Validation of cell volume determination for stereological studies of adrenocortical cells: comparison of values from semifine sections with those of dissociated zona fasciculata cells.

The average cell volume of rat adrenocortical zona fasciculata cells, determined using three different stains on semifine sections, was compared to that of dissociated, unfixed zona fasciculata cells. The maximum number of nuclei per area (NA) was obtained by counting nuclear profiles in semifine sections from nonosmicated adrenocortical tissue stained with toluidine blue. In osmicated adrenocortical tissue on the other hand, fewer nuclear profiles were seen and more of the small size classes were missing from the distribution of profiles. The skewness of the nuclear-profile distribution was much greater in osmicated than in nonosmicated tissue, reflecting the missing small profiles. Although the average diameter of nuclei (D) in nonosmicated tissue was smaller than that for osmicated tissue, profiles were in all likelihood more readily identified because they stained quite intensely. Three different methods for correcting nuclear-size profile distribution and determining nuclei per volume (NV) (Giger-Riedwyl, Cruz-Orive, and Weibel-Gomez) were generally in agreement for nonosmicated tissue. Considerable shrinkage occurred during preparation for electron microscopy, with the greater change occurring in nonosmicated tissue. Cell volume in osmicated tissue corrected for shrinkage varied considerably among the different methods, producing significantly greater values for cell volume than those in nonosmicated tissue. The cell volume of nonosmicated tissue, corrected for shrinkage during processing and for missing small nuclear profiles, did not differ significantly from that of freshly dissociated zona fasciculata cells. The one exception was nonosmicated tissue stained by Feulgen-methylene blue and corrected for missing small nuclear profiles by the method of Cruz-Orive. Use of nonosmicated adrenocortical tissue in stereological studies with an appropriate correction for shrinkage for each experiment is recommended for determining cell volume, although osmicated tissue should also be included to facilitate identification of intracellular membranes when electron microscopy is required.