Flow cytometry characterisation of Salmonella typhimurium mutants defective in proton translocating proteins and stationary-phase growth phenotype

We have shown that the growth, starvation and population heterogeneity of S almonella typhimurium and its isogenic nuoG and cydA mutants can be monitor ed by flow cytometry. Bacterial cells were analysed unstained, and after st aining with rhodamine 123, propidium iodide and acridine orange. In unstain ed cultures it was possible to distinguish flagellated and non-flagellated cells, nuoG and cydA mutants were less stained with rhodamine confirming th eir defects in generating membrane potential. Increase in propidium iodide staining associated with reduced membrane integrity was seen between day 4 and 14 in all the strains. Acridine orange staining showed that there was r etarded development in stationary phase in nuoG and cydA mutants. Furthermo re, up to day 28, a small portion of cells showed high RNA and DNA levels. To determine whether these cells represent a sub-population better adapted for long term survival, we measured the growth of the population by both OD values and viable counts. Because the OD values increased throughout the w hole study in both wild-type and mutant strains, while the viable counts gr adually decreased, we propose that even in very old cultures there must be a population of cells undergoing replication. (C) 2000 Elsevier Science B.V . All rights reserved.