Using Direct Epifluorescent Microscopic Count for Rapid Enumeration of Viable Yeast and Bacteria in Injured Conditions
CHIN-CHENG HUANG1*, SHWU-MEEI LIAU1, WEI-CHUNG TSAI1 AND HSI-HUA WANG2
1. Food Industry Research and Development Institute, Hsinchu, Taiwan, R.O.C.
2. Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan, R.O.C.
(Received: November 16, 2004; Accepted: January 24, 2005)
ABSTRACT
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.
Key words: live cell nucleic acid stain, direct epifluorescent microscopic count, direct microscopic count, viable yeast, injured cell, rapid enumeration