some marine bacteria. Modifications to the methodology overcome or circumvent some of 

 these problems, but uncertainty remains. Consequently, additional and independent indicators 

 of bacterial growth are needed. Leucine incorporation into protein has been used as a measure 

 of protein synthesis and bacterial biomass production, and is rapidly becoming a standard 

 method. A second alternative measurement is the use of dilution culturing, in which unfiltered 

 seawater is mixed with filtered seawater. This procedure decreases the efficiency of grazing 

 by microzooplankton in proportion to the dilution factor. By measuring the growth of bacteria 

 at various dilutions, it is possible to extrapolate to the theoretical condition of bacterial growth 

 in the complete absence of grazing. The primary disadvantage is that the incubations are 

 lengthy and subsequent analyses are labor-intensive, so that dilution culturing can only be used 

 for a small subset of all possible stations and depths sampled. Simultaneous measurements of 

 different combinations of these independent indices have been used successfully in marine 

 environments, and are proposed for studies of bacterial growth and production in the ocean 

 margin program. 



In addition to the above-described methods for determining bacterial production, an 

 entirely new approach using ribosomal RNA content and rRNA- specific oligonucleotide probes 

 will be used to estimate protein synthetic capacity and growth. Data from culture studies with 

 marine bacteria suggest that rRNA content measurements may estimate the community-average 

 growth rates as well as more conventional methods. Furthermore, the methodology can be 

 taxon-specific and cell-specific. At the species-specific level, rRNA content measurements 

 appear to be near-perfect predictors of growth rate when the rRNA-growth relationship is 

 determined empirically. The frequency distribution of rRNA among cells is the frequency 

 distribution of protein synthetic capacity and under appropriate conditions can be interpreted as 

 the frequency distribution of growth rate. Prior to the field year, DNA hybridization methods 

 will be employed to identify a subset of bacteria that dominate biomass and abundance in 

 coastal water and sediment of the study area. Probes specific to these dominants will be 

 constructed and used to establish empirical RNA-growth rate relationships for these bacteria in 

 axenic or mixed-community "natural" cultures (it is easier, but not necessary to isolate the 

 bacteria in axenic cultures). The goal is to develop and apply the capability to evaluate the 

 population dynamics of these dominant bacterial taxa in response to changes in environmental 

 conditions. It is important to recognize that a significant portion of the microbial activity 

 occurs in the bottom sediments. Because of uncertainties related to tracer adsorption onto 

 sediment surfaces, interference by particles in optical microscopy, and steep and easily 

 disturbed chemical and biological stratification, determining microbial activities and metabolic 

 rates in sediments represents a particularly difficult challenge. Chemical flux incubations must 

 be employed in conjunction with the more problematic tracer-based biomass production 

 techniques to quantitatively consttain the role of the sediment microbial community in the 

 shelf carbon cycle. 



During the OMP field year, bacterial abundance and production will be determined 

 routinely in water and sediment samples recovered along transects of the study area during 

 four seasonal cruises. Rates of DNA and protein synthesis, and rRNA content, will be 

 measured as independent indicators of bacterial production. A broad spatial coverage of the 

 study area, including vertical profiles, will be obtained so that estimates of heterotrophic 

 bacterial carbon production and mineralization can be compared with estimates of primary 



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