TITLE: Methodology used to examine bacterial abundance and production (leucine incorporation) RELEASE DATE May 17, 2005 AUTHOR(S):David Kirchman College of Marine 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 GENERAL COMMENTS 1. The term "bacteria" is used here, but the methods cannot distinguish bacteria from archaea or even very small (<1 um) eukaryotes. However, other data from fluoresencence in situ hybridization studies indicate that most of the cells in these samples are bacteria. 2. The samples were taken from CTD casts usually also used for 14C- primary production measurements. Bacterial abundance 1. Bacterial abundance was determined by direct epifluorescence microscopy using DAPI staining (Porter and Feig 1980). 2. Samples were preserved within 3 h of sampling in 2% (final concentration) formaldehyde overnight at 4 C before filtration. 3. Preserved seawater was filtered through polycarbonate filters and then the filter with sample was stained with DAPI. The volume varied with depth, from about 20 ml (surface) to >100 ml for deep samples to ensure adequate numbers of cells on the filter. 4. The filter was mounted on a glass slide as described by Cottrell and Kirchman (2003), and stored at -20 C until microscopic analysis. 5. Cells were then counted by epifluorescence microscopy using a semi-automated procedure (Cottrell and Kirchman 2003). 6. The mean and STD were calculated for each sample based on 10 microscopic fields of view. Bacterial production (leucine incorporation) 1. Leucine incorporation was used to estimate bacterial production (Kirchman et al. 1985). 2. Within one hour of subsampling from the CTD bottle, incubations for leucine incorporation were started, using the microcentrifuge method (Smith and Azam 1992). The final concentration of 3,4,5 3H-leucine was 20 nM. 3. Incubations were done within 0.5 C of the in situ temperature for about 2 hours. 4. Incubations were terminated by the addition of 5% trichloroacetic acid (TCA) (final concentration). 5. Samples were rinsed with TCA and then 80% ethanol. 6. After drying over night, scintillation cocktail (Ultima Gold) was added. 7. The samples were radioassayed by scintillation counting after 2-3 days. 8. The mean and STD of three replicates were calculated. REFERENCES 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. Kirchman, D. L., E. K'nees, and R. E. Hodson. 1985. Leucine incorporation and its potential as a measure of protein synthesis by bacteria in natural aquatic systems. Appl. Environ. Microbiol. 49: 599-607. Porter, K. G., and Y. S. Feig. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943-948. Smith, D. C., and F. Azam. 1992. A simple, economical method for measuring bacterial protein synthesis in seawater using 3H-leucine. Marine Microbial Food Webs 6: 107- 114.