High-Resolution sem and Clostridial Attachment

Abstract

Since the mid 1960’s when the TEM was a new and primary tool for elucidating the classification of the various strains of microorganisms, investigators around the world have been reporting morphological surface aberrations (such as hairs, ribbons, tubular appendages and feather-like structures) of the clostridial endospore that facilitate and verify the classification of these organisms. Yolton Pope, Samsonoff and others were unable to discern the function, of these appendages and stated that the “functional role of spore appendages remains obscure”.Today, there is still no information on the functional role, if any, of these “appendages” and “hairs”. For the past few years we have been examining the endospores from two clostridial species that are pathogenic in man and higher mammals,C.sporogenesATCC3584, andC.difficileATCC 9689and43594. These organisms cause wound infections, pseudomembranous colitis, and nosocomial diarrhea. Our previous work strongly suggested that the outer envelope of these spores, the exosporium, had a “morphological plasticity” that allowed the spores to attach to a surface that supported bacterial growth5,6. With current advances in high resolution SEM using LaB6 and field emission sources, it is now possible to study these extremely small and complex spore appendages at all stages of germination with spacial orientation capabilities not possible when the original studies were done. Over the past 2 yrs, the advances in software technology and improved SEM resolution and image enhancement capabilities, make it possible to work with fields of intact spores still attached to a nutritive surface and examine the exosporium and membrane contacts with the nutrient surface at resolution and magnifications formerly reserved for TEM. This investigation combines attachment studies using agar substrates (Trypticase Soy agar + 5% glucose, and Blood CDC agar) and human cells (lung fibroblasts and colon cells), with high resolution imaging of intact spores on their nutritive substrate (agar and human cell)to determine the ultrastructural changes that accomplish clostridial colonization. Studies were also done to determine if attachment was altered as a result of exposure to cation substitution, cation chelation, or colchicine treatment.