Method for hydrophytic plant sample preparation for light and electron microscopy (studies on Phragmites australis Cav.)

Nowadays there are no well-established, standard methods in electron microscopy despite its 50-year history. An excessive variety of research objects prompt researchers to modify and improve methodological approaches to sample preparation. One of the difficult objects to study by electron microscopy is hydrophytic plants, for example, Phragmites australis Cav. Traditional approaches to fixation and sample preparation do not give satisfactory results due to the peculiarities in structure and physiology of hydrophytic plants. The purpose of present research is modification description of the widespread method developed for double fixation of hydrophytic plant tissue for transmission electron microscopy. Suggested approach takes into account the features of hydrophyte plants. • The developed method allows improving the quality of plant samples by additional fixatives imbibition and removing of air bubbles from aerenchyma tissue using a vacuum. • The new step of sample preparation consisting in the layer-by-layer sample mixing in a special inclined mixer is applied for the embedding media penetrate sufficiently into the sample tissue. • The process of samples inclusion in polymeric resins is carried out in the flat-bottom capsules. Compare to standard conical capsules, flat-bottom capsules allow strictly defined orientation sample pieces, that is permit to produce a semi-thin and ultra-thin slices of perpendicular to the longitudinal structures of the plant. This is especially important to conduct an adequate comparative analysis of dimensions, shape, and electron density of fragments and parts of the studying samples.