Bioassay procedure for the evaluation of erythromycin activity in aquaculture environments

A new bioassay procedure was developed for the detection of erythromycin in aquaculture samples using a strain of a Stenotrophomonas as an indicator organism. Conventional disk‐plate and well‐plate radial diffusion assay procedures were developed, as well as a third procedure using the same indicator organism in Luria‐Bertani (LB) broth, supplemented with the indicator dye Brilliant Black (40 μg/mL) in a multi‐well microtiter plate. For both the disk‐plate and well‐plate radial diffusion assays, the response reflected in the size (width) of the growth inhibition zone, which was linear over the tested concentration range of 0.05 to 2.0 μg erythromycidml. The limit of quantitation of the bioassay was at 0.05 μg erythro‐mycidml. Among the three methods of assay tested with Stenotrophomonas sp., the semi‐quantitative dye reduction method is easy to read and is not diffusion dependent. This method allows for processing of more samples and more replication on a single titer plate. This new indicator organism is specific for erythromycin when tested in the presence of other antibacterial agents, i.e., oxytetracycline (Terramycin®) and/or Romet‐Ma. This new bioassay procedure is suitable for quantitation of low concentrations of erythromycin in aquaculture water and sediment samples.