Routes:

1- Chorioallontoic Membrane (CAM)

2- Allontoic Cavity

3- Amniotic Cavity

4- Yolk Sac

Materials:

Eggs: 9-day old or 10-day old embryonated eggs. Candle the eggs and mark the inoculation sites.

Eggs should be placed in an egg rack with the inoculation site uppermost.

Egg shell punch. Cotton wool. A 70 percent alcohol solution in water. Syringe 1 mL. Needles preferably 25 gauge, 16 mm. Stationery tape (also called cello or sticky tape) or melted wax to seal the

inoculation site. Inoculum. This must be free of microbial contamination. Discard tray. Biohazard Box

Inoculum: A substance (a virus or toxin or immune serum) that is introduced into the body to produce or increase immunity to a particular disease.

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.

Death of embryo may also occure.

Advantages:

Widely used method for the isolation of virus and growth. Ideal substrate for the viral growth and replication. Isolation and cultivation of many avian and few mammalian viruses. Cost effective and maintenance is much easier. Less labor is needed. The embryonated eggs are readily available. Sterile and wide range of tissues and fluids They are free from contaminating bacteria and many latent viruses. Specific and non specific factors of defense are not involved in embryonated

eggs. Widely used method to grow virus for some vaccine production.

Disadvantages: The site of inoculation for varies with different virus. That is, each virus

have different sites for their growth and replication. Major problem with cell cultures: The process requires trained technicians with experience in working on a full

time basis. State health laboratories and hospital laboratories do not isolate and identify

viruses in clinical work. Tissue or serum for analysis is sent to central laboratories to identify virus.

WAQAS NAWAZ

11-ARID-975

DVM 5th Semester