Electron Densitometry of Stained Virus Particles

Methods are described for determining the relative mass of particles in electron microscope specimens through the measurement of photographic densities in recorded images. These methods were applied to a quantitative study of the amounts of electron stains that could be associated with the particles of tomato bushy stunt virus (BSV) and tobacco mosaic virus (TMV). In the pH range above 2 where the viruses are stable, the amount of stain absorbed is too small to produce adequate contrast in the electron microscope. Maximum stain absorption was achieved at pH about 1 where with several reagents and combinations of reagents the mass of BSV could be increased to about four times that of the unstained particles. Optimum results were obtained with phosphotungstic acid alone or in combination with Pt, Th, or La ions. Since the pH conditions for high stain absorption are normally destructive, morphology is satisfactorily preserved only when the phosphotungstic acid is applied in concentrations of 10 per cent or greater or when the use of destructive reagents is preceded by a preliminary fixation under mild conditions. Maximum staining of TMV increased the mass of the particles to about two times that of the unstained. Estimates of the mass of heavily stained BSV particles indicate that their density is 3.3 gm./cm.(3) The high internal hydration of BSV probably accounts for the greater stain absorption and penetration compared to those of TMV which has very low or zero internal hydration. Anomalous images resulting from the use of electron stains are shown and discussed.