A technique for the freeze-fracture of tissue culture

Cultures of rat cerebellum grown on glass coverslips coated first with collodion and then collagen are fixed in glutaraldehyde to which ruthenium red has been added. After fixation, the collodion comes off the glass coverslips and the 3 layers: collodion-collagen-culture are handled as one piece. Following immersion in glycerol, selected areas are cut from the piece; the excised areas are a little smaller than those of the specimen carriers used in a Balzers apparatus. The ruthenium red, which stains all cells and processes, is a good indicator of specimen thickness. The explant which consists of several cell layers is redder than the peripheral monolayer of outgrowth. The square areas cut from either the explant or the outgrowth can be matched in thickness according to their shade of red. In frozen specimens, the red colour distinguishes the culture layer from the collodion-collagen support under it and from the white frost on top. During fracturing, the appearance of pink flakes on the edge of the knife indicates that the culture layer has been cleaved. The growth of cells on collodion-collagen and the fracture of the cultures with a knife, eliminate the restrictions inherent in the use of frozen 'metallic sandwiches'.