A Rapid Cytological Method for Determination of Mitotic and Fusion Indices in Mammalian Cell Cultures

Abstract

A quick and simple method for making semipermanent slides of unfixed cells taken directly from culture is described. This procedure yields preparations that reveal excellent nuclear and cytoplasmic morphology and allows accurate determination of both mitotic index and cell fusion index. After treatment with mitotic inhibitor of fusogen, the cells are centrifuged and resuspended at high concentration in culture medium 50% with respect to horse serum. A small drop of cell suspension is mixed with an equal volume of 2% lactic-acetic orcein (2 g synthetic orcein, 50.0 ml glacial acetic acid, 42.5 ml 85% lactic acid and 7.5 distilled water) and drawn into the tip of a Pasteur pipette. Staining time in the pipette depends upon the cell line being examined. A very small drop of stain mixture is placed on a slide, covered with a 22 mm2 No. 2 coverslip and squashed lightly to remove excess stain. The preparation is then immediately sealed with nail polish. Slides prepared in this way may be used at once or retained for 1-2 wk without significant deterioration. This method allows the establishment of an accurate mitotic index within 5 min after culture sampling; immediate determination of mitotic index is frequently important for subsequent decisions about experimental protocol. The reported staining schedule is applicable to cells of CHO K1 [Chinese hamster ovary K1 cells], HTC [rat liver HTC cells], mouse lymphoma L5178Y cells and various human lymphocytic lines.