Quantifying substrate uptake by individual cells of marine bacterioplankton by catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography.
Catalyzed reporter deposition fluorescence in situ hybridization combined with microautoradiography (MICRO-CARD-FISH) is increasingly being used to obtain qualitative information on substrate uptake by individual members of specific prokaryotic communities. Here we evaluated the potential for using this approach quantitatively by relating the measured silver grain area around cells taking up (3)H-labeled leucine to bulk leucine uptake measurements. The increase in the silver grain area over time around leucine-assimilating cells of coastal bacterial assemblages was linear during 4 to 6 h of incubation. By establishing standardized conditions for specific activity levels and concomitantly performing uptake measurements with the bulk community, MICRO-CARD-FISH can be used quantitatively to determine uptake rates on a single-cell level. Therefore, this approach allows comparisons of single-cell activities for bacterial communities obtained from different sites or growing under different ecological conditions.
Sintes
Eva
E
Department of Biological Oceanography, Royal Netherlands Institute for Sea Research (NIOZ), P.O. Box 59, 1790 AB Den Burg, Texel, The Netherlands. esintes@nioz.nl
Herndl
Gerhard J
GJ
D000818:Animals; D001345:Autoradiography / Q000379:methods*; D001419:Bacteria / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D002384:Catalysis; D003589:Cytophaga / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D041965:Flavobacteriaceae / Q000166:cytology / Q000254:growth & development / Q000378:metabolism; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007211:Indoles; D007930:Leucine / Q000378:metabolism*; D015345:Oligonucleotide Probes; D010933:Plankton / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D012623:Seawater / Q000382:microbiology*
D005456:Fluorescent Dyes; D007211:Indoles; D015345:Oligonucleotide Probes; C007293:DAPI; D007930:Leucine