Estimates of bacterial production using the leucine incorporation method are influenced by differences in protein retention of microcentrifuge tubes

Michael L. Pace, Paul del Giorgio, David Fischer, Robert Condon, Heather Malcom

Limnol. Oceanogr. Methods 2:55-61 (2004) | DOI: 10.4319/lom.2004.2.55

ABSTRACT: The most widely used methods to determine bacterial production involve measuring the incorporation of radioactive precursors such as leucine into macromolecular pools. The leucine method that involves incubation and extraction within a single microcentrifuge tube has become a widely used technique because of its relative convenience, precision and low cost. We observed a discrepancy in parallel determinations of leucine incorporation for the same water samples that lead us to explore aspects of the method including tube washing methods, operator differences, and differences among tube brands. Operators and washing methods had minimal effects on rate measurements but results were strongly dependent on tube brands. Differences in tube performance were observed consistently in comparisons from a variety of freshwater and marine environments. Microcentrifuge tubes differed in protein retention with the consequence that estimates of leucine incorporation in a given sample can vary by as much as 60% depending on the tube used. There was no simple relationship between tube plastics or manufacturer and tube performance. We advise researchers to check the protein retention of tubes and to use the same brand of tube during field studies to minimize this potential source of variation.