AME 19:57-66 (1999)  -  doi:10.3354/ame019057

ABSTRACT: Measuring bacterial productivity with radiolabeled substrates such as tritiated thymidine (³H-TdR) poses logistical difficulties and has high associated costs due to strict regulations on the transport, use, and disposal of radioactivity. The TdR analog 5-bromo-2'-deoxyuridine (BrdU) can be detected immunochemically and has been used for many years as a non-radioactive alternative for measuring DNA synthesis in cultures. The goal of this study was to determine whether a non-radioactive immunoassay for BrdU could be used to quantitatively measure bacterial productivity in natural aquatic samples. The first step was to determine the relative reliability of BrdU incorporation as an indicator of DNA synthesis in natural communities. Incorporation rates of ³H-BrdU and ³H-TdR in samples of coastal seawater and a freshwater lake were found to be highly correlated (r = 0.98, n = 50, p < 0.0001) with an average BrdU:TdR incorporation ratio of 0.71 ± 0.24 (mean ± SD). The results indicated that, despite an apparent kinetic discrimination, BrdU could accurately predict TdR incorporation over a wide range of bacterial productivity (0.45 to 349 pmol TdR l^-1 h^-1). A filter-based chemiluminescent immunoassay was then developed and used to estimate BrdU incorporation in natural seawater and freshwater samples non-radioactively. Estimated rates of BrdU incorporation were within 0.5 to 30% of ³H-TdR incorporation rates. The assay showed a linear chemiluminescent response spanning at least 1.5 orders of magnitude and a detection limit of <=7 fmol of incorporated BrdU. These results suggest that a BrdU-based immunoassay has the potential to serve as a simple, sensitive, and quantitative non-radioactive alternative to ³H-TdR for routine measurements of bacterial productivity in the field or laboratory.

KEY WORDS: Bacterial production · Tritiated thymidine · Bromodeoxyuridine · Non-radioactive · Immunoassay · Method