2023 Volume 70 Issue 1 Pages 1-9
The water freeze-drying method can be used to prepare samples for scanning electron microscopy (SEM) in which samples immersed in water are frozen and dried under vacuum. This method does not require chemical fixation of freshwater microorganisms, whose structures are easily disrupted during fixations, and mixed samples of different species can be processed simultaneously with minimal morphological damage. We optimised this method for plankton surveys using SEM. A membrane filter with a plastic frame was attached to the filtration device, and microorganisms in the collected water were filtered and concentrated. Immediately before the end of filtration, the framed filter was removed from the filtration device, whereas the concentrated microorganisms remained covered with water. The wet filter was frozen by contact with a cooled metal plate and vacuum-dried. After conductive coating of the filter, the microorganisms on the filter were identified and counted using SEM. This method is more time-consuming compared to conventional optical microscopy (OM) methods and provides no information on colours or internal structures, making species identification difficult. However, the number of microorganisms detected was two- to three-fold lager than that detected using OM, including some species that could not be detected using OM. This result can be explained as follows: there was almost no loss of microorganisms larger than the filter pore size, and the resolution of SEM is higher than that of OM. A further advantage of this method is that the measured samples can be stored for long periods without morphological damage, and the measurement results can be re-validated at a later date.
