A method for preserving and counterstaining vitally-stained cells. ∗

Abstract

Recently, in the course of some experiments on the viability of monocytes, 1 it became necessary to preserve, for further study, cells which had been vitally stained with neutral red. The cells consisted of various types of white blood cells present in the peritoneal exudate of the guinea pig. These cells had been under observation in hanging drop preparations such as are commonly used in tissue culture work, 2 and since the usual methods of preservation seriously affected or entirely removed the vital stain, it was necessary to apply a new method. The one finally devised, as described below, not only retains the vital dye but permits other staining methods, such as Wright's method for differentiating blood cells, to be superimposed upon the vitally-stained cells.

In brief, the method consists in taking the cover glass from a hanging drop preparation in which the cells have been well stained vitally, either by direct contact with neutral red on the cover slip, 3 or by exposure to a dye bath consisting of a few crystals of neutral red dissolved in unbuffered Ringer's solution in the incubator at 38° to 40°C, and immersing the preparation for one half to one hour at 38° to 40° C. in the Ringer-formalin fixative, suggested by Fischer, 4 which is buffered as noted below. The tissue is then washed in running water for three hours, rinsed with distilled water and permitted to dry. After drying, the preparation may be counterstained with a one-tenth of one per cent aqueous solution of methyl green, as a nuclear stain, or with Wright's blood stain. The preparation is again dried and may then be mounted in damar, or, if necessary, it may be cleared according to Fischer's 4 method, by subjecting it, before mounting, to passage for two minutes each through a graded series of acetone-xylol mixtures consisting of 5 per cent, 30 per cent, 70 per cent, xylol, and two changes of pure xylol.