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A cell spectrophotometer (Cytograf Model 6300 A, Bio/Physics Systems, Inc.) was tested in a cytotoxic assay using Ehrlich ascites tumor cells as a model system. Several cellular conditions associated with volume expansion, staining of cellular components and fixation of cells were applied and the magnitude of the scattering and extinction signals were tested in these diverse cellular conditions. The magnitude of the scattering pulse of a cell spectrophotometer was found to be greatly dependent on the staining color and intensity of cellular components with vital dyes or following osmium tetroxide fixation. When the absorption wavelength of the vital dye was close to the wavelength used in the cell spectrophotometer (about 630 nm), dead stained and living nonstained cell populations were completely separated from each other. The magnitude of the extinction pulse was greatly dependent on the state (normal, injured cells) and staining intensity (vital dye staining, osmium fixation) of cellular components. The magnitude of the extinction pulse was reduced from that in normal cells when cells were treated with p-chloromercuribenzene sulfonic acid or in a hypotonic solution that caused a marked volume expansion of injured cells. When cells were fixed with a mixture containing glutaraldehyde and osmium tetroxide the cellular components of normal and injured cells turned black and in these conditions the magnitude of the extinction signal was in a linear correlation to the cross-sectional area of cells. In the present study, the cell spectrophotometer proved to be an efficient method for estimation of cellular viability, based on different scattering properties of cells, offering the advantages of high speed and precision. Demonstration of the use of a variety of vital dyes having diverse extinction properties with capabilities to differentiate between living and dead cells has indicated the potential use of the cell spectrophotometer in cytotoxic assay. Modification of the magnitude of the extinction signal in the cell spectrophotometer also shows great promise for accurate automated size determination of both normal and injured cells. Previously, determination of the size of injured cells has been beset with methodologic errors.

Optical properties of normal and injured cells. Application of cytographic analysis to cell viability and volume studies.