Fixation Process
1. Remove the vitelline membrane from the blastoderm and discard it using the forceps and the camels hair brush. Then with a pipette, remove any excess saline solution from the watch glass. Gently position the blastoderm with a camels hair brush to keep its tissues flat and unwrinkled.
2. Fill the pipette with a fixative called Bouins fluid. Flow a few drops of fixative gently on to the embryo by holding the tip of the pipette at the edge of the blastoderm in the watch glass. Work carefully and slowly to avoid wrinkling the preparation. Do not disturb the embryo for ninety seconds and liberally flood it with Bouins fluid. Use a pair of dissecting needles to straighten the tissue in case wrinkling occurs. Fix one and one-half hours for a 33 to 48 hour embryo, a 72 hour embryo may have to be kept in the fixative for as long as four hours.
3. Pick up the embryo with a section lifter or a thin palette knife and then transfer it to a Petri dish coated with thin layer of paraffin on the bottom. Make sure that a few pipettefulls of fixative are flooded into the Petri dish before the transfer.
4. Cut away the excess peripheral tissue of the blastoderm for convenience in placing it in microscope glass slide. The nearer the cut, the more attractive the final mount may appear. Then run the preparation through an alcohol series. Thirty minutes in 35% alcohol, thirty minutes in 50% alcohol, and thirty minutes in 70% alcohol.
Staining and Mounting
1. To stain, flood the preparation with either Delafields hematoxylin or with Grenachers borax carmine. Staining time depends on the embryonic stage that is being prepared, about eight hours for a 48 hour embryo.
2. Dehydrate in an alcohol series again. Clear in oil of wintergreen for one hour.Using a section lifter, transfer the embryo to a clean microscope slide. Put the embryo on the slide and with of paper toweling, wipe away excess clearing fluid left in the specimen.
3. Apply a large drop of rather thick balsam. Warm gently the balsam if it is too thick to flow readily. Lower a microscope cover slip into place. The cover glass should be propped to prevent it from breaking of from crushing the specimen.
