Evaluation of cell activity and of methods for the cultivation of bacteria from a natural lake community

The viability and culturability of chemoheterotrophic bacteria in Zwischenahner Meer, a shallow eutrophic lake in Northern Germany, were assessed by different techniques. The fraction of metabolically active cells was measured by the CTC reduction method. At three sampling times, numbers of CTC-reducing cells represented only a minor fraction (less than or equal to 8%) of the total cell count. Only a slight stimulation of CTC reduction was observed upon the addition of single carbon substrates or substrate mixtures. A medium mimicking the low natural ion concentrations in freshwater was devised and used for the cultivation of bacteria in most probable number (MPN) series and on membrane filters. Similar to the number of CTC-reducing cells, the number of culturable cells as determined in MPN series never exceeded 7% of the total cell count with any of the carbon substrates tested. This indicated that the type of carbon substrate was not the major determinant of the rather low culturability. However, we observed a significant inhibition of bacterial growth in MPN series in which the typical R2A medium was used, or when phosphate buffer instead of HEPES was employed. In contrast to the low numbers of CTC-reducing cells and the low MPN values, up to 58% of the bacterial cells could divide on membrane filters incubated on top of the same type of medium that had been used in the MPN series. It is concluded that most of the bacteria in Zwischenahner Meer are able to grow and divide, but do not reach high cell densities even in improved culture media. Therefore, the majority of bacteria may be classified as viable but nonculturable, but would not be detected by the CTC reduction method. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.