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CENTRIFUGAL CYTOLOGY I. A QUANTITATIVE TECHNIQUE FOR THE PREPARATION OF GLUTARALDEHYDE-FIXED CELLS FOR THE LIGHT AND SCANNING ELECTRON MICROSCOPE

R. C. LEIF 1, H. N. EASTER JR. 2, R. L. WARTERS 3, L. A. DUNLAP 3, R. A. THOMAS 4, and M. F. AUSTIN

1 Department of Chemistry and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306
2 Department of Chemistry, Florida State University, Tallahassee, Florida 32306
3 Department of Biology, Florida State University, Tallahassee, Florida 32306
4 School of Engineering Science, Florida State University, Tallahassee, Florida 32306

A special swinging bucket rotor has been constructed which produces fixed and stained dispersions of cells obtained from dilute suspensions on conventional microscope slides. In each of eight sample chambers 12,500-250,000 cells are sedimented onto the slide. Then 0.1 ml of a 4% glutaraldehyde, 12% dimethyl sulfoxide fixing solution is layered over the cells and the centrifuge is restarted. The application of the centrifugal field during fixation flattens the cells and presumably minimizes cell losses from the slide. After 45 mm the bucket is disassembled and the cells are benzidine-stained for hemoglobin and then the slide is postfixed with ethanol, after which the cells are stained with Giemsa, dehydrated through the ethanols, stained with Sudan black and then mounted without air-drying at any intermediate step. Cover slip preparations, air-dried from xylene, are utilized for scanning electron microscopy. Quantitative cell counts of known concentrations of blood cells have demonstrated that 89.2% of the cells are recovered in the mounted preparation. A reconstruction experiment utilizing various synthetic mixtures of human and tadpole blood cells established that there is no selection artifact in the ratio of these cells recovered on the entire slide nor any artifacts due to nondiscriminate cell placement. Cell size determinations of glutaraldehyde-fixed erythrocytes were performed in an attempt to determine the isotonic concentration of glutaraldehyde.

Submitted on July 6, 1970


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