ORIGINAL PAPER Is Urografin density gradient centrifugation suitable to separate nonculturable cells from Escherichia coli populations? Ine ´s Arana Æ Maite Orrun ˜o Æ Carolina Seco Æ Alicia Muela Æ Isabel Barcina Received: 12 February 2007 / Accepted: 10 August 2007 / Published online: 29 August 2007 Ó Springer Science+Business Media B.V. 2007 Abstract The ability of Urografin or Percoll density gradient centrifugations to separate nonculturable subpopulations from heterogeneous Escherichia coli populations was analysed. Bacterial counts (total, active and culturable cells) and flow cytometric analyses were carried out in all recovered bands. After Urografin centrifugation, and despite the dif- ferent origin of E. coli populations, a common pattern was obtained. High-density bands were formed mainly by nonculturable cells. However, the increase in cell density would not be common to all nonculturable cells, since part of this subpopulations banded in low-density zones, mixed with culturable cells. Bands obtained after Percoll centrifugation were heterogeneous and culturable and nonculturable cells were recovered along the gradient. Thus, fractionation in Urografin cannot be only attributed to changes in buoyant densities during the transition from culturable to nonculturable state. Urografin density gradients allow us to obtain enriched fractions in nonculturable subpopulations from a heteroge- neous population, but working conditions should be carefully chosen to avoid Urografin toxicity. Keywords Density gradient centrifugation Á Escherichia coli Á Nonculturable subpopulation Á Urografin Abbreviations CC Culturable cells HD High-density band LD Low-density band NCC Nonculturable cells NVC Non viable cells VBNC Viable but nonculturable Introduction In adverse conditions, some bacterial populations gradually lose their ability to grow on culture media but they remain intact and metabolically active. This phenomenon is referred to as the viable but noncul- turable (VBNC) phenotype (Kjelleberg 1993; Barcina et al. 1997; Colwell and Gray 2000). An intense debate is going on about its biological significance: cells become nonculturable due to cellular deteriora- tion conducing to bacterial death (Bogosian et al. 2000; Nystro ¨m 2001) or this phenotype is a response similar to spore formation (McDougald et al. 1999; Kell and Young 2000)? Nystro ¨m (2003) suggests that loss of culturability is due to activation of genetic modules mediating programmed cell death. To dis- criminate between these options it is necessary to I. Arana Á M. Orrun ˜o Á C. Seco Á A. Muela Á I. Barcina (&) Departamento de Inmunologı ´a, Microbiologı ´a y Parasitologı ´a, Facultad de Ciencia y Tecnologı ´a, Universidad del Paı ´s Vasco, Apdo. 644, Bilbao 48080, Spain e-mail: isabel.barcina@ehu.es 123 Antonie van Leeuwenhoek (2008) 93:249–257 DOI 10.1007/s10482-007-9198-7