TITLE: SBI Bacteria Archaea RELEASE DATE December 12, 2005 AUTHOR(S): David Kirchman College of Marine and Earth Studies University of Delaware Lewes, DE 19958 302-645-4375 302-645-4028 (fax) kirchman@udel.edu FUNDING SOURCE AND GRANT NUMBER National Science Foundation OPP-0124733 DESCRIPTION This data file contains the data on prokaryotic community stricture, including the abundance of bacteria and archaea determined by fluorescence in situ hybridization (FISH) collected by Kirchman's group. TIME PERIOD SBI Spring and Summer Cruises 2002 and 2004 GENERAL COMMENTS Method used to examine prokaryotic community structure using fluorescence in situ hybridization (FISH): 1. The term "total prokaryotes" is used here to include all those cells identified with a general nucleic acid stain. "Bacteria" and "archaea" refer to cells identified by fluorescence in situ hybridization using probes specific for those groups. 2. The samples were taken from CTD casts usually also used for 14C-primary production measurements. Fluorescence in situ hybridization (FISH) 1. Samples were preserved within 3 h of sampling in 2% (final concentration) paraformaldehyde overnight at 4 C before filtration. 2. Preserved seawater was filtered through polycarbonate filters. The volume varied with depth, from about 20 ml (surface) to >100 ml for deep samples to ensure an adequate number of cells on the filter. 3. The filter was stored at -20 C until analysis in the lab at the University of Delaware. 4. Samples were analyzed by fluorescence in situ hybridization with Cy3-labeled oligonucleotide probes using hybridization buffer, wash buffer and temperature described in Kirchman et al. (2007). In brief, samples were prepared for FISH by incubating overnight with a hybridization solution containing the FISH probe. Unbound probe was then removed by incubating the sample in a wash buffer. Finally, the sample was stained with the general nucleic acid stain, DAPI (Porter and Feig, 1980), and then mounted on a glass slide. 5. Cells were then counted by epifluorescence microscopy using a semi-automated procedure (Cottrell and Kirchman 2003) to identify total prokaryotes stained with DAPI and probe-positive cells stained with Cy3. 6. FISH results were expressed as percentage of total prokaryotes and abundance (cells/mL) calculated by multiplying the percentage by the total prokaryote abundance. The mean and SD were calculated for each sample based on 10 microscopic fields of view. REFERENCES Kirchman, D. L., Elifantz, H, Dittel, A. I., Malmstrom, R. R. and M. T. Cottrell (2007) Standing stocks and activity of Archaea and Bacteria in the western Arctic Ocean. Limnol. Oceanogr. 52: in press Cottrell, M. T., and D. L. Kirchman. 2003. Contribution of major bacterial groups to bacterial biomass production (thymidine and leucine incorporation) in the Delaware Estuary. Limnol. Oceanogr. 48: 168-178. Porter, K. G., and Y. S. Feig. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943-948. 2