Effects of glutaraldehyde and critical point drying on the shape and size of erythrocytes in isotonic and hypotonic media

Abstract

SUMMARY Scanning electron microscopy (SEM) was applied to studies on osmotically swollen erythrocytes fixed by a method using increasing concentrations of glutaraldehyde (GA), OsO4 as a post fixative, and critical point drying (CPD). This fixation method prevented the osmotic effect of GA itself and preserved the cellular equilibrium shape intact in various media, i.e. discocytes in an isotonic medium and ellipsoids or spheres and ghosts in a hypotonic medium. The discocytes were reduced less in diameter and area than the ellipsoids and spheres during CPD, but the degree of volume shrinkage was of the same magnitude (approx. 73%) regardless of the cell shape. The shrinkage of the discocytes was advanced until the dry volume (approx. 29 μm3) was reached, while the shrinkage of the osmotically swollen cells ceased earlier. A miniaturization process coupled with solvent evaporation is suggested as an explanation for this. The osmotic swelling of erythrocytes was studied quantitatively with the aid of SEM micrographs. The increase in volume was about 60%, which is in agreement with the values obtained by light microscopy for unfixed and GA‐fixed cells in the present work and with those reported in the literature.

Effect of osmolality on erythrocyte rheology and perfusion of an artificial microvascular network

“…19 The persistence of a marked dimple area in an otherwise almost spherical shape is remarkable, it is not an artifact of RBC fixation by glutaraldehyde or the sample preparation. 20 The biconcave RBC shape is explained by the principle of least total curvature of the membrane, which means that the bending energy is minimized. 19 The biophysical properties of the membrane are considered to be uniform over the surface, 19 with dimple areas moving over the membrane surface during the tank-treading motion in shear flow.…”

Haematology studies during a 350‐metre dive

“…Long in vitro decompression procedures cause morphology changes by temperature fluctuations, intracellular decrease in pH and alteration of plasma osmolarity. The glutaraldehyde fixation medium employed in the dive to fix erythrocytes obtained at pressure had to rapidly penetrate the cell membrane to crosslink both proteins and lipids before in vitro decompression (19)(20)(21)(22)(23)(24)(25)(26)(27) and did so. The buffered-glutaraldehyde fixation system was also shown not to cause crenation in surface control samples, and always contained less echinocytes than reported for normal nondiving subjects (4).…”

Scanning electron microscopy of damage caused by Mesocyclops thermocyclopoides (Copepoda: Cyclopoidea) on larvae of the dengue fever vector Aedea aegypti (Diptera: Culicidae)

“…Size comparisons of the different structures analyzed were not done because those dimensions may have been altered during processing, particularly during the critical point drying, as has been previously described by various authors (Eskelinen andSaukko 1983, Beckett et al 1984).…”