SUMMARY Techniques for obtaining complementary replicas have already been shown to be valuable in aiding the interpretation of electron micrographs of replicas of specimens prepared by freeze‐etching and freeze‐fracturing techniques, and in the recognition of artefacts. This paper describes a simple and efficient method of obtaining complementary pairs of replicas of all types of specimen. Ordinary hollow brass rivets are used as specimen holders and are frozen in an end‐to‐end position using a special pair of forceps. Up to six rivets are placed in a device consisting of a hinged plate held together with clamps against the force of small springs. When the clamps are released, the pair of rivets are separated, fracturing the specimens. The device is easily adapted for use in any type of freeze‐etching or high vacuum apparatus. On the example of Saccharomyces cerevisiae the application of the technique to the detection of artefacts in complementary replicas are described. It was shown that the fibrils observed on cross‐fractured cell walls are produced by plastic deformation of cell wall components.

SUMMARY This paper describes a simple technique for obtaining complementary replicas of specimens fractured under liquid helium. Ordinary hollow brass rivets are used as specimen holders and are frozen in an end‐to‐end position. The pairs of rivets are placed in a device consisting basically of two concentric steel tubes which can be twisted and moved relative to each other guided by a peg‐in‐slot mechanism. The rivets are inserted while the device is under liquid nitrogen, and the device is then transferred to liquid helium and the tubes are moved so that the upper rivet is knocked away and falls into a collector. The device is then transferred back to liquid nitrogen, the fracture faces being protected from contamination throughout the process. Replicas are then obtained by any of the standard methods for specimens fractured under liquid nitrogen. The device has been tested using yeast cells as a model system, and some typical results are presented and discussed.

SUMMARY A cryogenic device, freely accessible from the top, is described which allows rapid cooling of specimens for electron microscopy by immersion into melting nitrogen.

An improved and rapid procedure for the freeze-fracture of cell cultures grown as monolayers is described. No additional equipment than normally used for freeze-fracturing is necessary and cell culture conditions are as usual on plastic dishes. Cells are grown on a plastic foil membrane so that they can be viewed with a light microscope. If necessary, while viewing with a binocular microscope, specimen holders are glued to the foil membrane over selected areas of the culture. The specimen holders with the attached cell culture are dissected out and processed for freeze-fracturing in a BIOETH 2005. Fracture planes occur along the cell surface as well as through entire cells. Analysis of corresponding fracture faces is possible.

Freeze-etching is now a well established method of biological electron microscopy but, despite its outstanding success in many areas, it still suffers from severe technical limitations when used to study heterogeneous or otherwise intractable tissues. These limitations are mainly related to the difficulty of retaining an intact replica during dissolution of the organic debris, and can be especially acute when dealing with structures composed of many different cell types, or where there are sharp boundaries between hard and soft parts of the specimen. Such difficulties have been described in more detail elsewhere, but they usually have the result that the replica breaks up into small pieces during thawing or cleaning so that it is impossible to locate specific areas of interest.One potential method of improving the retention of intact replicas is to provide an additional supporting layer that can be removed once the organic material has been cleaned away.