Novel techniques for the short-term culture and laboratory study of Didymosphenia geminata

Techniques for assessing the freshwater diatom Didymosphenia geminata in the laboratory under controlled conditions are described. Two methods for harvesting free, unattached cells from mats, and two methods for assessing cells were developed. Free cells were harvested from mats up to 28 days old using either a static method or a shaking method. Harvested cells were then assessed using two multi-well formats: a multiple- followed by a single-cell method. The multiple-cell method was found to be a rapid screening tool for fine-tuning parameter ranges to be assessed in the single-cell assay. The single-cell method, through careful monitoring, allowed quantitative assessment of cell processes. Attachment to a substratum was observed to be critical for survival and the division of free D. geminata cells. Although cells attached readily in the multiple-cell method, the inclusion of a hydrophobic substratum was essential for attachment to occur reliably when using the single-cell method. The improvement of the single-cell method through inclusion of a hydrophobic substratum was investigated, and results with and without the substratum were compared. A temperature of 18 degrees C was found to be more suitable than 12 degrees C for conducting the assay and the optimal pH range for the assay was found to be 7.6 to 8.2.