CHICK EMBRYOLOGY II
Materials needed
0.9% NaCl, several litres
1 mg/ml Nile blue sulfate in distilled water
100% ethanol
Acid-alcohol (1ml conc HCl in 100ml 70% ethanol)
90% ethanol
70% ethanol
Xylene or histosol
DPX or Permount
Formol-acetic-alcohol – 2 parts 40% formaldehyde, 1 part glacial acetic acid, 7 parts Spatulas, forceps, small scissors
Plastic petri dishes
Pipettes
Glass dishes (small)
Cavity slides
Finger bowls
1. Opening eggs for embryo harvest
Take an egg from each of the stages available. Carefully break open eggs into a finger bowl of 0.9% NaCl. Crack the egg, submerge the egg in the NaCl, and gently ease the two halves of the shell apart. Release the contents into the solution. The yolk will float to the surface and the blastoderm should be uppermost. If not, rotate the egg with a spatula.
Stain the embryo by applying a drop of neutral red (1% aqueous solution) on a glass rod or from a dropper. The stain will rapidly permeate the vitelline membrane and stain the embryo underneath. Stage the embryo from the Hamburger & Hamilton staging series (present in the lab).
Carefully cut round the perimeter of the area opaca with scissors without rupturing the yolk and clouding the solution. Pull the blastoderm plus vitelline membrane away with forceps and lift out with a spatula. Transfer to a small plastic petri dish with fresh NaCl, peel the vitelline membrane away from the embryo and remove any adherent yolk. Examine down the dissecting microscope and record the stage. Fix one embryo from each of the 3 stages available in formol-acetic-alcohol for 1 hr as described in section 3.
During this hour take another embryo from each of the 3 stages available, isolate the embryo from the egg as above and draw the embryo and identify all the embryonic structures present.
Do this with care and with speed and never let the embryo dry out at all – use the embryo in the next part of the lab (#2) to study cell death.
Repeat on each of the stages of embryos available.
2. Cell death in embryos
Collect one embryo from each of the stages available by the above method and ensure that all the membranes, the vitelline and amniotic, have been removed and place them together in 10mls of 0.9% NaCl in a plastic petri dish. Add 30ml of Nile Blue sulfate (NBS) made to a concentration of 1mg/ml in distilled water. Gently agitate the embryos on a shaker for 30 minutes at room temperature. Then remove the Nile Blue solution, replace with fresh 0.9% NaCl and incubate at the same conditions for up to 1 hr.
NBS is excluded from living cells and enters dead cells so regions of cell death can be seen. Record all the areas of cell death on drawings at each of the stages of embryogenesis available.
Keep these drawings available for next week.
What is the function of cell death, why is this occurring?
3. Carmine staining and mounting whole embryos
Harvest the embryos at the stages available by the method in 1 and place together in a Petri dish. Gradually remove the NaCl with a pipette using forceps to unwrinkled the blastoderm so that it flattens out dorsal side up as it approaches the bottom of the dish. Without letting the embryos dry out add fixative (formol-acetic-alcohol). Cover the dish and fix for 1hrs at room temperature.
Replace the fix with 70% ethanol, trim the blastoderm to the edge of the area pellucida. Replace 70% ethanol twice, 10 mins each change.
Replace with borax carmine solution for 10 mins.
Differentiate in acid-alcohol until the embryo assumes a bright transparent appearance.
Dehydrate by replacing stain with 90% ethanol (10 mins), then 2 changes of absolute ethanol (100%).
