Flow cytometry in fungi: comparison of method used

The use of FCM in zoology or plant research has long tradition and many of studies that compare methodical approaches have been published until now. The routine application of FCM in genome size estimation of microorganism is limited by methodical problems linked to their small genome size. In contrast to plant research, only a few methodical publications exist for the mycology field, and reviews are absent. Another problem is the absence of suitable standards to determine absolute genome size of fungi. Thus it is of high priority to compare methodical approaches used in flow cytometry studies in mycology and to find a new standard for FCM with fungi.
Conclusion:
Our results demonstrate the enormous effects of the methods used for FCM on genome size estimation. Fixation with methanol:glacial acetic acid (3:1 v/v), 10% DMSO, 0.1% Triton-X 100, and 5 mM EDTA in combination with Tris-MgCl2 buffer was selected as the best treatment. Aspergillus fumigatus CEA10 was found to be an appropriate standard for genome size estimation in fungi. CS&T beads (Becton Dickinson, New Jersey, USA) were used for application settings and quality control. Laser delay and laser alignment was calibrated by AlignFlow™ flow cytometry alignment beads, 2.5 μm for 488 nm excitation (Invitrogen, Eugene, USA).