A cryofixation method is presented which gives excellent ultrastructural preservation of monolayer cell cultures without any chemical pretreatments. Rat hepatocytes in primary culture were used in this study. The equipment needed is inexpensive and easy to manufacture. Cells are grown on a usual tissue culture support material (Thermanox plastic sheets). For cryofixation, samples are prepared essentially by a combined sandwich-cryogen-jet technique. 3 mm large discs are punched out and sandwiched with Cu-or Au-object holders of little mass; a 15 pm spacer is put in between. The viability of the cells is not impaired by the manipulations before freezing. The sandwich sample is quickly frozen by shooting a propane jet from a simple pressure chamber on to the metal object holder. The relevant parameters were optimized by parallel freeze-fracture analyses of 5" , , glycerol as a model system and by thermocouple measurements. Sandwich samples are then mounted in an appropriate double replication specimen table for further analysis by freeze-fracturing. It is possible to obtain a certain selectivity of the fracture plane with regard to apical, lateral or basal aspects of the cell layer. Alternatively, disc samples can be processed by chemical fixation methods (including freeze substitution to determine the freeze-fracture plane), since the support material Thermanox is insensitive to organic solvents and easy to cut. In each case the cells remain attached to their substratum throughout the whole procedure. Thus, the ultrastructural data can be directly correlated with parallel functional analyses obtained from the same cell cultures.

This paper describes the production and use of a new specimen carrier for the Balzers freeze-fracturing unit. The carriers are inexpensive, easily produced and have excellent mechanical and thermal properties. The rectangular form of the carriers provides easy handling and unequivocal orientation of anisotropic specimens.

The freeze-fracturing of various biological material is described with the aid of a modified Balzers specimen table. The advantages of this table are easier and faster handling of the specimens and a better thermal contact between the specimen and the cold stage.