以直接螢光顯微鏡計數法快速檢測受傷狀態的活酵母菌及活細菌菌數   全文下載

Using Direct Epifluorescent Microscopic Count for Rapid Enumeration of Viable Yeast and Bacteria in Injured Conditions

傳統的酵母菌計數以培養法需2～3天的時間，而直接顯微鏡計數法雖能快速的計數，但無法準確區別死菌和活菌，使其應用性降低。本研究以活細胞核酸染劑（live cell nucleic acid stain）染色酵母菌，除了與菌體的DNA結合而使細胞核和粒線體在螢光顯微鏡下呈現黃色螢光之外，該染劑還會使活菌的細胞質呈現綠色螢光，而死菌細胞質呈現橙色螢光。因此，一方面以直接顯微鏡計數總酵母菌數，另一方面以直接螢光染色顯微鏡計數法求得呈綠色螢光的活酵母菌數比率，兩者相乘即為活酵母菌數，其結果與傳統培養法所得結果之相關係數達0.96，並且只要30分鐘即可完成估算活酵母菌數。本方法也一樣可使用於活細菌菌數之快速檢測。

Traditional method for assessing cell count requires an incubation period of 2~3 days. The direct microscopic count (DMC) method gives rapid enumeration of yeast and bacteria. However, the application is limited, since it is not possible to distinguish accurately between viable and nonviable cell. In this report, several nucleic acid dyes from Molecular Probe Inc. were used to stain the cells for enumeration of live and dead yeast in injured conditions, in which the dye can stain the DNA of nucleus and mitochondria, which then fluoresce yellow under fluorescence microscope, as well as stain the cytoplasm of actively growing cells to fluoresce green, while the cytoplasm of inactive cells fluoresced orange. The direct microscopic count multiplied by the live cell count ratio obtained by this direct epifluoroscent microscopic count (DEMC) method gives the estimated viable yeast count. According to our results, the correlation coefficient (R2) between the cell count obtained by the DEMC method and that by the standard plate count was 0.91 for yeast and 0.96 for bacteria in frozen and heating conditions, respectively. Requiring only 30 min, this method can also be used for the rapid enumeration of viable bacteria in injured conditions.