微生物染色特性の時間依存性に関する検討：バイオエアロゾルオンライン計測のための基礎特性

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  • An Investigation of the Time Dependency for the Staining Process of Microbes: the Behavior of Microbes in relation to Bio-aerosol Online Monitoring

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Abstract

The staining of a bio-aerosol, or airborne biological particles that are suspended in the atmosphere in the form of an aerosol, represents a tool that can be used to evaluate the detailed characteristics, and the concentration, of such substances. This can be done online using available technology such as flow cytometry (Aerosol Flow Cytometry). Such an online tool could be useful for examining emission sources and the transport of bio-aerosols. Mixing a bio-aerosol and a mist containing fluorescence dye solution could be utilized as a possible "aerosol dying process". This is a process in which the time required to stain microbes with the dye solution would be a key factor both for staining efficiency and for the size of the instrument to be designed. In this study, the time dependency of the staining process was examined in relation to a combination of a typical type of microbe, or, yeast (Saccharomyces cerevisiae) suspended in water and a DAPI solution using a fluorescence spectrophotometer in conjunction with a dye concentration of 0.1-10 µg mr1 and a yeast concentration 0.1-100 µg mr1 • The study showed that the fluorescence intensity of a mixed solution (compared to that of the free dye) increased instantaneously after mixing under selected conditions and that 50 % of the maximum amount of stained yeast could be observed within less than 10 seconds. DAPI was confirmed to stain yeast cells in the order of seconds in the fluorescence spectrum and this could be observed as a red shift in the excitation and emission wavelengths of a mixed solution. By adjusting suitable conditions (e.g., DAPI 3 µg mr1 and yeast 2: 10 µg mr1 in the present case), the staining processes (<10 sec. 50 % staining) was rapid and the product remained stable. This suggests that the aerosol staining process has the potential for use in this area.

気中に浮遊する微生物（バ イオエアロゾル）のDNA を浮遊状態で気中染色できれば，既存のフロー サイ トメトリー 等の計測技術をエアロゾルに応用して （エアロゾルフロー サイ トメトリ ー ），微生物の種類・濃度などの詳細な情報をオンライン計測できる可能性がある。こうしたオンライン計測の実現は， バ イオエアロゾル発生源の特定や輸送変動を議論する上で有用なツールとなる。 バ イオエアロゾルと蛍光染料ミス トとの混合は気中染色法の候補の一 つであるが，染色液と接した薗のDNA染色に要する時間がエアロゾル保持時間・方法，装置寸法を決定する重要なファクタ ー となる。本研究では，イー スト菌と代表的な蛍光物質であるDAPI溶液の組み合わせを例として，様々な薗・蛍光物質濃度で菌分散液とDAPI溶液の混合直後からの秒単位のDNA蛍光強度の変化特性を蛍光分光光度計で計測し，混合条件と染色時間の関係に検討を加えた。この結果，適切な濃度条件（例えばDAPI 3µgml イ ー ス ト菌10 µg ml ) を設定することで再現性のある安定した時間特性が得られること，IO秒以下の極めて短時間にDNAの50%以上が染色されることを示し，気中染色法の実現可能性を示した。

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