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J Microbiol Methods. 2000 Aug;41(3):249-57.

Different methods for extracting bacteria from freshwater sediment and a simple method to measure bacterial production in sediment samples.

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Lab. de Limnologia, Depto. de Ecologia, Inst. de Biologia, Universidade Federal do Rio de Janeiro, Cidade Universitária, Ilha do Fundão, 21.941-590, Rio de Janeiro, Brazil.


The efficiency of different treatments was tested to extract bacterial cells from freshwater sediment samples. The influence of sonication, density gradient centrifugation, fixation by formalin and centrifugation speed on bacterial recovery was investigated. The method developed by Smith and Azam [Mar. Microb. Food Webs 6 (1992) 107] to measure microbial activity on bacterioplankton (3H-leucine incorporation), was also evaluated in sediment samples. After 1 min of sonication bacterial abundance was reduced by about 47% in diluted sediments with tetrasodium pyrophosphate. With the addition of Percoll after sonication, bacterial counts were not significantly different (P<0.05). Fixation by formalin increased bacterial counts using sonication. However, higher bacterial abundance was estimated in non-sonicated samples. Bacterial abundance in samples centrifuged at 7000xg with and without Percoll was not significantly different (P<0.05). Highest bacterial abundance was obtained after centrifugation at low speed (750xg). Bacterial abundance decreased with higher centrifugation speed (750, 1500 and 3000xg), the difference, however, was not significant. Bacterial production ranged from 0.10 microg C cm(-3) d(-1) in autoclaved sediment to 0. 27 microg C cm(-3) d(-1) in untreated sediment. The radioactivity measured in controls of both untreated and autoclaved sediment was high (70 and 91%, respectively), indicating a high level of leucine adsorption in sediment particles. In contrast, radioactivity in control samples previously centrifuged was markedly lower (6%). Despite the high values of radioactivity in the controls, bacterial production in untreated sediment was significantly higher than in centrifuged sediment (P<0.05).

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