The Use of DRAQ5 to Monitor Intracellular DNA in Escherichia coli by Flow Cytometry

Flow cytometry provides a rapid and high-content multiparameter analysis of individual microorganisms within a population. In the past years, several fluorescent stains were developed in order to monitor DNA content distribution and cell-cycle phases, mainly in eukaryotic cells. Recently, due to its low detection limits, several of these fluorescent stains were also applied to prokaryotic cells. In this study, the ability of a novel far-red fluorescent stain DRAQ5 in assessing intracellular DNA content distribution in Escherichia coli DH5 alpha was evaluated. The results showed that a DRAQ5-labelled live E. coli suspension can be obtained by incubation of 1 x 10(6) cells/mL with 5 A mu M DRAQ5 in PBS buffer supplemented with EDTA (pH = 7.4) during 30 min at 37 A degrees C. Flow cytometric analysis of fixed E. coli cells revealed that ethanol should be used in detriment of glutaraldehyde for DRAQ5 labelling. After the analysis of RNase and DNase digested samples, DRAQ5 was proven to be a specific DNA labelling stain. The present study demonstrates that the use of DRAQ5 as a DNA-labelling stain provides an easy assessment of intracellular DNA content and cell-cycle phases in Gram-negative bacteria such as E. coli.