Vigorous shaking of 50 ml of 0.2% (w/v) collodion-ethylacetate solution to which 0.33 ml of glycerol-water solution (86%, v/v) and 0.1 ml Lenor (fabric softener, Procter and Gamble Ltd) are added, produces a homogeneous suspension of small droplets. This suspension is used to deposit, by the conventional method (see Section II.B.2 above), a holey collodion film on grids. Note that collodion is used instead of Formvar. Because collodion is less stable than Formvar, for stabilization a thick layer of carbon should be evaporated onto the film (see Sections II.B.l and II.B.2 above). This thick carbon layer does not interfere with the investigation of the sample; the sample which is analysed is contained in the thin layer of ice spanning the holes. For further stabilization, this support is finally heated at 180°C for lOmin (Johannssen et al., 1979). A modified version of the procedure is described by Lunsdorf and Spiess (1986).
The surface of the support films has to be rendered hydrophilic by dipping the grid, shortly prior to use, into 96% ethanol. Hydrophilicity will be retained for several hours. An alternative procedure is glow discharge (Dubochet et al., 1971).
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