Conditions critical for optimal visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron microscopy

G Wendelschafer-Crabb, S L Erlandsen, D H Walker
1975 Journal of Virology  
The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of' infected cells which were fixed with glutaraldehyde followed by postfixation in Os04 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively
more » ... h surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of' infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of' resolving viral particles on cell surf'aces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used. The use of the scanning electron microscope (SEM) to identify surface features of virusinfected cells as virus particles has been reported by several investigators. De Harven et al. (3) described "knobs" visualized on murine Friend erythroleukemia cells as RNA tumor viruses which spontaneously bud from the cell line. This identification, based only on morphological criterias, was not wholly convincing since the cell surfaces in the specimens were not well preserved due to the air drying preparation of the specimens for SEM. Virus antigens have been localized by using nonspecific virus markers distinguishable by SEM. Nemanic et al. (6) developed a technique for the localization of antigens on budding mammary tumor virus by using the characteristic morphology of tobacco mosaic virus as a recognizable marker for SEM. A hapten sandwich technique in which tobacco mosaic virus was immunologically linked by an antibody bridge to mammary tumor virus antigens was used and resulted in several tobacco
doi:10.1128/jvi.15.6.1498-1503.1975 fatcat:nm625sez5zffjjwrmouvetqnem