Rapid diagnostic thin section electron microscopy of bacterial endospores

Emerging infectious diseases such as SARS and the bioterror attacks with anthrax spores that occurred after September 11th, 2001 have highlighted the need to be better prepared for the detection and management of infectious pathogens that threaten public health. Negative staining electron microscopy is one method used to screen environmental and clinical samples for relevant infectious pathogens. Unfortunately, bacterial endospores, like those of Bacillus anthracis, are difficult to identify using this method because of their density that prevents imaging of structural details. Thin section electron microscopy would be an alternative method but this usually requires a few days for preparation and diagnosis. In the present paper we describe the development of a rapid thin section protocol, using mainly Bacillus subtilis spores as a model, which allows an unequivocal diagnosis of endospores within 2 h. The protocol involves chemical fixation assisted by heat or microwaves, rapid dehydration, embedding in the low-viscosity resin LR White and chemically enhanced polymerization. Structural preservation of spores is comparable to preservation after standard Epon embedding. Immunolabeling experiments using B. atrophaeus spores and a specific antibody suggest that the protocol preserves significant antigenicity for on-section immunocytochemistry and therefore offers the possibility for the strain typing of spores using specific antibodies. Further experiments with vegetative bacteria, viruses and cell cultures indicate that the rapid thin section protocol not only preserves spores but also other biological structures. Because of its universality and speed the described protocol complements negative staining electron microscopy as a front line method for the morphology-based diagnosis of pathogens in environmental and clinical samples.