Abstract:

The co-cultured cells of Lactobacillus bμlgaricus1.1480 and Streptococcus thermophilus6038 were labeled with different concentrations of 5-(and) 6-carboxyfluorescein diacetate (5(6)-cFDA) in different solvents in order to explore the affecting factors of 5(6)-cFDA labeling. The initial percentages of lactic acid bacterial cells labeled with 5(6)-cFDA in double distilled water, PBS or dimethyl sulfoxide (DMSO) were 94.8%, 95.2% and 99.77%, respectively, which were decreased to 51.53%, 55.6% and 96.7% after 30 days. The cell suspension at a cell density of 1.0 × 106 cells/mL was added with different volumes of 5 mg/mL 5(6)-cFDA to desired final concentrations (1.09 × 10-7, 2.17 × 10-7, 4.35 × 10-7, 6.25 × 10-7, 8.7 × 10-7 and 10.87 × 10-7 mol/L). The diluted cell suspension containing 6.52×10-7 mol/L 5(6)-cFDA showed the best labeling efficiency and there was a linear relationship between cell labeling efficiency and the final concentration of 5(6)-cFDA ranging between 1.09 × 10-7 and 6.25 × 10-7 mol/L. It was also demonstrated that a cell density between 1×106 and 1×107 cells/mL was suitable for flow cytometric (FCM) determination of living cells, with a high labeling efficiency. The buffer at pH 7 had the smallest effect on cell viability and fluorescence intensity and the resulting labeling efficiency reached 98.3%.

Key words: 5-(and) 6-carboxyfluorescein diacetate, lactic acid bacteria cells, analysis