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

Abstract

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 (98%) 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. z

Multitude and temporal variability of ecological niches as indicated by the diversity of cultivated bacterioplankton

“…We chose a shallow eutrophic lake with a simple hydrodynamic structure for our investigations. Water samples were obtained from the head of a 20 m long pier located on the east shore of Zwischenahner Meer, a small eutrophic lake in northern Germany [16]. Zwischenahner Meer is a shallow lake (mean depth, 3.3 m).…”

“…Samples from 23 October 1997, 14 April 1998, and 20 May 1998 were used for the isolation of chemoheterotrophic bacteria. Tenfold serial dilutions of the water samples were plated onto solidified (1.2% agar) complex medium YPG which contains 0.075% (w/v) yeast extract, 0.15% (w/v) peptone, and 0.15% (w/v) glucose [16]. After 3 weeks of incubation at 20°C, the colonies from the highest positive dilutions were picked and restreaked at least three times until pure cultures had been obtained.…”

“…Synthetic freshwater [16] supplemented with low concentrations of organic substrates served as growth medium. Three different mixtures of carbon substrates were used: (1) low concentration YPG [16], (2) a fatty acid mixture containing formate, acetate and propionate (200 μM each), and (3) a mixture containing the seven amino acids alanine, arginine, asparagine, isoleucine, cysteine, tyrosine, glutamine (200 μM each), as well as glucose, pyruvate, citrate, 2‐oxoglutarate, succinate (200 μM each), Tween 80 (0.001% v/v) and the fatty acid mixture. The MPN series were set up in 10‐fold serial dilutions and in three parallels within polystyrene microtiter plates (Greiner, Frickenhausen, Germany).…”

Effectiveness of CSE to counterstain particles and dead bacterial cells with permeabilised membranes: application to viability assessment in waters

“…This fraction is much more important within marine communities and was generally confirmed by the DVC method. Although the viability of these active cells cannot be addressed by these direct techniques, there is more and more evidence that viable counts determined by cultural techniques may be underestimated, mainly in environments where bacteria are subjected to stressful conditions [27].…”

Pilot Test of Indigenous Microorganism Flooding in a Heavy Oil Reservoir

“…The number of bacteria of various physiological groups in the formation waters was determined by the serial dilution technique in duplicate by the most-probable-number method [5]. All media were self-prepared [6,7].…”