Rapid quantification of viable cells of the planktonic diatom Chaetoceros tenuissimus and associated RNA viruses in culture

Abstract

To understand the ecological relationships between diatoms and viruses in marine environments, extensive culture experiments under diverse environmental conditions are essential. Previous methods developed for enumeration of diatom cells and viruses in culture experiments, i.e., manual counting using a microscope and the most probable number (MPN) method, are time consuming and labour intensive. In this study, we developed a rapid enumeration method for cells of the planktonic diatom Chaetoceros tenuissimus and the genome copy numbers of the RNA virus CtenRNAV using image-based cytometry (IBC) and real-time quantitative polymerase chain reaction (qPCR), respectively. The live cell numbers counted using IBC were similar to those obtained by direct microscopic counts within the range from 10⁴ to 10⁶ mL⁻¹. The genome copy numbers of the viruses, as determined by qPCR, were almost 2–20 times higher than those determined by the MPN method owing to the presence of defective particles and virion aggregations in the samples. The dynamic patterns of virus abundances were similar for both methods; therefore, qPCR could be applied as an alternative method for enumeration of infectious virus units. The IBC and qPCR methods developed for diatom cells and viruses in this study were rapid and accurate and should therefore have applications as powerful tools for improving our understanding of diatom-virus ecological relationships.