Comparison of staining techniques for scanning electron microscopic detection of ultrastructural protuberances on cellulolytic bacteria

Cell surface protuberances found on cellulolytic bacteria, but not on noncellulolytic bacteria, can be detected by scanning electron microscopy. Cationized ferritin typically has been used as a stain to increase the microscopic resolution of these protuberances; however, as a cation it binds only to negatively charged molecules. Thus, binding of cationized ferritin to cell surface molecules can be affected by the cell's physiological state, We incubated the noncellulolytic bacterium, Clostridium beijerinckii, in different media at various temperatures to obtain cells with different growth rates and physiological states. Staining of these cells with cationized ferritin showed that slower growing cells exhibited more protuberant structures than faster growing cells. This provided a clear correlation of ultrastructural protuberances with physiological changes associated with growth rate. On the other hand, cells stained with osmium tetroxide exhibited no protuberant structures regardless of growth rate. Because various cations are known to induce aggregation of surface proteins on some cellulolytic Clostridia, we incubated the cellulolytic bacterium, Clostridium cellulovorans, in media containing glucose, cellobiose, or cellulose. Ultrastructural protuberances were evident on all cells stained with cationized ferritin, but extensive protuberances were detected only on cells grown in cellulose and stained with osmium tetroxide. For cells stained with cationized ferritin, the presence of ultrastructural protuberances was correlated with growth rate rather than induction of cellulolytic systems. By contrast, cells stained with osmium tetroxide showed a clear correlation between protuberant structures and cellulolytic activity.