L. Bernard et al., A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems, CYTOMETRY, 43(4), 2001, pp. 314-321
Background: Discrimination among viable, active, and inactive cells in aqua
tic ecosystems is of great importance to understand which species participa
te in microbial processes. In this study, a new approach combining flow cyt
ometry (FCM), cell sorting, and molecular analyses was developed to compare
the diversity of viable cells determined by different methods with the div
ersity of total cells and active cells.
Methods: Total bacteria were determined by SYBR-II staining. Viable bacteri
a were determined in water samples from different sites by plate count tech
niques and by the direct viable count (DVC) method. Substrate-responsive ce
lls (i.e., DVC+ cells) were distinguished from nonresponsive cells (i.e., D
VC- cells) by FCM and sorted. The genetic diversity of the sorted cell frac
tion was compared with the diversity of the total microbial community and w
ith that of the culturable cell fraction by denaturing gradient gel electro
phoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rDNA fragm
ents. The same approach was applied to a seawater sample enriched with nutr
ients. In this case, actively respiring cells (CTC+) were also enumerated b
y FCM, sorted, and analyzed by DGGE.
Results: The diversity of viable cells varied depending on the methods (tra
ditional culture or DVC) used for viability assessment. Some phylotypes det
ected in the fraction of viable cells were not detectable at the community
level (from total DNA). Similar results were found for actively respiring c
ells. Inversely, some phylotypes found at the community level were not foun
d in viable and active cell-sorted fractions. It suggests that diversity de
termined at the community level includes nonactive and nonviable cells.
Conclusion: This new approach allows investigation of the genetic diversity
of viable and active cells in aquatic ecosystems. The diversity determined
from sorted cells provides relevant ecological information and uncultured
organisms can also be detected. New investigations in the field of microbia
l ecology such as the identification of species able to maintain cellular a
ctivity under environmental changes or in the presence of toxic compounds a
re now possible. Cytometry 43:314-321, 2001. (C) 2001 Wiley-Liss. Inc.