JP5936879B2 - Antibacterial treatment method of culture plate - Google Patents
Antibacterial treatment method of culture plate Download PDFInfo
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- JP5936879B2 JP5936879B2 JP2012043204A JP2012043204A JP5936879B2 JP 5936879 B2 JP5936879 B2 JP 5936879B2 JP 2012043204 A JP2012043204 A JP 2012043204A JP 2012043204 A JP2012043204 A JP 2012043204A JP 5936879 B2 JP5936879 B2 JP 5936879B2
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- 230000000844 anti-bacterial effect Effects 0.000 title claims description 50
- 238000000034 method Methods 0.000 title claims description 27
- 229920003002 synthetic resin Polymers 0.000 claims description 17
- 239000000057 synthetic resin Substances 0.000 claims description 17
- 229920003023 plastic Polymers 0.000 claims description 11
- 239000004033 plastic Substances 0.000 claims description 11
- -1 polyethylene Polymers 0.000 claims description 11
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 10
- 239000001301 oxygen Substances 0.000 claims description 10
- 229910052760 oxygen Inorganic materials 0.000 claims description 10
- 239000011521 glass Substances 0.000 claims description 9
- 229920002396 Polyurea Polymers 0.000 claims description 8
- 239000012298 atmosphere Substances 0.000 claims description 7
- 238000007740 vapor deposition Methods 0.000 claims description 7
- 238000006116 polymerization reaction Methods 0.000 claims description 6
- 239000004698 Polyethylene Substances 0.000 claims description 5
- 239000004642 Polyimide Substances 0.000 claims description 5
- 229920000573 polyethylene Polymers 0.000 claims description 5
- 239000005020 polyethylene terephthalate Substances 0.000 claims description 5
- 229920000139 polyethylene terephthalate Polymers 0.000 claims description 5
- 229920001721 polyimide Polymers 0.000 claims description 5
- 239000004677 Nylon Substances 0.000 claims description 4
- 238000003618 dip coating Methods 0.000 claims description 4
- 229920001778 nylon Polymers 0.000 claims description 4
- 238000004528 spin coating Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000003242 anti bacterial agent Substances 0.000 description 8
- 239000000758 substrate Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 244000063299 Bacillus subtilis Species 0.000 description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000588747 Klebsiella pneumoniae Species 0.000 description 2
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 2
- 241000607720 Serratia Species 0.000 description 2
- 206010041925 Staphylococcal infections Diseases 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 239000000919 ceramic Substances 0.000 description 2
- QFTYSVGGYOXFRQ-UHFFFAOYSA-N dodecane-1,12-diamine Chemical compound NCCCCCCCCCCCCN QFTYSVGGYOXFRQ-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 208000015688 methicillin-resistant staphylococcus aureus infectious disease Diseases 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- XSCLFFBWRKTMTE-UHFFFAOYSA-N 1,3-bis(isocyanatomethyl)cyclohexane Chemical compound O=C=NCC1CCCC(CN=C=O)C1 XSCLFFBWRKTMTE-UHFFFAOYSA-N 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 229920000426 Microplastic Polymers 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- BZHJMEDXRYGGRV-UHFFFAOYSA-N Vinyl chloride Chemical compound ClC=C BZHJMEDXRYGGRV-UHFFFAOYSA-N 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004898 kneading Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006254 polymer film Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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Description
本発明は、特定の情報を持つ細胞を、生きた状態で分離し（セルソーター）、薬剤の効果を実証するなどで用いられる培養プレートの抗菌処理方法に関するものである。 The present invention relates to a method for antibacterial treatment of a culture plate used for separating cells having specific information in a living state (cell sorter) and demonstrating the effect of a drug.
特定の情報を持つ細胞を、生きた状態で分離し（セルソーター）、薬剤の効果を実証するなどで用いられる培養プレートでは分離した細胞が培養プレートに付着した雑菌によって死滅するなどの大きな影響を受けるため培養プレート自身に抗菌性が求められつつある。 Cells with specific information are isolated in a living state (cell sorter), and culture plates used to demonstrate the effects of drugs are greatly affected by the fact that the separated cells are killed by bacteria attached to the culture plate. Therefore, antibacterial properties are being demanded on the culture plate itself.
培養プレートは抗菌性の度合いにより菌増殖の程度が大きく影響を受ける。
このような培養プレートは、耐久性を考慮して、プラスチックや金属、セラミックス等が材料とされる場合が多い。
Culture plates are greatly affected by the degree of bacterial growth depending on the degree of antibacterial activity.
Such a culture plate is often made of plastic, metal, ceramics or the like in consideration of durability.
プラスチックから培養プレートを製造して抗菌性を付与する方法としては、ポリエチレン、ポリスチレン、塩化ビニル、ポリプロピレン等のプラスチックを加熱溶融し、５〜３０％の濃度で抗菌剤を含有したプラスチックペレットであるマスターバッチを添加することにより、抗菌剤を練り込む方法が一般に行われている。尚、この方法の場合、最終製品への抗菌剤の添加量は０．３〜２％程度となる。前記プラスチックには、その種類や加工方法により若干の差があるものの通常は２５０〜３００℃以上の耐熱性が要求され、また、抗菌剤には、この温度において、抗菌剤成分の揮発、分解、含有水分の放出等による抗菌剤の重量変化、抗菌効果の低下や変色等を生じないことが必要とされるため、プラスチックや抗菌剤の材料の選定に制限が生じるという問題があった。 As a method for producing an antibacterial property by producing a culture plate from plastic, a master is a plastic pellet containing an antibacterial agent at a concentration of 5 to 30% by heating and melting a plastic such as polyethylene, polystyrene, vinyl chloride, or polypropylene. A method of kneading an antibacterial agent by adding a batch is generally performed. In this method, the amount of antibacterial agent added to the final product is about 0.3 to 2%. The plastics are required to have a heat resistance of 250 to 300 ° C. or more, although there are some differences depending on the type and processing method. In addition, antibacterial agents volatilize, decompose, decompose, There is a problem that the selection of the material of the plastic or the antibacterial agent is limited because it is necessary to prevent the weight change of the antibacterial agent due to the release of the contained moisture, the decrease in the antibacterial effect, and the discoloration.
一方、金属やセラミックスへ抗菌性を付与する方法としては、予め成形した部材表面に貼着剤により抗菌剤を塗布したプラスチックフィルムを貼着する方法が一般に行われている（例えば、特許文献１参照）。
しかしながら、培養プレートのように部材の形状が凹凸で複雑である場合には、かかる部材表面の全面に均一に抗菌性を付与することは困難であるという問題があった（特許文献２参照）。
On the other hand, as a method for imparting antibacterial properties to metals and ceramics, a method of sticking a plastic film in which an antibacterial agent is applied to a previously molded member surface with an adhesive is generally performed (see, for example, Patent Document 1). ).
However, when the shape of the member is uneven and complex like a culture plate, there is a problem that it is difficult to uniformly impart antibacterial properties to the entire surface of the member (see Patent Document 2).
このような凹凸形状を有する多孔質部材の表面に抗菌性を付与する方法として蒸着重合膜を部材表面に被覆した後、オゾン処理によって抗菌性を付与する方法がある（特許文献３参照）。しかしながら、オゾン処理によって抗菌性を付与した培養プレートは、オゾン処理後経時変化が激しく約1ヶ月で抗菌性が無くなるという問題があった。 As a method for imparting antibacterial properties to the surface of a porous member having such a concavo-convex shape, there is a method of imparting antibacterial properties by ozone treatment after coating the vapor deposition polymer film on the surface of the member (see Patent Document 3). However, the culture plate to which antibacterial properties are imparted by ozone treatment has a problem that the antibacterial properties are lost after about one month because the change with time after ozone treatment is severe.
本発明は、前記従来技術の問題点を解消し、抗菌性を付与する対象となる培養プレート全面に、いわゆる抗菌剤を用いることなく抗菌性を付与することを目的とする。 The object of the present invention is to solve the problems of the prior art and to impart antibacterial properties to the entire culture plate to be imparted with antibacterial properties without using a so-called antibacterial agent.
本発明者等は前記課題を解決するべく鋭意検討の結果、培養プレートの表面にポリ尿素、ナイロン、ポリエチレン、ポリエチレンテレフタレート、ポリイミド等の合成樹脂被膜を形成後、真空紫外光照射処理することにより前記培養プレートに優れた抗菌性を付与することできることを見出し、本発明を完成した。 As a result of intensive studies to solve the above-mentioned problems, the present inventors formed a synthetic resin film such as polyurea, nylon, polyethylene, polyethylene terephthalate, and polyimide on the surface of the culture plate, and then subjected to vacuum ultraviolet light irradiation treatment. The present inventors have found that an excellent antibacterial property can be imparted to the culture plate and completed the present invention.
即ち、本発明は以下の態様を有する。
[態様１]
培養プレートの表面に合成樹脂被膜を形成後、酸素雰囲気で真空紫外光照射処理することにより前記培養プレートに抗菌性を付与することを特徴とする、培養プレートの抗菌処理方法。
[態様２]
ディップコート、スピンコート、又は蒸着重合により合成樹脂被膜を形成する、態様１記載の方法。
[態様３]
合成樹脂がポリ尿素、ナイロン、ポリエチレン、ポリエチレンテレフタレート又はポリイミドである、態様１又は２記載の方法。
[態様４]
前記培養プレートがガラス又はプラスチックであることを特徴とする態様１〜３のいずれか一項に記載の方法。
[態様５]
態様１〜４のいずれか一項に記載の方法で得られる、抗菌性培養プレート。
[態様６]
前記培養プレートがガラス又はプラスチックであることを特徴とする態様５に記載の抗菌性培養プレート。
That is, this invention has the following aspects.
[Aspect 1]
An antibacterial treatment method for a culture plate, comprising forming a synthetic resin film on the surface of the culture plate and then applying antibacterial properties to the culture plate by performing vacuum ultraviolet light irradiation treatment in an oxygen atmosphere.
[Aspect 2]
The method according to
[Aspect 3]
The method according to
[Aspect 4]
The method according to any one of
[Aspect 5]
The antibacterial culture plate obtained by the method as described in any one of aspects 1-4.
[Aspect 6]
6. The antibacterial culture plate according to
本発明の方法によれば、ディップコート、スピンコート、蒸着重合等により合成樹脂被膜を形成して抗菌性を付与するため、培養プレートを構成する材料及び培養プレートの形状等について制限されることなく、合成樹脂被膜の形成後に、真空紫外光照射処理を短時間することによって、培養プレート表面に均一に、黄色ブドウ球菌、大腸菌、肺炎かん菌、緑膿菌、Ｏ−１５７、枯草菌、セラチア及びＭＲＳＡ等に対する優れた抗菌性を付与することができる。 According to the method of the present invention, since a synthetic resin film is formed by dip coating, spin coating, vapor deposition polymerization, etc. to impart antibacterial properties, the material constituting the culture plate and the shape of the culture plate are not limited. After the synthetic resin coating is formed, vacuum ultraviolet light irradiation treatment is performed for a short period of time, so that the surface of the culture plate is uniformly mixed with Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, O-157, Bacillus subtilis, Serratia and Excellent antibacterial properties against MRSA and the like can be imparted.
本発明の抗菌処理方法は、まず、培養プレートの表面にディップコート、スピンコート、蒸着重合等の当業者に公知の任意の方法から、培養プレートの材質などに応じて適宜選択した方法によって合成樹脂被膜を形成する。 In the antibacterial treatment method of the present invention, first, a synthetic resin is selected from any method known to those skilled in the art, such as dip coating, spin coating, vapor deposition polymerization, etc., on the surface of the culture plate according to the method appropriately selected according to the material of the culture plate. Form a film.
前記培養プレートとしては、ガラス、金属、プラスチック等を使用することができ、この中でもガラス又はプラスチックを使用することが好ましい。因みに、培養プレートの大きさ及び形状等に特に制限はない。 As the culture plate, glass, metal, plastic or the like can be used, and among these, glass or plastic is preferably used. Incidentally, there is no particular limitation on the size and shape of the culture plate.
前記合成樹脂被膜は、ポリ尿素、ナイロン、ポリエチレン、ポリエチレンテレフタレート、ポリイミド等の当業者に公知の任意の材質を使用することができる。尚、前記合成樹脂被膜の厚みは、合成樹脂被膜及び培養プレートの種類などに応じて、０．１μｍ以上の適当な厚さとすることが好ましい。０．１μｍ未満であると、抗菌性能が不足するからである。 The synthetic resin film may be made of any material known to those skilled in the art, such as polyurea, nylon, polyethylene, polyethylene terephthalate, and polyimide. The thickness of the synthetic resin film is preferably an appropriate thickness of 0.1 μm or more depending on the types of the synthetic resin film and the culture plate. It is because antibacterial performance is insufficient when it is less than 0.1 μm.
上記の培養プレートの表面に合成樹脂被膜を形成された後に、該皮膜に真空紫外光照射処理し、抗菌性を付与する。前記真空紫外光照射処理に関しては、前記合成樹脂被膜を酸素雰囲気で真空紫外光照射させることができれば特に温度や時間等の条件、紫外光照射のための装置及び照射出力等についての制限はなく、当業者が適宜選択することができる。通常、合成樹脂被膜が形成された培養プレートを室温、又は、約１００℃まで、例えば、６０〜８０℃の範囲に加熱した後、 １００００〜７００００Pa程度の範囲の真空度に酸素を導入し、かかる酸素雰囲気で１０〜６０分程度真空紫外光照射させるようにすればよい。 After a synthetic resin film is formed on the surface of the culture plate, the film is subjected to vacuum ultraviolet light irradiation treatment to impart antibacterial properties. Regarding the vacuum ultraviolet light irradiation treatment, there are no restrictions on the conditions such as temperature and time, the apparatus for irradiation with ultraviolet light, the irradiation output, etc., as long as the synthetic resin film can be irradiated with vacuum ultraviolet light in an oxygen atmosphere. Those skilled in the art can select as appropriate. Usually, after the culture plate on which the synthetic resin coating is formed is heated to room temperature or about 100 ° C., for example, in the range of 60 to 80 ° C., oxygen is introduced to the degree of vacuum in the range of about 10,000 to 70000 Pa, and this is applied. What is necessary is just to irradiate vacuum ultraviolet light for about 10 to 60 minutes in oxygen atmosphere.
上記方法により、処理された培養プレートは、抗菌性を有する培養プレートとして機能することになる。 By the above method, the treated culture plate functions as a culture plate having antibacterial properties.
抗菌処理室１には図示しないが真空排気系が接続されて任意の真空度に調整自在となっている。抗菌処理室１にはエキシマ紫外線レーザー照射装置２が設置されていて成膜処理された培養プレート３を抗菌処理室１に設置した後、真空排気し、酸素ガス導入機構４から任意の真空度に酸素を導入しエキシマ紫外線レーザー照射ができるように設置されている。また、前記抗菌処理室１の中央下部に基板上下機構５が配置され、この基板上下機構５の上には被抗菌処理物である培養プレート３が設置され、最適条件下で抗菌処理ができるようにしてある。
Although not shown, the
以下、実施例を参照して本発明を説明する。尚、本発明の技術的範囲はこれら実施例の記載に限定されるものではなく、これら記載に基づき当業者が適宜変更・修正したものも本発明に含まれる。 Hereinafter, the present invention will be described with reference to examples. It should be noted that the technical scope of the present invention is not limited to the description of these examples, and modifications and corrections appropriately made by those skilled in the art based on these descriptions are also included in the present invention.
（実施例１）
ポリ尿素膜の成膜原料物質としては、1,12-ジアミノドデカン（DAD）と、1,3-ビス（イソシアナートメチル）シクロヘキサン(H6XDI)を用いた。また、非成膜処理物としては、ガラス製培養プレートを用いた。まず、特許文献３の実施例１に記載されている蒸着重合装置及び方法を用いてガラス製培養プレート（ウェルの径：35mm、ウェルの深さ：1.45〜1.55mm、プレート外寸：8.5×12.7×2.0cm、http://www.phoenixsci.co.jp/product/product07/を参照）にポリ尿素膜（厚さ：０．２μｍ）を形成させた。その後、図１に示した抗菌処理装置を用いて、下記の処理条件で抗菌性付与処理を行った。
Example 1
As raw materials for forming the polyurea film, 1,12-diaminododecane (DAD) and 1,3-bis (isocyanatomethyl) cyclohexane (H6XDI) were used. In addition, a glass culture plate was used as the non-film-formed product. First, a glass culture plate (well diameter: 35 mm, well depth: 1.45 to 1.55 mm, outer plate size: 8.5 × 12.7) using the vapor deposition polymerization apparatus and method described in Example 1 of
具体的な真空紫外光照射条件は、抗菌処理室１の基板上下機構５上に設置した後、基板上下機構５によりエキシマ紫外線レーザー光源の窓６から培養プレート３までの距離を１００ｍｍに調整しバルブ７を閉じた。
Specific vacuum ultraviolet light irradiation conditions are as follows: the distance from the excimer ultraviolet laser
次に前記抗菌処理室１の真空排気を停止し、酸素導入機構４から５×１０４Paの真空度に酸素を導入し、室温にて、エキシマ紫外線レーザー照射（照射出力４０W）、を２０分間行った。
Next, evacuation of the
尚、エキシマ紫外線レーザー光源としてはウシオ電機株式会社製光化学実験用エキシマ光照射ユニット（型式SUS06）を用いた。 As an excimer ultraviolet laser light source, an excimer light irradiation unit (model SUS06) for photochemical experiment manufactured by USHIO INC. Was used.
エキシマ紫外線レーザー照射終了後、抗菌処理室１に窒素ガスを大気圧になるまで導入し培養プレート３を取り出した。
After completion of the excimer ultraviolet laser irradiation, nitrogen gas was introduced into the
（実施例２）
まず、実施例１と同様の蒸着重合法によってガラス製培養プレートにポリエチレンテレフタレート被覆被膜（厚さ：０．２μｍ）を形成させた。該培養プレートを、抗菌処理室１内で１００℃に加熱した後、実施例1同じ真空紫外光照射処理条件で培養プレートに抗菌処理した。
(Example 2)
First, a polyethylene terephthalate-coated film (thickness: 0.2 μm) was formed on a glass culture plate by the same vapor deposition polymerization method as in Example 1. After the culture plate was heated to 100 ° C. in the
（抗菌性試験１）
こうして得られた本発明の培養プレートに加えて、実施例１と同様にポリ尿素膜を形成させた後、上記真空紫外光照射が施されていないポリ尿素膜被覆培養プレートを比較例１とした。更に、特許文献３に記載の方法で抗菌処理を施した培養プレート（ポリイミド被覆）を比較例2とした。これらの各培養プレートを、大気中に1週間放置し、その後、ＪＩＳ Ｚ ２８０１に従った試験方法で抗菌性の試験を行った。上記試験の結果を下記表１に示す。
(Antimicrobial test 1)
In addition to the culture plate of the present invention thus obtained, a polyurea film was formed in the same manner as in Example 1, and then the polyurea film-coated culture plate not subjected to vacuum ultraviolet light irradiation was used as Comparative Example 1. . Furthermore, a culture plate (polyimide coating) subjected to antibacterial treatment by the method described in
（抗菌性試験１）
更に、抗菌処理後1ヶ月大気に暴露した各培養プレートについて、ＪＩＳ Ｚ ２８０１に従った試験方法で抗菌性の試験を行った。上記試験の結果を下記表２に示す。
(Antimicrobial test 1)
Further, each culture plate exposed to the atmosphere for 1 month after antibacterial treatment was subjected to an antibacterial test by a test method according to JIS Z 2801. The results of the above test are shown in Table 2 below.
以上の表に示された結果から、実施例１及び２の処理がされた本発明の培養プレートは、黄色ブドウ球菌、大腸菌、肺炎かん菌、緑膿菌、Ｏ−１５７、枯草菌、セラチア及びＭＲＳＡのいずれに対しても、比較例２と比べても極めて優れた抗菌性を示すことがわかった。 From the results shown in the above table, the culture plates of the present invention treated in Examples 1 and 2 were S. aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, O-157, Bacillus subtilis, Serratia and It was found that the antibacterial property was extremely excellent as compared with Comparative Example 2 for any of MRSA.
本発明の抗菌処理方法は様々な大きさ及び形状等を有する各種の培養プレートに適用することが可能である。 The antibacterial treatment method of the present invention can be applied to various culture plates having various sizes and shapes.
１ 抗菌処理室
２ エキシマ紫外線レーザー照射装置
３ 培養プレート
４ 酸素導入機構
５ 基板上下機構
６ エキシマ紫外線レーザー光源の窓
７ バルブ
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