RAPID CYTOLOGICAL METHOD FOR DETERMINATION OF MITOTIC AND FUSION INDEXES IN MAMMALIAN-CELL CULTURES

A quick and simple method for ding 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 mimic index and all fusion index. After treatment with mitotic inhibitor or 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, G. T. Curr; 50.0 ml glacial acetic acid, 42.5 ml 85% lactic acid, and 75 ml distilled water) and drawn into the tip of a Pasteur pipette. Staining time in the pipette depends up 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 scaled with nail polish. Slides prepared in this way may be used at once or retained for 1-2 weeks 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 KI, HTC, L5178Y and various human lymphocytic lines. © 1979 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.