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 lyzed, establishing that 1sothermal freeze fixation 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 fraction i.n the tissue leading to modifications in size of ice crystals and to changes in the histological structure

Light Microscopy Measurements of Ice Recrystallization in Frozen Corn Starch Pastes Using Isothermal Freeze Fixation

Isothermal freeze fixation was used to analyze ice recrystallization by light microscopy in a 10 % (W/W) frozen corn starch paste during storage at temperatures in the range of -5 to -20 °C. Different formulations were tested in order to obtain a suitable fixative for this method of indirect observation of the ice crystals. A solution of formaldehyde, ethanol and water (10:45:45 V:V) was selected because it minimized substitution-induced distortion and contraction of the matrix. The diffusion coefficients of the selected fixative in the frozen system were measured at different temperatures in conditions of unidirectional mass transfer in a semi - infinite medium. The activation energy for diffusion was determined (Ea = 95.11 ± I. 15 KJ/mol). Fixation times for the frozen starch paste at different temperatures were predicted from a mathematical model for unidirectional mass transfer with a discontinuous diffusion coefficient. Matrix contraction during the different stages of the freeze fixation method was evaluated. Recrystallization of ice in frozen corn starch pastes during storage was analyzed by the measurement of the changes in ice crystal equivalent diameters on the micrographs. A kinetic equation for recrystallization was fitted to the experimental data to obtain the corresponding parameters. Contraction of the matrix affects the kinetic constants but has no effect on activation energy. The effect of recrystallization during fixation on ice crystal measurements was not significant

Histologic analysis of ice crystals in pork frozen by liquid N2 and high pressure-assisted freezing

Proceedings of SCANNING 97: Monterey, California, USA.Peer reviewe