Fixing Conditions in the Freeze Substitution Technique for Light Microscopy Observation of Frozen Beef Tissue

The freeze substitution histological technique allows the indirect light microscopicobservation and the quantitative evaluation of ice crystal size in frozen tissues. Theuseofchemical fixatives in substitu ting fluids improves morphological and histochemical preservation of the tissue. Fixation conditions become important since this step can introduce modifications in crystal sizes . Effects of temperature on: a) diffusion rate of fixing solution in tissue, b) recrystallization rate of ice in frozen beef and c) variations of the froze~ water fraction were ana l yzed, establishing that 1sothermal freeze fixatio n constitutes an appropriate method for histological observation. Fixing at temperatures lower than that of the sample, in an attempt to reduce recrystallization effects , involves an increase of the frozen water f~action i.n the tissue leading to modifications in s12e of 1ce crysta ls and to changes in the histological structure. Initial paper received August 12 1985 Manuscript received Mny 7 1986 Direc t inquiries to N. E. Zaritzky Telephone number: 54 21 36096