Vital Staining of Virus Lesions on Chorio-Allantoic Membranes by Trypan Blue

In the study of the early focal lesions of viruses on chick chorio-allantoic membranes, some difficulty is often experienced in recognizing the earlier focal lesions on direct examination of the membrane even with moderate magnification. The recognition of the foci and the preservation of such membranes for more leisurely study under the microscope has been greatly facilitated by staining the lesions with trypan blue, and, after formalin fixation, mounting them in glycerin gelatin.

In this method, 1 cc of a 0.5% aqueous suspension of trypan blue is employed. It is placed directly upon the membrane when ready for examination through the window in the shell. Gentle rotation of the egg facilitates the general distribution of the stain, after which the shell is closed and the egg placed in the incubator for from 10 to 30 minutes depending on the age and extent of the lesions. The more extensive lesions require a shorter time to take up the stain and may become too intensely colored for satisfactory examination if the stain is left on too long.

The membrane is then removed in the usual manner, washed gently in physiological saline to remove the excess of trypan blue, and fixed flat in 10% formalin for a few minutes. After draining away the excess liquid the membrane is flattened on a 2 × 2.5 inch glass slide and mounted in a glycerin gelatin containing 50% glycerin, 5% gelatin, and 1 % phenol. The gelatin should be soaked in water about 2 hours before adding the glycerin and phenol, then heated gently for a few minutes while stirring. For mounting the membrane, the glycerine jelly is melted and while still hot (about 70°C) dropped on the membrane until it is well covered, flaming the slide gently but not enough to produce bubbles.