Involvement of RNA and DNA in the staining of Escherichia coli by SYTO 13

To assess the extent to which DNA and RNA bacterial content contributes to fluorescent response of SYTO 13.

RNA and DNA of Escherichia coli 536 cells were extracted and fluorimetrically quantified to compare the different contents, throughout a 24 h culture, with their SYTO 13 fluorescence emission when analysed by the cytometer. SYTO 13 fluorescence varied depending on the stage of bacterial growth and in accordance with both DNA and RNA content. RNA content accounted for at least two-thirds of the total fluorescence of a cell. Escherichia coli cells were treated with chloramphenicol to improve their RNA content. With this treatment, both nucleic acids remained constant but there was a clear improvement in fluorescent emission. SYTO 13 fluorescence was also studied in E. coli X-1488 minicells.

Although both nucleic acids are implicated, RNA accounts for a major part of SYTO 13 fluorescence. The fluorescence cannot be considered as a direct reflection of nucleic acid content. Other factors, such as topology or supercoiling, need to be considered.

The results confirm the efficacy of SYTO 13 for labelling bacteria and for assessing the distinct physiological status. A better knowledge of the parameters implicated in its fluorescence emission has been achieved.