The changes in volume, weight and specific gravity of tissue samples during fixation in osmium tetroxide or formaldehyde and subsequent infiltration with methacrylate or paraffin were studied in more than 600 tissue specimens. Substantial changes in volume and weight were found, and as a rule the weight changes closely parallelled the volume changes. The influence of the histological procedures on the specific gravity was not so pronounced. During fixation in 1 per cent osmium tetroxide in Tyrode's solution at pH 7.2, a rapid increase of volume took place. Half of the final value was reached after only 15 minutes and the swelling was practically complete after 4 hours. After 24 hours it amounted to 30 per cent. Fixation in formaldehyde likewise caused swelling, which was inversely proportional to the concentration (0.5–16 per cent). With 4 per cent formaldehyde in Krebs-Ringer solution the swelling amounted to 18 per cent after 12 hours, and half of this value was reached in about 30 minutes. Specimens left in the fixation fluids for long periods displayed secondary shrinkage at a rate apparently independent of the concentration of fixative. During dehydration in ethanol, marked shrinkage occurred that averaged 23 per cent for osmium-fixed and 33 per cent for formaldehyde-fixed material. Stepwise dehydration caused less abrupt volume changes, although the end-result differed but little. Among other dehydrating agents, miscible with methacrylate (methanol, acetone, 2-ethoxy-ethanol, dioxane, triethylphosphate), only methanol appeared to give better results than ethanol. Infiltration with methacrylate caused only slight shrinkage, around 7 per cent, regardless of the preceding fixative. During polymerization, however, methacrylate undergoes a 20 per cent reduction of its volume. As far as can be judged from planimetric observations, this shrinkage is to a great extent transferred to the specimens. Paraffin infiltration caused shrinkage of around 30 per cent. With osmium-fixed specimens the fixation temperature influenced the shrinkage during infiltration. Lower fixation temperature gave greater shrinkage. In other stages the temperature had little influence on volume changes. The swelling observed during fixation was found to be due, not to the fixing agents themselves, but to the aqueous media in which they were dissolved. When specimens were placed in saline, Tyrode's, Krebs-Ringer's and sucrose solutions, swelling took place in all of them, including the strongly hypertonic ones. The swelling was inversely proportional to the concentration and was less pronounced in sucrose than in the other medias. Addition of the fixatives, osmium tetroxide or formaldehyde, reduced the swelling to an extent that cannot be explained merely by osmotic action. The addition of macromolecules such as gelatin or dextran was found to reduce the swelling during fixation. The swelling phenomenon could be completely abolished if dextran and sucrose were added in suitable amounts to the osmium tetroxide solution. From the results obtained the following conclusions can be drawn. During conventional preparation procedures for histological specimens, major changes in volume, weight and specific gravity occur. Doubtless, these changes considerably affect the original geometry of the tissues constituents. It is possible, however, to reduce the volume alterations e.g. by addition of colloid-osmotically active substances.
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