Materials Required:

1. Eggs: 9-day old or 10-day old embryonated eggs.

2. Egg shell punch/Carborundum disc

3. Cotton

4. Spirit

5. 1ml disposable syringe

6. Stationery tape (also called cello or sticky tape)

7. Viral suspension in Beijenox bottle

8. Biohazard

Procedure:

. Swab the end of the eggs to be inoculated with 70% ethanol. Allow the alcohol to evaporate.

. Place used cotton wool in discard tray.

. Candle the egg with high intensity LED torch and mark “X” over the embryo’s eye.

. Draw a line on the shell marking the air space.

. With a sterile egg shell puncher or carborundum disc pierce a hole in the end of the egg at the marked inoculation site.

. Attach a needle to 1 ml syringe.

. Draw 0.1ml of inoculums (Viral suspension) into 1 ml syringe.

. Place the needle through the hole in the eggshell keep the needle and syringe vertical. The needle will need to penetrate approximately 16 mm into the egg to reach specific site of inoculation.

. Inject 0.1 ml of inoculums into the egg.

. Withdraw the needle from the egg.

. Seal the hole in the shell with stationery tape or melted wax.

. Discard the used needles and syringes in a biohazard box.

. Place the inoculated eggs into a second incubator. Check the temperature and humidity of incubator.

. In the case of amniotic inoculation, when the needle reaches the embryo a thrust will strike the embryo and the embryo will move away from the needle. Then inject the virus suspension.

Expected Results:

The virus suspension are succesfully inoculated into the eggs. The growth of the virus can be detected by visible pocks in the egg.