JP5160157B2 - Microbial culture medium and microbiological testing instrument for oily samples - Google Patents
Microbial culture medium and microbiological testing instrument for oily samples Download PDFInfo
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Description
本発明は、油性試料の微生物検査用培地および微生物検査器具に関する。
The present invention relates to a microorganism testing medium and a microorganism testing instrument for oily samples .
従来、微生物検査用の培地として、寒天培地が一般的に使用されている。また、寒天培地よりも多量の試料を塗抹可能なものとして、各成分を所定量より多い溶解水に溶解して固化し、その固化した培地を乾燥して溶解水の量を所定量の７０乃至２００％に調整し、製造された固体培地がある（例えば、特許文献１参照）。 Conventionally, an agar medium is generally used as a medium for microorganism testing. Further, it is assumed that a larger amount of sample can be smeared than the agar medium, each component is dissolved and dissolved in more than a predetermined amount of dissolved water, the solidified medium is dried, and the amount of dissolved water is reduced to a predetermined amount of 70 to 70. There is a solid medium manufactured by adjusting to 200% (for example, see Patent Document 1).
しかしながら、一般的な寒天培地や特許文献１記載の固体培地は、５〜１０℃程度の低温で保管しなければならないため、生産現場では大型保冷庫を使用し、流通過程では保冷輸送する必要があり、保冷処置の費用が嵩むという課題があった。また、水分量が多く、油性試料との親和性が悪いという課題もあった。長期間培養する場合、乾燥が進み、菌の培養ができなくなるという課題もあった。ｐＨが５．０以下になると軟化するため、それよりも低いｐＨでの培養が困難であるという課題もあった。 However, since a general agar medium and the solid medium described in Patent Document 1 must be stored at a low temperature of about 5 to 10 ° C., it is necessary to use a large cool box at the production site and transport it by cold transport in the distribution process. There was a problem that the cost of the cold insulation treatment increased. There is also a problem that the amount of water is large and the affinity with the oily sample is poor. In the case of culturing for a long period of time, there has been a problem that drying has progressed and bacteria cannot be cultured. Since it softens when pH becomes 5.0 or less, there also existed the subject that culture | cultivation in pH lower than that was difficult.
本発明は、このような課題に着目してなされたもので、親油性で保水性が高く、常温での保管・流通が可能で、長期間の培養および低ｐＨでの培養が可能な油性試料の微生物検査用培地および微生物検査器具を提供することを目的としている。
The present invention has been made paying attention to such problems, and is an oleophilic sample that is lipophilic, has high water retention, can be stored and distributed at room temperature, and can be cultured for a long period of time or at a low pH. and its object is to provide a culture medium for microbiological tests and microbial inspection tool.
上記目的を達成するために、本発明に係る油性試料の微生物検査用培地は、水分総質量に対し寒天を１．０乃至１．３質量％、ジェランガムを０．１乃至０．１５質量％含有することを、特徴とする。なお、ジェランガムは、水溶性の天然高分子多糖類であり、増粘安定剤として各種食品に利用されている。なお、ここで、各質量％は、乾燥質量％である。
In order to achieve the above object, the microbial test medium for an oily sample according to the present invention contains 1.0 to 1.3% by mass of agar and 0.1 to 0.15% by mass of gellan gum based on the total mass of water. It is characterized by doing. Gellan gum is a water-soluble natural polymer polysaccharide and is used in various foods as a thickening stabilizer. Here, each mass% is dry mass%.
本発明に係る微生物検査用培地は、寒天とジェランガムとを含有しているため、寒天のみを含有した寒天培地に比べて、保水性が高く、常温での保管・流通が可能である。また、寒天培地より多量の試料を短時間に吸収することができ、検出精度を高めることができる。寒天培地に比べて表面の弾力性が高いため、画線時や塗抹時に傷つきにくい。ジェランガムは親油性であるため、水性試料だけでなく、低濃度の油成分を含む試料も吸収することができる。ｐＨが５．０以下でも軟化しないため、低ｐＨでの培養が可能である。 Since the culture medium for microorganism testing according to the present invention contains agar and gellan gum, it has higher water retention and can be stored and distributed at room temperature than an agar medium containing only agar. In addition, a larger amount of sample than the agar medium can be absorbed in a short time, and the detection accuracy can be improved. Compared to agar medium, the surface has high elasticity, so it is hard to be damaged during painting or smearing. Because gellan gum is lipophilic, it can absorb not only aqueous samples but also samples containing low concentrations of oil components. Since it does not soften even at a pH of 5.0 or less, culture at a low pH is possible.
本発明に係る微生物検査用培地は、保水性が高いため、例えば１週間以上におよぶ長期間の培養でも、乾燥せずに培養が可能である。また、５５℃程度の高温の培養でも、数日間は乾燥せずに培養が可能である。このように、高い保水性により、培養時に乾燥しにくく、硬さも維持されるため、寒天培地に比べて培養する菌に対する影響を小さくすることができる。 The culture medium for microbiological examination according to the present invention has high water retention, so that it can be cultured without drying, for example, even for long-term culture over one week. Further, even at a high temperature of about 55 ° C., the culture can be performed without drying for several days. Thus, since it is hard to dry at the time of culture | cultivation and hardness is maintained by high water retention, the influence with respect to the microbe to culture | cultivate can be made small compared with an agar medium.
本発明に係る微生物検査用培地は、寒天とジェランガムとを乾燥状態で含有していても、水分を含んだゲル状態で含有していてもよい。また、本発明に係る微生物検査用培地は、一般の培地で使用される、酵母エキス、デンプン、ブドウ糖などの栄養分や、塩化ナトリウムなどの塩を含んでいることが好ましい。酸性培地の場合には、硫酸などの酸でｐＨを調整することが好ましい。
本発明に係る微生物検査用培地は、水分総質量に対し寒天を１．０乃至１．３質量％、ジェランガムを０．１乃至０．１５質量％含有しているため、特に、培地として適度な硬さに形成され、試料の吸収性が高い。
The culture medium for microorganism testing according to the present invention may contain agar and gellan gum in a dry state or in a gel state containing moisture. Moreover, it is preferable that the culture medium for microorganism test | inspection which concerns on this invention contains nutrients, such as yeast extract, starch, and glucose, and salts, such as sodium chloride, which are used with a common culture medium. In the case of an acidic medium, it is preferable to adjust the pH with an acid such as sulfuric acid.
The microorganism testing medium according to the present invention contains 1.0 to 1.3% by mass of agar and 0.1 to 0.15% by mass of gellan gum with respect to the total water mass. It is formed to be hard and the sample has high absorbency.
本発明に係る微生物検査用培地は、カチオンを含有することが好ましい。この場合、カチオンにより、ジェランガムをゲル化させることができる。また、カチオンの価数や量により、ゲルの状態を調整することができる。カチオンの含有量は、適度な硬さのゲル化のため、水分総質量に対し１価のカチオンで０．１乃至１質量％、２価のカチオンで０．００２乃至０．０２質量％であることが好ましい。カチオンは、塩として培地の水分に含有されることが好ましい。塩が塩化ナトリウムの場合、水分総質量に対し０．１質量％乃至１質量％、特に、０．１質量％含有されることが好ましい。この場合、培地として特に適度な硬さに形成される。 The culture medium for microbial examination according to the present invention preferably contains a cation. In this case, gellan gum can be gelled by cations. Moreover, the state of a gel can be adjusted with the valence and quantity of a cation. The cation content is 0.1 to 1% by mass for monovalent cations and 0.002 to 0.02% by mass for divalent cations, based on the total mass of water, for gelation with an appropriate hardness. It is preferable. The cation is preferably contained as a salt in the water of the medium. When the salt is sodium chloride, it is preferably contained in an amount of 0.1% by mass to 1% by mass, particularly 0.1% by mass, based on the total mass of water. In this case, the medium has a particularly appropriate hardness.
本発明に係る微生物検査用培地は、ｐＨ調整剤を含有することが好ましい。この場合、培地のｐＨを調整することができる。本発明に係る微生物検査用培地で、前記ｐＨ調整剤は、例えば、硫酸または水酸化ナトリウムから成る。硫酸から成る場合、ｐＨ４．０などの低ｐＨに調整することができる。水酸化ナトリウムから成る場合、高ｐＨに調整することができる。
本発明に係る微生物検査器具は、前述のいずれかの微生物検査用培地を容器に収容し、表面を内部より乾燥させたことを、特徴とする。容器は、シャーレが好ましい。表面を内部より乾燥させることにより、液状試料の吸収量を高めることができる。
本発明に係る微生物検査用培地および微生物検査器具は、油性試料の微生物検査用培地として優れた効果を発揮する。
The microorganism testing medium according to the present invention preferably contains a pH adjuster. In this case, the pH of the medium can be adjusted. In the microorganism testing medium according to the present invention, the pH adjuster comprises, for example, sulfuric acid or sodium hydroxide. When it consists of sulfuric acid, it can be adjusted to a low pH such as pH 4.0. When it consists of sodium hydroxide, it can be adjusted to a high pH.
The microorganism testing instrument according to the present invention is characterized in that any one of the aforementioned microorganism testing media is housed in a container and the surface is dried from the inside. The container is preferably a petri dish. The amount of liquid sample absorbed can be increased by drying the surface from the inside.
The microorganism testing medium and the microorganism testing instrument according to the present invention exhibit excellent effects as a microorganism testing medium for oily samples.
本発明によれば、親油性で保水性が高く、常温での保管・流通が可能で、長期間の培養および低ｐＨでの培養が可能な油性試料の微生物検査用培地およびその微生物検査用培地を用いた微生物検査器具を提供することができる。
According to the present invention, a microbial test medium for an oleaginous sample that can be stored and distributed at room temperature, can be stored and distributed at room temperature, and can be cultured for a long period of time and at a low pH, and its microbial test medium The microbe inspection instrument using can be provided.
以下、本発明の実施の形態について説明する。
本発明の実施の形態の油性試料の微生物検査用培地は、水分と寒天とジェランガムと酵母エキスとデンプンとブドウ糖と塩化ナトリウムと硫酸とを含有している。なお、硫酸は、酸性培地を作成する場合に使用する。
Embodiments of the present invention will be described below.
The culture medium for microbiological examination of the oily sample of the embodiment of the present invention contains water, agar, gellan gum, yeast extract, starch, glucose, sodium chloride, and sulfuric acid. In addition, sulfuric acid is used when preparing an acidic culture medium.
寒天は、水分総質量に対し乾燥質量で０．５乃至１．５質量％の割合で含有されている。なお、寒天は、水分総質量に対し１．０質量％以上で固化し、１．５質量％以上になると硬くなりすぎる傾向を示す。
ジェランガムは、水分総質量に対し乾燥質量で０．０５乃至０．２質量％の割合で含有されている。ジェランガムには、市販製品（例えば、商品名「ゲルライト（ＧＥＬＲＩＴＥ；登録商標）」（発売元；三栄源エフ・エフ・アイ株式会社）や、商品名「ケルコゲル（登録商標）」（発売元；三晶株式会社）など）を使用することができる。なお、ジェランガムは、水分総質量に対し０．０５質量％以上で固化するが、０．５質量％以上の寒天と混合されなければ、塗抹に必要な硬さにならない。また、ジェランガムは、水分総質量に対し０．２質量％以上含むと、水分の吸収性が極度に低下する。
Agar is contained at a ratio of 0.5 to 1.5 mass% in terms of dry mass with respect to the total mass of moisture. In addition, agar solidifies at 1.0 mass% or more with respect to the total water mass, and tends to become too hard when it becomes 1.5 mass% or more.
Gellan gum is contained in a proportion of 0.05 to 0.2% by mass on a dry mass basis with respect to the total mass of moisture. Gellan gum includes commercially available products (for example, trade name “GELITE (registered trademark)” (seller: Saneigen FFI Co., Ltd.) and trade name “Kelcogel (registered trademark)” (seller: 3). Akira Co., Ltd.) etc. can be used. Gellan gum solidifies at 0.05% by mass or more with respect to the total water mass, but does not have the hardness required for smearing unless it is mixed with 0.5% by mass or more of agar. Moreover, when gellan gum contains 0.2 mass% or more with respect to the total moisture, the water absorbability is extremely lowered.
塩化ナトリウムは、水分総質量に対し０．１質量％の割合で含有されている。塩化ナトリウムは、水分中で１価のカチオンを生じる。
硫酸は、ｐＨ調整剤として含有されている。なお、硫酸は、耐熱性好酸性菌（ＴＡＢ）の検査用培地などで、一般的に使用される。
微生物検査用培地は、硫酸により、ｐＨが３．８になるよう調整される。
微生物検査器具は、容器、例えば、直径９０ミリ、厚さ１５ミリのシャーレに微生物検査用培地１０乃至３０ミリリットルを収容して成る。収容された微生物検査用培地は、表面を内部より乾燥させてある。
Sodium chloride is contained at a ratio of 0.1% by mass with respect to the total mass of water. Sodium chloride generates monovalent cations in moisture.
Sulfuric acid is contained as a pH adjuster. Sulfuric acid is generally used in a culture medium for heat-resistant acidophilic bacteria (TAB).
The microorganism testing medium is adjusted to a pH of 3.8 with sulfuric acid.
The microorganism testing instrument is configured by containing 10 to 30 ml of a microorganism testing medium in a container, for example, a petri dish having a diameter of 90 mm and a thickness of 15 mm. The contained microorganism culture medium has its surface dried from the inside.
次に、作用について説明する。
微生物検査用培地は、寒天とジェランガムとを含有しているため、寒天のみを含有した寒天培地に比べて、保水性が高く、常温での保管・流通が可能である。これにより、長期保存したとき、培地から離水して水蒸気になり上皿に水滴が結露する現象を緩和することができる。
Next, the operation will be described.
Since the microorganism testing medium contains agar and gellan gum, it has higher water retention and can be stored and distributed at room temperature than an agar medium containing only agar. Thereby, when it preserve | saves for a long time, it can relieve water from a culture medium, it becomes water vapor | steam, and the phenomenon that a water droplet dew condensation on an upper plate can be eased.
また、寒天培地より多量の試料を短時間に吸収することができ、検出精度を高めることができる。例えば、寒天培地では、添加可能な試料の量が０．１ミリリットルであるのに対し、微生物検査用培地では、製造時に表面を２ミリリットルの水分量だけ乾燥させることにより、１ミリリットルの試料を添加・吸収することができる。また、このときの吸収時間が１０〜１５分であり、寒天培地の約半分の時間で吸収することができる。 In addition, a larger amount of sample than the agar medium can be absorbed in a short time, and the detection accuracy can be improved. For example, in an agar medium, the amount of sample that can be added is 0.1 ml, whereas in a microorganism testing medium, 1 ml of sample is added by drying the surface by 2 ml of moisture during manufacture.・ Can be absorbed. Moreover, the absorption time at this time is 10 to 15 minutes, and can be absorbed in about half the time of the agar medium.
ジェランガムは親油性であるため、微生物検査用培地は、水性試料だけでなく、低濃度の油成分を含む試料も吸収することができる。このため、寒天培地では、油性試料を添加したとき培養中に油が遊離して浮き出してくる現象が発生していたが、微生物検査用培地では、このような油が遊離する現象を防ぐことができる。また、実質的に水に不溶の成分を含有する試料にも親和性があり、吸収可能である。 Since gellan gum is lipophilic, the microorganism testing medium can absorb not only aqueous samples but also samples containing low concentrations of oil components. For this reason, in an agar medium, when an oily sample was added, a phenomenon occurred in which the oil was released during the cultivation, and the phenomenon that the oil was released was prevented in the microorganism testing medium. it can. In addition, a sample containing a component substantially insoluble in water has affinity and can be absorbed.
微生物検査用培地は、保水性が高いため、例えば１週間以上におよぶ長期間の培養でも、乾燥せずに培養が可能である。また、５５℃程度の高温の培養でも、数日間は乾燥せずに培養が可能である。このように、高い保水性により、培養時に乾燥しにくく、硬さも維持されるため、寒天培地に比べて、培養する菌に対する影響を小さくすることができる。 The culture medium for microbiological examination has high water retention, so that it can be cultured without drying, for example, even for long-term culture over one week. Further, even at a high temperature of about 55 ° C., the culture can be performed without drying for several days. Thus, since it is hard to dry at the time of culture | cultivation and hardness is maintained by high water retention, the influence with respect to the microbe to culture | cultivation can be made small compared with an agar medium.
微生物検査用培地は、塩化ナトリウムが生じる１価のカチオンにより、ジェランガムをゲル化させて、培地として適度な硬さに形成されている。寒天培地に比べて表面の弾力性が高いため、画線時や塗抹時に傷つきにくい。ｐＨが４．０でも軟化しないため、培養が可能である。
本発明の実施の形態の微生物検査用培地および微生物検査器具は、油性試料の微生物検査用培地として優れた効果を発揮する。
The culture medium for microbiological examination is formed into a suitable hardness as a medium by gelling gellan gum with monovalent cations that generate sodium chloride. Compared to agar medium, the surface has high elasticity, so it is hard to be damaged during painting or smearing. Since it does not soften even at a pH of 4.0, culture is possible.
The microorganism testing medium and the microorganism testing instrument according to the embodiment of the present invention exhibit excellent effects as a microorganism testing medium for oily samples.
[実施例1]
微生物検査用寒天培地を用いた微生物検査器具を以下の方法で作製して水分の吸収状況を観察した。
[ジェランガム添加寒天培地]
１０００ミリリットルの水に粉末寒天を１２．０ｇ、乾燥ジェランガムを１．２ｇ、酵母エキスを２．５g、ポリペプトンを５．０g、およびブドウ糖を１．０g加えて混合し、１２１℃で１５分間、高圧蒸気滅菌器にて滅菌した。その後、６０℃まで冷却してｐHが７．０±０．１の微生物検査用標準寒天培地を作製した。直径９０ミリ、厚さ１５ミリのシャーレに、この標準寒天培地を２０ミリリットル注ぎ、２５℃まで冷やして固めた。この培地をエアーフィルター付き乾燥機にて、計量しながら２g軽くなるまで乾燥させた。
このようにして作製された寒天培地を使用した場合、希釈液１ミリリットルの吸収時間は、２０分前後であった。
[Example 1]
A microorganism inspection instrument using an agar medium for microorganism inspection was prepared by the following method, and the moisture absorption state was observed.
[Agar medium supplemented with gellan gum]
Add 12.0 g of powdered agar, 1.2 g of dried gellan gum, 2.5 g of yeast extract, 5.0 g of polypeptone, and 1.0 g of glucose to 1000 milliliters of water and mix at high pressure at 121 ° C. for 15 minutes Sterilized with a steam sterilizer. Thereafter, the mixture was cooled to 60 ° C. to prepare a standard agar medium for microbiological examination having a pH of 7.0 ± 0.1. 20 ml of this standard agar medium was poured into a petri dish having a diameter of 90 mm and a thickness of 15 mm, and cooled to 25 ° C. and solidified. This medium was dried with a dryer equipped with an air filter while weighing until 2 g was lightened.
When the agar medium prepared in this way was used, the absorption time of 1 ml of the diluted solution was around 20 minutes.
[実施例２]
アリシクロバシルス検査用寒天培地を用いた微生物検査器具を以下の方法で作製した。
[ジェランガム添加寒天培地]
１０００ミリリットルの水に粉末寒天を１２．０ｇ、乾燥ジェランガムを１．２ｇ、酵母エキスを２．０ｇ、可溶性デンプンを２・０g、およびブドウ糖を１．０g加えて混合し、１２１℃で１５分間、高圧蒸気滅菌器にて滅菌した。その後６０℃まで冷却し、これに１０％硫酸溶液を１．０5ミリリットル添加してｐHが３．７±０．２のアリシクロバシルス検査用寒天培地を作製した。直径９０ミリ、厚さ１５ミリのシャーレに、この寒天培地２０ミリリットルを注ぎ、２５℃に冷やして固めた。この培地をエアーフィルター付き乾燥機にて、計量しながら２g軽くなるまで乾燥させた。
このようにして作製された寒天培地に、アリシクロバシルスを添加した試料１ミリリットルを塗抹して実験を実施した。
[Example 2]
A microorganism testing instrument using an agar medium for testing Alicyclobacillus was prepared by the following method.
[Agar medium supplemented with gellan gum]
Add 12.0 g of powdered agar, 1.2 g of dried gellan gum, 2.0 g of yeast extract, 2.0 g of soluble starch, and 1.0 g of glucose to 1000 ml of water and mix at 121 ° C. for 15 minutes. Sterilized with a high-pressure steam sterilizer. Thereafter, the solution was cooled to 60 ° C., and 1.05 ml of a 10% sulfuric acid solution was added thereto to produce an agar medium for testing Alicyclobacillus having a pH of 3.7 ± 0.2. Into a petri dish having a diameter of 90 mm and a thickness of 15 mm, 20 ml of this agar medium was poured, and cooled to 25 ° C. and hardened. This medium was dried with a dryer equipped with an air filter while weighing until 2 g was lightened.
The experiment was carried out by smearing 1 ml of the sample added with alicyclobacillus on the agar medium thus prepared.
[ジェランガム無添加寒天培地]
比較のため、ジェランガム無配合の培地も作製した。１０００ミリリットルの水に粉末寒天を１２．０ｇ、酵母エキスを２．０ｇ、可溶性デンプンを２・０g、およびブドウ糖を１．０g加えて混合し、１２１℃で１５分間、高圧蒸気滅菌器にて滅菌した。その後、６０℃まで冷却し、これに１０％硫酸溶液を１．０5ミリリットル添加してｐHが３．７±０．２のアリシクロバシルス検査用寒天培地を作製した。直径９０ミリ、厚さ１５ミリのシャーレに、この寒天培地２０ミリリットルを注ぎ、２５℃に冷やして固めた。この培地をエアーフィルター付き乾燥機にて、計量しながら２g軽くなるまで乾燥させた。
[Agar medium without gellan gum]
For comparison, a medium without gellan gum was also prepared. Add 12.0 g of powdered agar, 2.0 g of yeast extract, 2.0 g of soluble starch, and 1.0 g of glucose to 1000 ml of water, mix and sterilize at 121 ° C for 15 minutes in a high-pressure steam sterilizer did. Thereafter, the solution was cooled to 60 ° C., and 1.05 ml of a 10% sulfuric acid solution was added thereto to produce an agar medium for testing Alicyclobacillus having a pH of 3.7 ± 0.2. Into a petri dish having a diameter of 90 mm and a thickness of 15 mm, 20 ml of this agar medium was poured, and cooled to 25 ° C. and hardened. This medium was dried with a dryer equipped with an air filter while weighing until 2 g was lightened.
[比較試験]
このようにして作製された寒天培地に試料１ミリリットルを塗抹した。試料には、耐熱性好酸性菌のアリシクロバシルス アシドテレストリス（以下、「アリシクロバシルス」）102濃度の検体希釈液を使用した。
ジェランガム添加寒天培地および無添加寒天培地の両方を培養器に入れ、50℃で６日間培養を行った。
その結果、ジェランガム添加培地と無添加培地のどちらも培養前の寒天の厚みは５ミリであったものが３日目には添加培地は３ミリ、無添加培地は２ミリになった。また、ジェランガム無添加培地はシャーレと培地の間に亀裂がはいりだし、寒天から水分が放出されシャーレの蓋が水滴でいっぱいになった。
６日後には、ジェランガム添加培地は厚さが１ミリになり、寒天の厚みはなくなったものの６日目の時点においても表面がほとんど乾燥せず、アリシクロバシルスの培養も可能であった。ジェランガム無添加寒天培地は厚さが０．５ミリとなり、培地の深部まで乾燥していて、アリシクロバシルスの培養は困難な状態であった。
[Comparative test]
One milliliter of the sample was smeared on the agar medium thus prepared. The sample used was a dilute specimen of 10 2 concentration of heat-resistant acidophilic bacterium, Alicyclobacillus acid telestris (hereinafter, “Alicyclobacillus”).
Both the gellan gum-added agar medium and the non-added agar medium were placed in an incubator and cultured at 50 ° C. for 6 days.
As a result, in both the gellan gum-added medium and the non-added medium, the agar thickness before culture was 5 mm, but on the third day, the added medium was 3 mm and the non-added medium was 2 mm. In addition, the gellan gum-free medium was cracked between the petri dish and the medium, moisture was released from the agar, and the petri dish's lid was filled with water droplets.
After 6 days, the gellan gum-added medium had a thickness of 1 mm, and although the agar thickness disappeared, the surface was hardly dried even on the 6th day, and alicyclobacillus could be cultured. The gellan gum-free agar medium had a thickness of 0.5 mm and was dried to the deep part of the medium, making it difficult to culture alicyclobacillus.
[実施例３]
微生物検査用寒天培地を用いた微生物検査器具を以下の方法で作製した。
[ジェランガム添加寒天培地]
１０００ミリリットルの水に粉末寒天１２．０ｇ、乾燥ジェランガム１．２ｇ、およびSCDLPブイヨン培地を３５．０ｇ加えて混合し、１２１℃で１５分間、高圧蒸気滅菌器にて滅菌した。その後、６０℃まで冷却してｐHが７．２±０．１の微生物検査用SCDLP培地を作製した。９０ミリ、厚さ１５ミリのシャーレに、この寒天培地２０ミリリットルを注ぎ、２５℃に冷やして固めた。この培地をエアーフィルター付き乾燥機にて、計量しながら２g軽くなるまで乾燥させた。
[Example 3]
A microorganism testing instrument using an agar medium for microorganism testing was prepared by the following method.
[Agar medium supplemented with gellan gum]
Powdered agar (12.0 g), dried gellan gum (1.2 g), and SCDLP bouillon medium (35.0 g) were added to 1000 ml of water, mixed, and sterilized at 121 ° C. for 15 minutes in a high-pressure steam sterilizer. Then, it cooled to 60 degreeC and produced SCDLP culture medium for microorganisms test | inspection with pH 7.2 +/- 0.1. 20 ml of this agar medium was poured into a petri dish having a thickness of 90 mm and a thickness of 15 mm, and was cooled to 25 ° C. and hardened. This medium was dried with a dryer equipped with an air filter while weighing until 2 g was lightened.
[ジェランガム無添加寒天培地]
比較のため、ジェランガム無添加の寒天培地も作製した。１０００ミリリットルの水に粉末寒天を１２．０ｇとSCDLPブイヨン培地を３５．０ｇ加えて混合し、１２１℃で１５分間、高圧蒸気滅菌器にて滅菌した。６０℃まで冷却してｐHが７．２±０．１の微生物検査用SCDLP培地を作製した。直径９０ミリ、厚さ１５ミリのシャーレに、この寒天培地２０ミリリットルを注ぎ、２５℃に冷やして固めた。この培地をエアーフィルター付き乾燥機にて、計量しながら２g軽くなるまで乾燥させた。
[Agar medium without gellan gum]
For comparison, an agar medium without gellan gum was also prepared. Powdered agar (12.0 g) and SCDLP broth medium (35.0 g) were added to 1000 ml of water and mixed, and sterilized at 121 ° C. for 15 minutes in a high-pressure steam sterilizer. After cooling to 60 ° C., an SCDLP medium for microbiological examination having a pH of 7.2 ± 0.1 was prepared. Into a petri dish having a diameter of 90 mm and a thickness of 15 mm, 20 ml of this agar medium was poured, and cooled to 25 ° C. and hardened. This medium was dried with a dryer equipped with an air filter while weighing until 2 g was lightened.
[比較試験]
SCDLPブイヨンにサラダ油をそれぞれ１％から３０％加えたものを用意して油性試料とした。油性試料は、１、５、１０、１５、２０、２５、３０％の７パターン用意した。これらの油性試料を、準備したジェランガム添加培地と無添加培地の両方にそれぞれ１ミリリットルずつ塗抹した。
試料の吸収時間は、15分前後であった。ジェランガム無添加の培地は、油性試料の油分が１５％以上の試料では、水分のみが培地に染込み、油分が表面に残りその油分が寒天培地になじまず、油分の流動が見られ培養が困難であった。ジェランガム添加の寒天培地は、油性試料が寒天表面になじみやすく、無添加培地に比べて油分も小さく分散され油分の流動は確認されなかった。また、ジェランガム添加培地は、無添加培地より表面の油分層も薄いことが確認された。しかしながら、油性分が３０％になると、ジェランガム添加培地においても、寒天表面の油分の流動が多少見られ始めた。ジェランガム添加寒天培地は、２5％以下の油分であれば培養に使用できる。
[Comparative test]
Oil samples were prepared by adding 1 to 30% salad oil to SCDLP broth. Seven patterns of oily samples of 1, 5, 10, 15, 20, 25, 30% were prepared. One milliliter of each of these oily samples was smeared on both the prepared gellan gum-added medium and non-added medium.
The absorption time of the sample was around 15 minutes. In the medium with no gellan gum added, if the oil content of the oily sample is 15% or more, only moisture is infiltrated into the medium, the oil remains on the surface, and the oil does not conform to the agar medium. Met. In the agar medium supplemented with gellan gum, the oily sample was easily adapted to the surface of the agar, and the oil content was smaller than that of the non-added medium, and the oil content was not confirmed. Moreover, it was confirmed that the gellan gum-added medium has a thinner oil layer on the surface than the non-added medium. However, when the oily content reached 30%, some of the oil content on the agar surface started to be observed even in the gellan gum-added medium. An agar medium supplemented with gellan gum can be used for culturing if the oil content is 25% or less.
[実施例４]
微生物検査器具を以下の方法で作製した。
２０℃の水１０００ミリリットルに乾燥寒天１２．０ｇと乾燥ジェランガム１．２ｇと酵母エキス２．０ｇとデンプン２．０ｇとブドウ糖１．０ｇと塩化ナトリウム１．０ｇを加えて混合、１２１℃で１５分間、高圧蒸気滅菌した。６０℃まで冷却後、１Ｎ硫酸０．１ミリリットルを添加し、ｐＨ３．８の微生物検査用培地を作製した。その微生物検査用培地２０ミリリットルを直径９０ミリ、厚さ１５ミリのシャーレに注ぎ、２５℃に冷やして固めた。固まった後、計量しながら２ｇ軽くなるまで、エアーフィルター付乾燥機にて培地表面を乾燥させた。こうして、微生物検査器具を作製した。
作製した微生物検査器具の培地の上に、耐熱性好酸性菌であるアリシクロバチルス属菌を含む試料１ミリリットルを塗沫した。試料の吸収時間は、１０分であった。
作製した微生物検査器具を培養器に入れ、５５℃で１週間培養を行った。その結果、１週間後でも、培地の表面が乾燥せず、耐熱性好酸性菌を培養可能であった。
[Example 4]
A microorganism testing instrument was produced by the following method.
Add 12.0 g of dried agar, 1.2 g of dried gellan gum, 2.0 g of yeast extract, 2.0 g of starch, 1.0 g of glucose and 1.0 g of sodium chloride to 1000 ml of water at 20 ° C., and mix at 121 ° C. for 15 minutes. And autoclaved. After cooling to 60 ° C., 0.1 ml of 1N sulfuric acid was added to prepare a pH 3.8 culture medium for microorganism testing. 20 ml of the microorganism testing medium was poured into a petri dish having a diameter of 90 mm and a thickness of 15 mm, and cooled to 25 ° C. and solidified. After solidifying, the surface of the medium was dried with a dryer equipped with an air filter until the weight was reduced by 2 g while weighing. In this way, a microorganism testing instrument was produced.
On the culture medium of the prepared microorganism testing instrument, 1 ml of a sample containing Alicyclobacillus genus which is a heat-resistant acidophilic bacterium was smeared. The absorption time of the sample was 10 minutes.
The produced microorganism testing instrument was placed in an incubator and cultured at 55 ° C. for 1 week. As a result, even after one week, the surface of the medium did not dry, and the heat-resistant acidophilic bacteria could be cultured.
実施例４の微生物検査器具の培地の上に油性試料１ミリリットルを塗沫し、吸収させた。油性試料は、アリシクロバシルス102濃度の検体希釈液１０ミリリットルにオリーブオイル１滴を滴下して準備した。試料の吸収時間は、１０分であった。その微生物検査器具を培養器に入れ、５５℃で１週間培養を行った。その結果、１週間後でも、培地の表面が乾燥せず、耐熱性好酸性菌を培養可能であった。また、培養中に油が遊離して浮き出してくることはなかった。 1 ml of an oily sample was smeared on the culture medium of the microbiological test device of Example 4 and absorbed. An oily sample was prepared by dropping one drop of olive oil into 10 ml of a sample dilution of 10 2 concentration of Alicyclobacillus. The absorption time of the sample was 10 minutes. The microorganism testing instrument was placed in an incubator and cultured at 55 ° C. for 1 week. As a result, even after one week, the surface of the medium did not dry, and the heat-resistant acidophilic bacteria could be cultured. In addition, oil was not released during the culture.
Claims (7)
A microbiological test instrument for an oily sample characterized by containing the microbiological test medium for an oily sample according to any one of claims 1 to 6 in a container and drying the surface from the inside.
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