Flattening and shrinkage of bacteriophage T7 after preparation for electron microscopy by negative staining

A technique for using stereo electron microscopy to determine the degree of flattening and volume of particles embedded in negative stain is developed. Using this technique, it is shown that bacteriophage T7 particles, which have been prepared for electron microscopy by negative staining with uranyl acetate, are considerably flattened. The edge-to-edge diameter of negatively stained phage is 545 ± 20 . Some fields in negatively stained specimens contain T7 phage whose DNA is positively stained, which are surrounded by little or no negative stain. These positively stained phage particles are shown to be less flattened than the negatively stained phage and consequently have a smaller edge-to-edge diameter (457 ± 17 ). The volumes of negatively and positively stained phage (excluding the phage tail) are equal within experimental error to the anhydrous phage volume (4.99 ± 0.15 × 10^-17 ml) and are significantly less than the hydrated phage volume (1.14 ± .02 × 10^-16 ml, (Ref. 1). Thus, both negatively and positively stained T7 phage have undergone a decrease in volume during preparation for electron microscopy.