Purpose: To define filament-inducing conditions and timeframes for several strains of Salmonella, to characterize and compare morphological and physiological features of Salmonella filamented under different conditions, and to develop a quantitative, flow cytometry (FCM)-based method for monitoring septation and division of filaments upon removal of salt- or cold-stress.
Methods: Five Salmonella strains (ATCC 14028, ATCC 13311, ATCC 13076 and two peanut butter outbreak-associated isolates) and an E. coli control (ATCC 25922) were grown under four filament-inducing conditions of salt- or cold-stress in liquid or solid media. Additional Salmonella controls (overnight growth in TSB and extended growth in TSB) were also prepared. The resulting cultures were characterized using fluorescence microscopy (FM) and FCM in conjunction with LIVE/DEAD staining. Time course FCM analysis was used to monitor differentiation of filaments into individual cells after removal of stress conditions.
Results: All Salmonella strains formed filaments in response to salt-stress (within 5-6 d) or cold-stress (within 10-16 d) and the extent of filamentation was examined and compared across strains and conditions using both FM and FCM. We found that post-stress transition from filaments to single cells could be monitored quantitatively over a period of 6 h using time course FCM.
Significance: This study provides single-cell approaches for physiological characterization of stress-filamented Salmonella populations that may help elucidate the practical impact of filamentation on detection of this pathogen in foods.