Studies on the cell surface of zoospores and cysts of the fungus Phytophthora cinnamomi: The influence of fixation on patterns of lectin binding

Abstract

The study of the surface properties of zoospores and cysts of the fungus Phytophthora cinnamomi required a fixation regimen that would preserve the cells adequately and not interfere with binding and detection of probes on the cell surface. When they were fixed in 4% formaldehyde (F), specific binding of concanavalin A-fluorescein isothiocyanate and rhodamine-labeled soybean agglutinin was obtained. However, electron microscopy showed that preservation was so poor that intracellular binding sites had become exposed. By contrast glutaraldehyde (G), even at concentrations as low as 0.05%, gave good preservation of the zoospores but induced high levels of nonspecific fluorescence, making its use impractical for studies using fluorescent probes. Addition of 1-4% F to 0.05-0.8% G reduced the level of G-induced fluorescence while not diminishing the quality of ultrastructural preservation. This effect was evaluated quantitatively and an optimum fixation regime for the fungal cells, namely, 0.2% G and 2-4% F in 50 mM PIPES buffer, was determined. This combined fixative facilities correlated fluorescence and ultrastructural labeling with lectins and immunocytochemical probes.