Separation of active and inactive fractions from starved culture of Vibrio parahaemolyticus by density dependent cell sorting

Abstract

The co-existence of physiologically different cells in bacterial cultures is a general phenomenon. We have examined the applicability of the density dependent cell sorting (DDCS) method to separate subpopulations from a long-term starvation culture of Vibrio parahaemolyticus. The cells were subjected to Percoll density gradient and separated into 12 fractions of different buoyant densities, followed by measuring the cell numbers, culturability, respiratory activity and leucine incorporation activity. While more than 78% of cells were in lighter fractions, about 95% of culturable cells were present in heavier fractions. The high-density subpopulations also had high proportion of cells capable of forming formazan granules. Although this was accompanied by the cell specific INT-reduction rate, both leucine incorporation rates and INT-reduction rates per cell had a peak at mid-density fraction. The present results indicated that DDCS could be used to separate subpopulations of different physiological conditions.

Is Urografin density gradient centrifugation suitable to separate nonculturable cells from Escherichia coli populations?

“…Percoll gradient centrifugation has been used to fractionate E. coli and V. parahaemolyticus cultures during growth phase transition and following starvation, with a variety of results (Makinoshima et al 2002(Makinoshima et al , 2003Nishino et al 2003;Nayak et al 2005). Makinoshima et al (2002Makinoshima et al ( , 2003 indicated that E. coli cultures can be fractionated into more than 20 cell subpopulations and they attributed the cell separation to the increase in buoyant density from exponential to stationary phase of growth.…”

“…Percoll is regarded as non-toxic to cells (Pertoft 2000;Nayak et al 2005). However, Urografin has been described as toxic to bacterial cells (Kim and Lachman 1982).…”