This document discusses viral inoculation methods using embryonated eggs. It provides advantages of using eggs such as their ability to isolate and cultivate avian and some mammalian viruses. It also discusses important considerations for the source of eggs including using specific pathogen free flocks to minimize risks. Different routes for inoculating viruses into eggs are described, including allantoic sac, chorioallantoic membrane, and yolk sac routes. Tools, procedures, and incubation conditions for successful egg inoculation are outlined.

1. Viral inoculation methods
Dr. Hassaan Bin Aslam

2. Advantages
• Isolation and cultivation of many avian and few
mammalian viruses
• Ideal receptacle for the virus to grow
• Sterile and wide range of tissue and fluids
• Cost-effective
• Less labour intensive
• Readily available
• Easy maintenance

3. Source of eggs
• There is a theoretical risk of contaminating
vaccines with pathogens that are transmitted
through embryonated eggs.
• The use of embryos from a Specific Pathogen
Free (SPF) flock minimizes this risk.
• However, in many countries, embryonated
eggs from an SPF flock are unavailable. In
this case it is practical to use embryonated
eggs from a healthy local flock.

4. Consider following points
• Does the flock appear healthy and free from infectious disease?
• Is there any serological testing performed on the flock?
• What is the vaccination regime used in the flock?
• Is the flock free of Salmonella pullorum?
• Are the eggs clean?
• What is the percentage of fertile eggs?
• Is the hatching rate acceptable?
• Are the newly hatched chicks healthy?
• Do not purchase eggs during an outbreak of any disease in the flock
supplying the eggs.
• If eggs are purchased from a commercial hatchery at 8 or 9 days old,
candle at the hatchery to select eggs with viable embryos. Keep the eggs
warm during transport to the vaccine production centre.
• Eggs with white shells are preferable as they are easier to candle

5. Recording details of egg purchases
• Date when the eggs are ordered and the name of the person who
received the order.
• Number and age of the eggs ordered.
• Date and number of the eggs received.
• Colour and appearance of the eggs received.
• Number of eggs damaged during transport.
• Date and number of eggs placed in incubator.
• Number of viable eggs after candling prior to inoculation.

6. Storage and cleaning of eggs
• Do not buy dirty eggs.
• Eggs that are stained can be disinfected by washing in a
warm (37°C) solution of 0.1 percent Chloramin B (benzine
sulfonamide sodium salt) or wiped with a 70 percent alcohol
solution.
• Fertile eggs that have not been incubated should be
purchased.
• They can then be placed in an incubator when they are
delivered. Alternatively, they can be stored for several days in
cool conditions (16°C to 18°C) prior to incubation.
• This may reduce the number of viable embryos, as some
embryos may not develop after storage.

7. Incubation of eggs before inoculation
• Before inoculation
• Many vaccine production centres will already have large commercial incubators installed. Smaller
incubators are available and are suitable for the small-scale production of vaccine.
• Incubation temperature = 38°C to 39°C.
• Humidity should be maintained at 60 to 65 percent. A tray filled with water and placed in the bottom
of the incubator is usually sufficient to maintain this level of humidity.
• Place the eggs in the incubator with the air sac on top.
• Eggs should be turned three times a day.
• After inoculation:
• Inoculated eggs contain virus and should be placed in a different incubator. Eggs inoculated with virulent strains of
Newcastle disease virus should not be incubated in the same incubator as used for eggs inoculated with the avirulent
I-2 strain of Newcastle disease virus.
• Inoculated eggs are incubated under the same conditions as un-inoculated eggs but do NOT turn the eggs.

8. Cleaning of incubators
• Keep surfaces clean by wiping
out with a wet cloth and
disinfecting with 70 percent
alcohol solution or a non-
corrosive disinfectant.

9. Egg inoculation
• The egg is usually inoculated by the shell usually
by making hole or a small window in the egg shell.
• The viral fluid or suspected viral suspension is
injected into the fluid of egg.
• The exact tissue that is inoculated in guided by
the type of virus being cultivated and the goals of
experiment.

10. Structure of fertilised chicken egg
Prerequisites:
• Sample preparaion
Homogenisation of tissue
Centrifugation of homogenate
Filtration of inoculum
• Addition of antibiotics

11. Various routes of egg inoculation

12. Allantoic sac route
• Eggs: 9-day old or 10-day old embryonated eggs.
Candle the eggs and mark the inoculation sites as
described in Section 5. 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.

13. Allantoic sac route
• Use cotton wool and 70 percent alcohol to swab the end of the eggs to be inoculated. Allow the
alcohol to evaporate.
• Swab the eggshell punch with 70 percent alcohol solution. Place used cotton wool in discard tray.
• Pierce a hole in the end of the egg at the marked inoculation site.
• Attach needle to 1 mL syringe.
• Draw inoculum into 1 mL syringe.
• Keeping the needle and syringe vertical, place the needle through the hole in the eggshell. The
needle will need to penetrate approximately 16 mm into the egg to reach the allantoic cavity.
• Inject 0.1 mL of inoculum 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.
• Place the inoculated eggs into a second incubator. Check the temperature and humidity of
incubator.

14. Tools for piercing a hole
• A dental drill can be used if it is available. In most
laboratories a tool called an eggshell punch can be
improvised using materials that are cheap and easy
to procure.
• How to make
• A hole is made in the middle of the piece of
copper plate, which is then curved to the
shape of an egg.
• A file is used to create a small spike at the end
of the rod or wire, which is then pushed
through the hole in the copper until the point
extends one mm beyond the copper.
• The rod or wire is then brazed in place where it
passes through the copper. The other end of
the steel wire or rod is bent to form a handle.
• Insert the needle into the rubber stopper so
that only 1 mm of the tip is showing. Use and
store carefully as the tip of the needle will be
very sharp.

15. Chorio-allantoic membrane inoculation
• Age:
• Pox virus (fowl pox etc)
• Keep the egg upright
• Drill the hole in the top mid of marked air sac area
• Lay down the egg with embryo side down
• Carefully drill a hole in the middle of lateral
(longitudinal axis) side of the egg
• Apply negative pressure inside the egg by sucking
out air with the help of syringe attached with
rubber tubing (or vacuum pump)
• Place a drop of sterile saline on the lateral side
hole
• Dropping of water inside the shell indicated the
dropping of membrane.
• Inject 0.1-0.2 ml of inoculum after membrane is
dropped
• Seal both holes with wax

16. Yolk sac inoculation
• Age: 6-7 days old
• Avian encephalomyelitis virus
• Turkey rhinotracheitis virus
• Egg-upright position
• Drill hole in the top middle of egg shell
• Inject inoculum (0.2ml) straight into egg up
to the hilt of needle (23Gx12)
• Seal the hole with varnish/wax and incubate
• Discard needle in the sharp box

17. Applications
• Isolation of virus
• Vaccine production
• Estimation of infectivity of virus
• Estimation of infectivity titter of virus