A simple and highly efficient fixation method for Chrysochromulina polylepis (Prymnesiophytes) for analytical flow cytometry

BACKGROUND: To study the fragile Prymnesiophyte species Chrysochromulina polylepis by flow cytometry (FC), we needed an effective fixation method. This method must guarantee a high yield of fixed cells to achieve acceptable measurement times by FC and to allow quick processing of many samples. Moreover, we wanted a method that allows for storage of fixed samples when FC analysis cannot be done immediately. METHODS: Different aldehydes and methanol were tested at different final concentrations. Gravity sedimentation and centrifugation were applied to achieve higher cell concentrations. Storage of fixed samples was tested under different conditions. RESULTS: 0.25% glutaraldehyde (GA) fixation yielded a recovery rate of about 90%. The signals obtained by FC analysis were excellent. It is possible to centrifuge GA-fixed cells and to store them for several weeks. CONCLUSIONS: GA is the fixative of choice for FC analysis of C. polylepis (and possibly other small delicate species) because it yielded highly significant recovery rates and high-quality FC signals. Cells can be centrifuged to increase the cell concentration, thereby achieving short measurement times with FC. The possibility of long-term storage of fixed cells presents an additional advantage if FC analysis cannot be done immediately.