Fixation of Chattonella antiqua and C. marina (Raphidophyceae) using Hepes-buffered paraformaldehyde and glutaraldehyde for flow cytometry and light microscopy

Chattonella antiqua and C. marina are harmful algal species that cause massive Fish kills in coastal environments. Generally, Chattonella cells cannot be preserved well by fixation because of their fragile nature. In the present study, we developed a new fixative for Chattonella. Phosphate-buffered saline, which is generally used as it buffer for Fixative, caused precipitation after fixation. In contrast, Hepes or sodium cacodylate prevented the precipitation. Moreover, these buffers worked well in the fixation to preserve cell morphology. Since Hepes is not as noxious its cacodylate, we selected Hepes as the buffer for the fixative. Cell counting revealed that the decrease in cell numbers by fixation was negligible and did not start until at least 8 days after fixation. We successfully analysed the DNA amount in fixed cells of Chattonella by flow cytometry. The present Study demonstrated that Hepes-buffered paraformaldehyde and glutaraldehyde are superior to other fixatives for flow cytometry and light microscopy.