Bromodeoxyuridine as an alternative to H-3-thymidine for measuring bacterial productivity in aquatic samples

Measuring bacterial productivity with radiolabeled substrates such as triti ated thymidine (H-3-TdR) poses logistical difficulties and has high associa ted costs due to strict regulations on the transport, use, and disposal of radioactivity. The TdR analog 5-bromo-2'-deoxyuridine (BrdU) can be detecte d immunochemically and has been used for many years as a non-radioactive al ternative for measuring DNA synthesis in cultures. The goal of this study w as to determine whether a non-radioactive immunoassay for BrdU could be use d to quantitatively measure bacterial productivity in natural aquatic sampl es. The first step was to determine the relative reliability of BrdU incorp oration as an indicator of DNA synthesis in natural communities. Incorporat ion rates of H-3-BrdU and H-3-TdR in samples of coastal seawater and a fres hwater lake were found to be highly correlated (r = 0.98, n = 50, p < 0.000 1) with an average BrdU:TdR incorporation ratio of 0.71 +/- 0.24 (mean i- S D). The results indicated that, despite an apparent kinetic discrimination, BrdU could accurately predict TdR incorporation over a wide range of bacte rial productivity (0.45 to 349 pmol TdR l(-1) h(-1)). A filter-based chemil uminescent immunoassay was then developed and used to estimate BrdU incorpo ration in natural seawater and freshwater samples non-radioactively. Estima ted rates of BrdU incorporation were within 0.5 to 30% of H-3-TdR incorpora tion rates. The assay showed a linear chemiluminescent response spanning at least 1.5 orders of magnitude and a detection limit of less than or equal to 7 fmol of incorporated BrdU. These results suggest that a BrdU-based imm unoassay has the potential to serve as a simple, sensitive, and quantitativ e non-radioactive alternative to H-3-TdR for routine measurements of bacter ial productivity in the field or laboratory.