An alternative SEM drying method using hexamethyldisilazane (HMDS) for microbial cell attachment studies on sub-bituminous coal

The use of hexamethyldisilazane (HMDS) as a drying agent was investigated in the specimen preparation for scanning electron microscopy (SEM) imaging of bacterial surface colonization on sub-bituminous coal. The ability of microbes to biofragment, ferment and generate methane from coal has sparked interest in the initial attachment and colonization of coal surfaces. HMDS represents an attractive alternative to critical point drying (CPD) in the imaging of cells on coal, negating the need for expensive equipment. Coal is easily fragmented into sub-micron particles, which can be problematic in critical point drying procedures. In this study, both individual and aggregated cells appeared well shaped with minimal occurrence of flattened cells, signifying the suitability of HMDS in cell attachment studies on sub-bituminous coal. In the absence of glucose, microcolonies of short and long cells showed similar positive results using this method. EPS shrinkage found in microcolonies was inevitable, though this enabled observation of points of attachment between cells and with coal, which would be less effective if the EPS was intact. Overall the use of HMDS drying is preferred over the more commonly used CPD method as it is safer, cheaper and more practical.