃、倒置にて２０時間保存した後、２５℃、倒置にてシャーレを３０分間放置し、その後、培地入りシャーレの重量を電子天秤にて測定した。続いてシャーレを縦置きにして３０分間放置して培地表面に滲み出た水を濾紙によって拭き取った後、再び培地入りシャーレの重量を測定し、（滲み出た水の拭き取り前のシャーレの重量）－（滲み出た水を拭き取った後のシャーレの重量）を凝水量として算出した（凝水量測定日は２０１７年３月２２日）。
なお、低分子量寒天ＳＷの各添加量につき９枚のシャーレを凝水試験対象とし、重量の測定はシャーレ１枚毎に行った。
(3) Measurement of the amount of coagulated water The petri dish containing the medium prepared in (1) and (2) was stored at 10 ° C. in an inverted position for 3.5 months (both the medium preparation date and the storage start date are December 8, 2016). Then, after storing in an inverted position at 4 ° C. for 20 hours, the petri dish was left in an inverted position at 25 ° C. for 30 minutes, and then the weight of the petri dish containing the medium was measured with an electronic balance. Subsequently, the petri dish was placed vertically and left for 30 minutes to wipe off the water exuded on the surface of the medium with a filter paper, and then the weight of the petri dish containing the medium was measured again (the weight of the petri dish before wiping off the exuded water). -(Weight of petri dish after wiping off the exuded water) was calculated as the amount of water coagulation (the date of measurement of the amount of coagulation was March 22, 2017).
Nine petri dishes were subject to the water coagulation test for each addition amount of the low molecular weight agar SW, and the weight was measured for each petri dish.
（４）結果
培地の凝水量の測定結果を表８に示す。
(4) Results Table 8 shows the measurement results of the amount of coagulated water in the medium.
表８に示した様に、１０℃にて３．５ヶ月保存後において、低分子量寒天ＳＷの添加培地の凝水量は、良好に抑制されていた。 As shown in Table 8, the amount of coagulated water in the medium to which the low molecular weight agar SW was added was well suppressed after storage at 10 ° C. for 3.5 months.
（ドリガルスキー改良培地における凝水試験）
ドリガルスキー改良培地（BTB乳糖寒天培地）を基礎培地とし、低分子量寒天ＳＷ（ＳＳＫセールス株式会社製、重量平均分子量：約１０万、ゼリー強度：１４０～１５０ｇ／ｃｍ２）を添加して凝水抑制効果を調べた。
(Water coagulation test in Drigalski improved medium)
Drigalski improved medium (BTB lactose agar medium) is used as the basal medium, and low molecular weight agar SW (manufactured by SSK Sales Co., Ltd., weight average molecular weight: about 100,000, jelly strength: 140-150 g / cm 2 ) is added to coagulate water. The inhibitory effect was investigated.
（１）培地の準備
以下の表９に示した培地成分４を秤量し、精製水に懸濁後、１２１℃で１５分間高圧滅菌した。滅菌後、５０℃に冷却した後、培地を１８ｍＬずつシャーレに分注して固化した。なお、従来の微生物培養用の寒天と低分子量寒天を区別するため、表９において、従来の微生物培養用の寒天を寒天Ａ、低分子量寒天ＳＷを寒天Ｂと記載する。
(1) Preparation of medium The medium component 4 shown in Table 9 below was weighed, suspended in purified water, and sterilized under high pressure at 121 ° C. for 15 minutes. After sterilization, the medium was cooled to 50 ° C., and 18 mL of the medium was dispensed into a petri dish and solidified. In order to distinguish between the conventional agar for culturing microorganisms and the low molecular weight agar, in Table 9, the conventional agar for culturing microorganisms is referred to as agar A and the low molecular weight agar SW is referred to as agar B.
（２）凝水量の測定
（１）で調製した培地入りシャーレを４℃、倒置にて２０時間保存した後、２５℃、倒置にてシャーレを３０分間放置し、その後、培地入りシャーレの重量を電子天秤にて測定した。続いてシャーレを縦置きにして３０分間放置して培地表面に滲み出た水を濾紙によって拭き取った後、再び培地入りシャーレの重量を測定し、（滲み出た水の拭き取り前のシャーレの重量）－（滲み出た水を拭き取った後のシャーレの重量）を凝水量として算出した。
なお、低分子量寒天ＳＷの各添加量につき１０枚のシャーレを凝水試験対象とし、重量の測定はシャーレ１枚毎に行った。
(2) Measurement of the amount of coagulated water The petri dish containing the medium prepared in (1) was stored at 4 ° C. for 20 hours in an inverted position, then left at 25 ° C. for 30 minutes in an inverted position, and then the weight of the petri dish containing the medium was weighed. Measured with an electronic balance. Subsequently, the petri dish was placed vertically and left for 30 minutes to wipe off the water exuded on the surface of the medium with a filter paper, and then the weight of the petri dish containing the medium was measured again (the weight of the petri dish before wiping off the exuded water). -(Weight of petri dish after wiping off the exuded water) was calculated as the amount of water coagulation.
In addition, 10 petri dishes were subject to the water coagulation test for each addition amount of the low molecular weight agar SW, and the weight was measured for each petri dish.
（３）結果
低分子量寒天ＳＷを添加した培地の平均凝水量を、低分子量寒天ＳＷを添加していない培地の平均凝水量と比較した凝水削減量および凝水削減率とともに表１０に示す。
(3) Results The average coagulation amount of the medium to which the low molecular weight agar SW was added is shown in Table 10 together with the water coagulation reduction amount and the water coagulation reduction rate compared with the average coagulation amount of the medium to which the low molecular weight agar SW was not added.
表１０に示した様に、ドリガルスキー改良培地（BTB乳糖寒天培地）を基礎培地とした場合も、低分子量寒天ＳＷの添加による凝水抑制効果が認められた。 As shown in Table 10, even when the improved Drigalsky medium (BTB lactose agar medium) was used as the basal medium, the effect of suppressing water coagulation by the addition of the low molecular weight agar SW was observed.
（カンジダ鑑別用寒天培地における凝水試験）
カンジダ鑑別用寒天培地を基礎培地とし、低分子量寒天ＳＷ（ＳＳＫセールス株式会社製、重量平均分子量：約１０万、ゼリー強度：１４０～１５０ｇ／ｃｍ２）を添加して凝水抑制効果を調べた。
(Water coagulation test on agar medium for Candida differentiation)
Using agar medium for Candida differentiation as a basal medium, low molecular weight agar SW (manufactured by SSK Sales Co., Ltd., weight average molecular weight: about 100,000, jelly strength: 140-150 g / cm 2 ) was added to investigate the effect of suppressing water coagulation. ..
（１）培地の準備
以下の表１１に示した培地成分５を秤量し、精製水に懸濁後、１００℃で４０分間加温溶解した。５０℃に冷却した後、培地を１８ｍＬずつシャーレに分注して固化した。なお、従来の微生物培養用の寒天と低分子量寒天を区別するため、表１１において、従来の微生物培養用の寒天を寒天Ａ、低分子量寒天ＳＷを寒天Ｂと記載する。
(1) Preparation of medium The medium component 5 shown in Table 11 below was weighed, suspended in purified water, and then heated and dissolved at 100 ° C. for 40 minutes. After cooling to 50 ° C., 18 mL of the medium was dispensed into a petri dish and solidified. In order to distinguish between the conventional agar for culturing microorganisms and the low molecular weight agar, Table 11 describes the agar for conventional culturing as agar A and the low molecular weight agar SW as agar B.
（２）凝水量の測定
（１）で調製した培地入りシャーレを４℃、倒置にて２０時間保存した後、２５℃、倒置にてシャーレを３０分間放置し、その後、培地入りシャーレの重量を電子天秤にて測定した。続いてシャーレを縦置きにして３０分間放置して培地表面に滲み出た水を濾紙によって拭き取った後、再び培地入りシャーレの重量を測定し、（滲み出た水の拭き取り前のシャーレの重量）－（滲み出た水を拭き取った後のシャーレの重量）を凝水量として算出した。
なお、低分子量寒天ＳＷの各添加量につき１０枚のシャーレを凝水試験対象とし、重量の測定はシャーレ１枚毎に行った。
(2) Measurement of the amount of coagulated water The petri dish containing the medium prepared in (1) was stored at 4 ° C. for 20 hours in an inverted position, then left at 25 ° C. for 30 minutes in an inverted position, and then the weight of the petri dish containing the medium was weighed. Measured with an electronic balance. Subsequently, the petri dish was placed vertically and left for 30 minutes to wipe off the water exuded on the surface of the medium with a filter paper, and then the weight of the petri dish containing the medium was measured again (the weight of the petri dish before wiping off the exuded water). -(Weight of petri dish after wiping off the exuded water) was calculated as the amount of water coagulation.
In addition, 10 petri dishes were subject to the water coagulation test for each addition amount of the low molecular weight agar SW, and the weight was measured for each petri dish.
（３）結果
低分子量寒天ＳＷを添加した培地の平均凝水量を、低分子量寒天ＳＷを添加していない培地の平均凝水量と比較した凝水削減量および凝水削減率とともに表１２に示す。
(3) Results The average coagulation amount of the medium to which the low molecular weight agar SW was added is shown in Table 12 together with the water coagulation reduction amount and the water coagulation reduction rate compared with the average coagulation amount of the medium to which the low molecular weight agar SW was not added.
表１２に示した様に、カンジダ鑑別用寒天培地を基礎培地とした場合も、低分子量寒天ＳＷの添加による凝水抑制効果が認められた。 As shown in Table 12, even when the agar medium for Candida differentiation was used as the basal medium, the effect of suppressing water coagulation by the addition of the low molecular weight agar SW was observed.
（低分子量寒天の種類の検討１）
羊血液寒天培地を基礎培地とし、低分子量寒天であるＳＷ（ＳＳＫセールス株式会社製、重量平均分子量：約１０万、ゼリー強度：１４０～１５０ｇ／ｃｍ２）またはウルトラ寒天ＢＸ－３０（伊那食品工業株式会社製、重量平均分子量：２万～５万程度、ゼリー強度：３０～６０ｇ／ｃｍ２）を添加して凝水抑制効果を調べた。
(Examination of types of low molecular weight agar 1)
SW (manufactured by SSK Sales Co., Ltd., weight average molecular weight: about 100,000, jelly strength: 140-150 g / cm 2 ) or ultra agar BX-30 (Ina Food Industry Co., Ltd.), which uses sheep blood agar medium as the basal medium and is a low molecular weight agar. Manufactured by Co., Ltd., weight average molecular weight: about 20,000 to 50,000, jelly strength: 30 to 60 g / cm 2 ) was added, and the effect of suppressing water coagulation was investigated.
（１）培地の準備
以下の表１３に示した培地成分６を秤量し、以下の（２）で羊血液を添加した後の培地量が１Ｌとなるように精製水に懸濁後、１２１℃で１５分間高圧滅菌した。従来の微生物培養用の寒天と低分子量寒天を区別するため、表１３において、従来の微生物培養用の寒天を寒天Ａ、低分子量寒天ＳＷまたはウルトラ寒天ＢＸ－３０を寒天Ｂと記載する。
(1) Preparation of medium Weigh the medium component 6 shown in Table 13 below, suspend it in purified water so that the amount of medium after adding sheep blood in (2) below is 1 L, and then record at 121 ° C. Was sterilized under high pressure for 15 minutes. In order to distinguish between conventional agar for culturing microorganisms and low molecular weight agar, in Table 13, agar for conventional culturing is referred to as agar A, and low molecular weight agar SW or ultra agar BX-30 is referred to as agar B.
（２）羊血液の添加
高圧滅菌後、培地を５０℃に冷却した後、予め無菌的に採取した羊脱繊維血液を、培地１Ｌ当たり５０ｍＬとなるように添加した。その後、培地を１８ｍＬずつシャーレに分注して固化した。
(2) Addition of sheep blood After high-pressure sterilization, the medium was cooled to 50 ° C., and then aseptically collected sheep defibered blood was added so as to be 50 mL per 1 L of the medium. Then, 18 mL of the medium was dispensed into a petri dish and solidified.
（３）凝水量の測定
（１）、（２）で調製した培地入りシャーレを４℃、倒置にて２０時間保存した後、２５℃、倒置にてシャーレを３０分間放置し、その後、培地入りシャーレの重量を電子天秤にて測定した。続いてシャーレを縦置きにして３０分間放置して培地表面に滲み出た水を濾紙によって拭き取った後、再び培地入りシャーレの重量を測定し、（滲み出た水の拭き取り前のシャーレの重量）－（滲み出た水を拭き取った後のシャーレの重量）を凝水量として算出した。
なお、低分子量寒天の各添加量につき９枚のシャーレを凝水試験対象とし、重量の測定はシャーレ１枚毎に行った。
(3) Measurement of the amount of coagulated water The petri dish containing the medium prepared in (1) and (2) was stored at 4 ° C. for 20 hours in an inverted position, then left at 25 ° C. for 30 minutes in an inverted position, and then contained in a medium. The weight of the petri dish was measured with an electronic balance. Subsequently, the petri dish was placed vertically and left for 30 minutes to wipe off the water exuded on the surface of the medium with a filter paper, and then the weight of the petri dish containing the medium was measured again (the weight of the petri dish before wiping off the exuded water). -(Weight of petri dish after wiping off the exuded water) was calculated as the amount of water coagulation.
Nine petri dishes were used for the water coagulation test for each amount of low molecular weight agar added, and the weight was measured for each petri dish.
（４）培地のゼリー強度の測定
（１）、（２）で調製した培地を上記（３）の凝水量測定用のものとは別に用意し、２０℃の恒温槽にて２０時間保存した後、レオメーター（ＦＵＤＯＨ ＲＨＥＯ ＭＥＴＥＲ ＲＴＣ－３００２Ｄ）（株式会社レイテック製）の測定部が培地に１ｍｍ侵入する強度をゼリー強度として測定した。なお、低分子量寒天の各添加量につきシャーレ１枚をゼリー強度測定の対象とし、１枚のシャーレの３箇所を測定して平均値をゼリー強度として算出した。
(4) Measurement of jelly strength of medium The medium prepared in (1) and (2) is prepared separately from the one for measuring the amount of coagulation in (3) above, and stored in a constant temperature bath at 20 ° C. for 20 hours. , The strength at which the measuring unit of the Leometer (FUDOH RHEO MTER RTC-3002D) (manufactured by Raytec Co., Ltd.) penetrated the medium by 1 mm was measured as the jelly strength. For each amount of low molecular weight agar added, one petri dish was targeted for jelly strength measurement, three points of one petri dish were measured, and the average value was calculated as the jelly strength.
（５）結果
培地の凝水量およびゼリー強度の測定結果を表１４に示す。凝水量の測定結果は、寒天Ｂ（低分子量寒天ＳＷまたはウルトラ寒天ＢＸ－３０）の各添加量における平均凝水量を、寒天Ｂを添加していない培地の平均凝水量と比較した凝水削減量および凝水削減率とともに表示し、また、ゼリー強度は、寒天Ｂを添加していない培地のゼリー強度からの上昇値とともに表示している。
(5) Results Table 14 shows the measurement results of the amount of coagulated water and the jelly strength of the medium. The measurement result of the amount of coagulation is that the average amount of coagulation in each addition amount of agar B (low molecular weight agar SW or ultra agar BX-30) is compared with the average amount of coagulation of the medium to which agar B is not added. And, it is displayed together with the water coagulation reduction rate, and the jelly intensity is displayed together with the increase value from the jelly intensity of the medium to which agar B is not added.
表１４に示した様に、低分子量寒天ＳＷを添加した場合、ウルトラ寒天ＢＸ－３０を添加した場合ともに良好な凝水抑制効果が認められた。また、ウルトラ寒天ＢＸ－３０は、培地１Ｌ当たりの添加量が４ｇ、５ｇの場合もゼリー強度の上昇値が１０ｇ／ｃｍ２と小さいため、この程度の添加濃度では発育した菌のコロニー形成状態（拡がり状態）には影響しないと考えられた。 As shown in Table 14, a good water coagulation suppressing effect was observed both when the low molecular weight agar SW was added and when the ultra agar BX-30 was added. Further, in Ultra Agar BX-30, even when the addition amount per 1 L of the medium is 4 g and 5 g, the increase value of the jelly strength is as small as 10 g / cm 2 , and therefore, the colonization state of the grown bacteria at this addition concentration ( It was thought that it would not affect the spread state).
（低分子量寒天の種類の検討２）
羊血液寒天培地を基礎培地とし、低分子量寒天であるＳＷ（ＳＳＫセールス株式会社製、重量平均分子量：約１０万、ゼリー強度：１４０～１５０ｇ／ｃｍ２）、ウルトラ寒天ＢＸ－３０（伊那食品工業株式会社製、重量平均分子量：２万～５万程度、ゼリー強度：３０～６０ｇ／ｃｍ２）またはウルトラ寒天ＢＸ－１００（伊那食品工業株式会社製、重量平均分子量：７万～１０万程度、ゼリー強度：１００～１５０ｇ／ｃｍ２）を添加して凝水抑制効果を調べた。
(Examination of types of low molecular weight agar 2)
SW (manufactured by SSK Sales Co., Ltd., weight average molecular weight: about 100,000, jelly strength: 140-150 g / cm 2 ), ultra-agar BX-30 (Ina Food Industry Co., Ltd.) using sheep blood agar medium as the basal medium. Made by Co., Ltd., weight average molecular weight: about 20,000 to 50,000, jelly strength: 30 to 60 g / cm 2 ) or Ultra Agar BX-100 (manufactured by Ina Food Industry Co., Ltd., weight average molecular weight: about 70,000 to 100,000, Jelly strength: 100-150 g / cm 2 ) was added and the effect of suppressing water coagulation was investigated.
（１）培地の準備
以下の表１５に示した培地成分７を秤量し、以下の（２）で羊血液を添加した後の培地量が２Ｌとなるように精製水に懸濁後、１２１℃で１５分間高圧滅菌した。従来の微生物培養用の寒天と低分子量寒天を区別するため、表１５において、従来の微生物培養用の寒天を寒天Ａ、低分子量寒天ＳＷ、ウルトラ寒天ＢＸ－３０またはウルトラ寒天ＢＸ－１００を寒天Ｂと記載する。
(1) Preparation of medium The medium component 7 shown in Table 15 below is weighed, suspended in purified water so that the amount of medium after addition of sheep blood in (2) below is 2 L, and then 121 ° C. Was sterilized under high pressure for 15 minutes. In order to distinguish between agar for conventional microbial culture and low molecular weight agar, in Table 15, agar for conventional microbial culture is referred to as agar A, low molecular weight agar SW, ultra agar BX-30 or ultra agar BX-100 as agar B. It is described as.
（２）羊血液の添加
高圧滅菌後、培地を５０℃に冷却した後、予め無菌的に採取した羊脱繊維血液を、培地１Ｌ当たり５０ｍＬとなるように添加した。その後、培地を１８ｍＬずつシャーレに分注して固化した。
(2) Addition of sheep blood After high-pressure sterilization, the medium was cooled to 50 ° C., and then aseptically collected sheep defibered blood was added so as to be 50 mL per 1 L of the medium. Then, 18 mL of the medium was dispensed into a petri dish and solidified.
（３）凝水量の測定
（１）、（２）で調製した培地入りシャーレを４℃、倒置にて２０時間保存した後、２５℃、倒置にてシャーレを３０分間放置し、その後、培地入りシャーレの重量を電子天秤にて測定した。続いてシャーレを縦置きにして３０分間放置して培地表面に滲み出た水を濾紙によって拭き取った後、再び培地入りシャーレの重量を測定し、（滲み出た水の拭き取り前のシャーレの重量）－（滲み出た水を拭き取った後のシャーレの重量）を凝水量として算出した。
なお、低分子量寒天の各添加量につき１０枚のシャーレを凝水試験対象とし、重量の測定はシャーレ１枚毎に行った。
(3) Measurement of the amount of coagulated water The petri dish containing the medium prepared in (1) and (2) was stored at 4 ° C. in an inverted position for 20 hours, then left at 25 ° C. in an inverted position for 30 minutes, and then contained in a medium. The weight of the petri dish was measured with an electronic balance. Subsequently, the petri dish was placed vertically and left for 30 minutes to wipe off the water exuded on the surface of the medium with a filter paper, and then the weight of the petri dish containing the medium was measured again (the weight of the petri dish before wiping off the exuded water). -(Weight of petri dish after wiping off the exuded water) was calculated as the amount of water coagulation.
In addition, 10 petri dishes were subject to the water coagulation test for each addition amount of low molecular weight agar, and the weight was measured for each petri dish.
（４）培地のゼリー強度の測定
（１）、（２）で調製した培地を上記（３）の凝水量測定用のものとは別に用意し、２０℃の恒温槽にて２０時間保存した後、レオメーター（ＦＵＤＯＨ ＲＨＥＯ ＭＥＴＥＲ ＲＴＣ－３００２Ｄ）（株式会社レイテック製）の測定部が培地に１ｍｍ侵入する強度をゼリー強度として測定した。なお、低分子量寒天の各添加量につきシャーレ１枚をゼリー強度測定の対象とし、１枚のシャーレの３箇所を測定して平均値をゼリー強度として算出した。
(4) Measurement of jelly strength of medium The medium prepared in (1) and (2) is prepared separately from the one for measuring the amount of coagulation in (3) above, and stored in a constant temperature bath at 20 ° C. for 20 hours. , The strength at which the measuring unit of the Leometer (FUDOH RHEO MTER RTC-3002D) (manufactured by Raytec Co., Ltd.) penetrated the medium by 1 mm was measured as the jelly strength. For each amount of low molecular weight agar added, one petri dish was targeted for jelly strength measurement, three points of one petri dish were measured, and the average value was calculated as the jelly strength.
（５）結果
培地の凝水量およびゼリー強度の測定結果を表１６に示す。凝水量の測定結果は、寒天Ｂ（低分子量寒天ＳＷ、ウルトラ寒天ＢＸ－３０またはウルトラ寒天ＢＸ－１００）の各添加量における平均凝水量を、寒天Ｂを添加していない培地の平均凝水量と比較した凝水削減量および凝水削減率とともに表示し、また、ゼリー強度は、寒天Ｂを添加していない培地のゼリー強度からの上昇値とともに表示している。
(5) Results Table 16 shows the measurement results of the amount of coagulated water and the jelly strength of the medium. The measurement result of the amount of coagulation is that the average amount of coagulation in each addition amount of agar B (low molecular weight agar SW, ultra agar BX-30 or ultra agar BX-100) is the average amount of coagulation of the medium to which agar B is not added. It is displayed together with the compared amount of water coagulation reduction and water coagulation reduction rate, and the jelly intensity is displayed together with the increase value from the jelly intensity of the medium to which agar B is not added.
表１６に示した様に、低分子量寒天ＳＷを添加した場合、ウルトラ寒天ＢＸ－３０を添加した場合、ウルトラ寒天ＢＸ－１００を添加した場合ともに良好な凝水抑制効果が認められた。また、ウルトラ寒天ＢＸ－３０、ウルトラ寒天ＢＸ－１００は培地１Ｌ当たりの添加量が４ｇの場合もゼリー強度の上昇値がそれぞれ１０ｇ／ｃｍ２、２０ｇ／ｃｍ２と小さいため、この程度の添加濃度では発育した菌のコロニー形成状態（拡がり状態）には影響しないと考えられた。 As shown in Table 16, a good water coagulation suppressing effect was observed when the low molecular weight agar SW was added, when the ultra agar BX-30 was added, and when the ultra agar BX-100 was added. Further, in the case of Ultra Agar BX-30 and Ultra Agar BX-100, even when the addition amount per 1 L of the medium is 4 g, the increase values of the jelly strength are as small as 10 g / cm 2 and 20 g / cm 2 , respectively, so that the addition concentration is about this level. It was considered that the colonization state (spread state) of the developed bacteria was not affected.
（低分子量寒天添加培地の経時変化試験２）
羊血液寒天培地を基礎培地とし、低分子量寒天ＳＷ（ＳＳＫセールス株式会社製、重量平均分子量：約１０万、ゼリー強度：１４０～１５０ｇ／ｃｍ２）を添加して調製後、１０℃、倒置にて３．５ヶ月保存した培地における凝水試験を行った。
(Time-dependent change test of low molecular weight agar-added medium 2)
Using sheep blood agar medium as the basal medium, add low molecular weight agar SW (manufactured by SSK Sales Co., Ltd., weight average molecular weight: about 100,000, jelly strength: 140-150 g / cm 2 ) to prepare, and then invert at 10 ° C. A water coagulation test was performed on a medium stored for 3.5 months.
（１）培地の準備
以下の表１７に示した培地成分８を秤量し、以下の（２）で羊血液を添加した後の培地量が１Ｌとなるように精製水に懸濁後、１２１℃で１５分間高圧滅菌した。従来の微生物培養用の寒天と低分子量寒天を区別するため、表１７において、従来の微生物培養用の寒天を寒天Ａ、低分子量寒天ＳＷを寒天Ｂと記載する。
(1) Preparation of medium Weigh the medium component 8 shown in Table 17 below, suspend it in purified water so that the amount of medium after adding sheep blood in (2) below is 1 L, and then record at 121 ° C. Was sterilized under high pressure for 15 minutes. In order to distinguish between the conventional agar for culturing microorganisms and the low molecular weight agar, Table 17 describes the agar for conventional culturing as agar A and the low molecular weight agar SW as agar B.
（２）羊血液の添加
高圧滅菌後、培地を５０℃に冷却した後、予め無菌的に採取した羊脱繊維血液を、培地１Ｌ当たり５０ｍＬとなるように添加した。その後、培地を１８ｍＬずつシャーレに分注して固化した。
(2) Addition of sheep blood After high-pressure sterilization, the medium was cooled to 50 ° C., and then aseptically collected sheep defibered blood was added so as to be 50 mL per 1 L of the medium. Then, 18 mL of the medium was dispensed into a petri dish and solidified.
（３）凝水量の測定
（１）、（２）で調製した培地入りシャーレを１０℃、倒置にて３．５ヶ月保存した（培地調製日、保存開始日とも２０１７年６月１３日）。その後、４℃、倒置にて２０時間保存した後、２５℃、倒置にてシャーレを３０分間放置し、その後、培地入りシャーレの重量を電子天秤にて測定した。続いてシャーレを縦置きにして３０分間放置して培地表面に滲み出た水を濾紙によって拭き取った後、再び培地入りシャーレの重量を測定し、（滲み出た水の拭き取り前のシャーレの重量）－（滲み出た水を拭き取った後のシャーレの重量）を凝水量として算出した（凝水量測定日は２０１７年９月２７日）。
なお、低分子量寒天ＳＷの各添加量につき９枚のシャーレを凝水試験対象とし、重量の測定はシャーレ１枚毎に行った。
(3) Measurement of the amount of coagulated water The petri dish containing the medium prepared in (1) and (2) was stored at 10 ° C. in an inverted position for 3.5 months (both the medium preparation date and the storage start date were June 13, 2017). Then, after storing in an inverted position at 4 ° C. for 20 hours, the petri dish was left in an inverted position at 25 ° C. for 30 minutes, and then the weight of the petri dish containing the medium was measured with an electronic balance. Subsequently, the petri dish was placed vertically and left for 30 minutes to wipe off the water exuded on the surface of the medium with a filter paper, and then the weight of the petri dish containing the medium was measured again (the weight of the petri dish before wiping off the exuded water). -(Weight of petri dish after wiping off the exuded water) was calculated as the amount of water coagulation (the date of measurement of the amount of coagulation was September 27, 2017).
Nine petri dishes were subject to the water coagulation test for each addition amount of the low molecular weight agar SW, and the weight was measured for each petri dish.
（４）結果
培地の凝水量の測定結果を表１８に示す。
(4) Results Table 18 shows the measurement results of the amount of coagulated water in the medium.
表１８に示した様に、１０℃にて３．５ヶ月保存後において、低分子量寒天ＳＷの添加培地の凝水量は、良好に抑制されていた。 As shown in Table 18, the amount of coagulated water in the medium to which the low molecular weight agar SW was added was well suppressed after storage at 10 ° C. for 3.5 months.
本発明による微生物培養用の固体培地は、固化成分として従来の微生物培養用の寒天に加えて、重量平均分子量が２万～１５万である低分子量寒天を使用することにより培地表面をコロニー形成に適切な状態に保ち、凝水を抑制可能である。また、特別な操作を要することなく、容易に培地を製造することができる。そのため、本発明の固体培地は、臨床をはじめ、食品、環境などの幅広い領域において有用である。 The solid medium for culturing microorganisms according to the present invention uses low molecular weight agar having a weight average molecular weight of 20,000 to 150,000 in addition to the conventional agar for culturing microorganisms as a solidifying component to form a colony on the surface of the medium. It is possible to keep it in an appropriate state and suppress water coagulation. In addition, the medium can be easily produced without requiring a special operation. Therefore, the solid medium of the present invention is useful in a wide range of fields such as clinical practice, food, and environment.
Claims (4)
（ａ）重量平均分子量が３０万～４５万である寒天、
（ｂ）重量平均分子量が１５万以下である低分子量寒天。 A flat plate solid medium used for culturing microorganisms, which comprises at least (a) and (b) as a solidifying component.
(A) Agar having a weight average molecular weight of 300,000 to 450,000,
(B) Low molecular weight agar having a weight average molecular weight of 150,000 or less.
（ａ）重量平均分子量が３０万～４５万である寒天、
（ｂ）重量平均分子量が１５万以下である低分子量寒天。 A method for producing a flat plate solid medium used for culturing a microorganism, which comprises using (a) and (b) as solidifying components.
(A) Agar having a weight average molecular weight of 300,000 to 450,000,
(B) Low molecular weight agar having a weight average molecular weight of 150,000 or less.
（ａ）重量平均分子量が３０万～４５万である寒天、
（ｂ）重量平均分子量が１５万以下である低分子量寒天。 A medium used for culturing microorganisms, which comprises at least (a) and (b) as a solidifying component.
(A) Agar having a weight average molecular weight of 300,000 to 450,000,
(B) Low molecular weight agar having a weight average molecular weight of 150,000 or less.
（ａ）重量平均分子量が３０万～４５万である寒天、
（ｂ）重量平均分子量が１５万以下である低分子量寒天。 A method for producing a culture medium used for culturing a microorganism, which comprises using (a) and (b) as solidifying components.
(A) Agar having a weight average molecular weight of 300,000 to 450,000,
(B) Low molecular weight agar having a weight average molecular weight of 150,000 or less.
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| JP2011125263A (en) * | 2009-12-17 | 2011-06-30 | Nissui Pharm Co Ltd | Blood agar medium and method for preserving the same |
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| JP2011125263A (en) * | 2009-12-17 | 2011-06-30 | Nissui Pharm Co Ltd | Blood agar medium and method for preserving the same |
| WO2016167373A1 (en) * | 2015-04-16 | 2016-10-20 | 日産化学工業株式会社 | Culture medium additive, culture medium composition, and method for culturing cells or tissue using same |
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