Suitability of the thawed algae for transmission electron microscopy study: Ultrastructural investigation on Coccomyxa melkonianii SCCA 048

Morphological and ultrastructural investigations are crucial for the identification and characterization of species such as microalgae, microorganisms that greatly change their morphology and physiology during their life cycle. Transmission electron microscopy (TEM) is an excellent tool for the ultrastructural observation of cells and their components. To date, limited ultrastructural studies have been carried out on microalgae, due to the difficulties in sample preparation. The aim of this work is to establish an appropriate fixation method that allows to better preserve the algal ultrastructure and test the suitability of the thawed algae for TEM observation. Fresh and thawed algae (Coccomyxa melkonianii SCCA 048) were fixed with different TEM fixation methods (a mix of glutaraldehyde and paraformaldehyde for several incubation times, sometimes preceded by a prefixation in cold methanol). The ultrastructural images obtained from fresh algae were compared to those obtained from frozen biomass. The best morphological results were achieved by fixing fresh algae in 1% paraformaldehyde and 1.25% glutaraldehyde for 5 hr. Pretreating with frozen methyl alcohol reduced fixation time to 2 hr. Both fresh and frozen algae ultrastructure were rather well preserved also with 1% paraformaldehyde and 1.25% glutaraldehyde for 2 hr. Ultrastuctural morphological images of the thawed algae demonstrated that also frozen samples can be used in TEM research, widening specimen suitability by means of this technique.