Improved preservation of bacterila capsule for electron microscopy

✍ Scribed by Jacques, Mario ;Graham, Lori

• Publisher: Wiley (John Wiley & Sons)
• Year: 1989
• Tongue: English
• Weight: 215 KB
• Volume: 11
• Category: Article
• ISSN: 0741-0581
• DOI: 10.1002/jemt.1060110212

✦ Synopsis

Conventional fixation for thin-section electron microscopy is insufficient to preserve bacterial capsular material, a highly hydrated structure which collapses during dehydration and embedding. Boyles et al. (1984Boyles et al. ( , 1985) ) have recently introduced a modified aldehyde fixation process in which the addition of a primary amine was found to improve the preservation of actin filaments, the basement membrane material and cell surface glycocalyx of m u s e tissues. To the best of our knowledge, the application of diamines to the analysis of bacterial ultrastructure has not been reported in the literature. In the present study, excellent preservation of bacterial capsular material was seen when a diamine (lysine) was used together with glutaraldehyde in conventional embedding procedures. Methodology. Strains of W u r e l l a m u l t o c i d a (types A and D), m i e l l a pneumoniae, n o b w pleuropneumo njag (serotypes 1 and 8), Rhodococcus a, Fscher ichia a 08:KX105 and 09:K30:H12 (and their acapsular mutants),lichen iformis, and slimeproducing m h v l o c o w gpidermidis and Pseudomonas aruainosa were used in this study.

Bacterial cells, cultured on the appropriate solid media, were harvested, and suspended in freshly-prepared fixative consisting of 2.5% glutaraldehyde, 0.075% ruthenium red, 50 mM lysine, in 0.1 M cacodylate buffer (pH 7.0), for 20 min at room temperature. Cells were sedimented by