Inter-laboratory validation of the serial dilution culture—most probable number method for enumerating viable phytoplankton

The most probable number (MPN) assay is used to enumerate viable phytoplankton in the 10–50 μm size range for Type Approval of ballast water management systems. However, its validation has been questioned by the United States Coast Guard, which does not accept it as an alternative to its required method. To address key elements of method validation, three research laboratories used the same experimental protocols to enumerate viable organisms from uniformly viable and heat-killed cultures of three species of phytoplankton. Statistical analysis of the ratio of MPN estimates of viable organisms to measurements from flow cytometry was consistent with predictions based on perfect adherence to the method’s assumptions. These include reliable detection of all viable organisms and no false-positive results. The validation confirmed established estimates of the MPN method’s precision and demonstrated reproducibility, having been transferred to and implemented by two of the laboratories for the first time.