The preservation of surface-associated micro-organisms prepared for scanning electron microscopy

Scanning electron microscopy (SEM) has become a popular means of studying micro-organisms which associate with surfaces. However, as yet no detailed examination has been made of the influence of specimen preparation on the number of organisms finally seen on the SEM screen. In this investigation critical assessment is made of the influence of a wide range or preparative factors on the preservation of filamentous bacteria associated with the epithelial surfaces of rat intestine. Organisms were quantitated using a rigorous counting method (transect line analysis); statistical testing of these counts enabled the comparison of different preparative factors. The composition of the fixative was found to significantly influence the number of organisms preserved; of the fifteen fixatives studied, Karnovsky's fixative with ruthenium red best preserved surface-associated organisms. The influence of other factors on the number and appearance of preserved organisms was also examined. These factors included the washing of specimens prior to fixation, the storage of fixed specimens, and the handling and storage of critical point dried specimens. The results are discussed with reference to the optimal methods for preparing specimens for SEM.