EGG INOCULATION Principles Practice and Vaccine Development

Dr.T.V.Rao MD
Dr.T.V.Rao MD
Towards Developing Better
Skills in Microbiology
Egg inoculation continues to be a
Important Student Exercise in Several
Post Graduate Examinations in
Medical Microbiology for evaluation.
The Students should develop the
Necessary skills to be familiar with
the exercise in Virology
Dr.T.V.Rao MD
Viruses are Different From
Other Microbes
Viruses are obligate intracellular parasites.
They depend totally on their host cells for
their existence. Their total host dependence
makes it extremely difficult to get good
insight of them natural conditions, because
the internal characteristics of the host cells
are likely to interfere with the observations.
Due to these reasons, it has been found
desirable that viruses are cultivated or grown
Dr.T.V.Rao MD
in the laboratory itself.
Difficulties in Diagnosis of
Viral Infections
Can not be seen under light microscope
Can not be cultivated easily
Do not grow on culture media
Treatment was not available
Changed situation
Rapid techniques have emerged
Screening for Blood transfusion
Treatment available
Dr.T.V.Rao MD
Techniques used
Detection of Viral Antigen
Growing and detecting viruses in
Tissue / Organ / Cell culture
Fertilized hen’s egg
Laboratory animal inoculation eg mice
Detection of antibody in serum
IgG – Rising titer in paired sample
IgM – Indicates current / recent infection
Dr.T.V.Rao MD
Electron Microscope 
/ Immune Electron
Light microscope –
Inclusion bodies eg
Negri Body in
Microscope Fluorescent
antibody technique
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Demonstration of Viral
Precipitation on gel eg HBsAg
Counter Immuno Electro
Phoresis (CIEP)
Enzyme Linkes Immuno
Sorbant Assay (ELISA)
Dr.T.V.Rao MD
Isolation of Virus
 Laboratory animals
 Fertilized Hen’s Egg
 Chorioallantoic
 Allantoic cavity
 Amniotic cavity
 Yolk sac
 Organ/Tissue/Cell
 Growth identified by
serological method like
Dr.T.V.Rao MD
Virus Culture
Embryonated Egg
Chorioallantioc membrane (CAM)
Allantoic cavity
Amniotic cavity
Yolk Sac
Cell Lines/
Tissue cultures
Diploid/ Secondary
Animal inoculation
Suckling mice
Dr.T.V.Rao MD
Embryonated Hen’s Egg
Cultivation of Viruses and Bacteria
Chorioallantoic membrane (CAM) – visible
lesions called pocks. Each infectious virus
particle forms one pock. e.g. Variola, Vaccinia
Allantoic cavity – Influenza virus (vaccine
production) & paramyxoviruses
 Amniotic cavity – primary isolation of Influenza virus
Yolk sac – Chlamydia, Rickettsia & some viruses
Dr.T.V.Rao MD
Embryonated eggs:
 The Embryonated hen’s egg was first used for
cultivation of viruses by Good Pasteur and Burnet
(1931). Cultivation of viruses in organized tissues
like chick embryo necessitates a different type of
approach.. For all practical purposes they all
themselves behave as tissue cultures. The process of
cultivation of viruses in embryonated eggs depends
on the type of egg which is used. The egg used for
cultivation must be sterile and the shell should be
intact and healthy.
Dr.T.V.Rao MD
Burnet as Director of the
Hall Institute, 1944-1965
F.M. Burnet in
the laboratory in
the early 1950's,
experimenting on
influenza virus
genetics, using
the developing
hen's egg.
Dr.T.V.Rao MD
Wins Nobel Prize
 Burnet was confirmed
by the award of the
1960 Nobel Prize to him
and Peter Medawar for
the discovery of
tolerance, a discovery
in immunology of
minor importance
compared with the
clonal selection theory.
Dr.T.V.Rao MD
Only Embryonated Eggs Are
Suitable for Growing Virus
eggs are
candled daily
to see the
embryos inside.
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Eggs are Used for Mass Vaccine
Production in Influenza
 Animals and chick
embryo were the first
method that was used to
cultivate virus. This
method is rarely used as
it is not convenient.
However, when
preparing for bulk virus,
(e.g. antigen or vaccine
production) the usage of
chick embryo is useful.
Dr.T.V.Rao MD
Advantages of Fertile Eggs
 Fertile chicken eggs
provide a convenient,
space-saving incubator
for many kinds of animal
viruses. Different
viruses can be injected
into an egg at different
sites and the egg can be
easily observed for viral
replication throughout
the development of the
chicken embryo.
Dr.T.V.Rao MD
Advantages of Using
Embryonated Eggs
 Isolation and cultivation of
many avian and few
mammalian viruses
 Ideal receptacle for virus to
 Sterile & wide range of
tissues and fluids
 Cost- much less
 Maintenance-easier
 Less labor
 Readily available
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Advantages of Fertilized Eggs
 Free from bacteria
and many latent
 Free from specific
and non specific
factors of defense.
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Structure and Utility of
Fertilized Egg
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Routes of Injecting the Fertilized
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Cultivation of Virus in Eggs
 To cultivate viruses in
eggs, the procedure
adopted should be very
simple. The eggs are kept
in incubator and embryos
of 7-12 days old are used.
The egg containing
embryo usually has an air
apace at the larger end.
The position of this sac is
first determined.
Dr.T.V.Rao MD
Begin you Exercise with
Candling Eggs
 Candling is the process of
holding a strong light above
or below the egg to observe
the embryo. A candling
lamp consists of a strong
electric bulb covered by a
plastic or aluminum
container that has a handle
and an aperture. The egg is
placed against this aperture
and illuminated by the light.
If you do not have a
candling lamp, improvise.
Try using a torch.
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Marking the
inoculation site:
 1. Hold the blunt end of the
egg against the aperture of
the candling lamp and note
the position of the head of
the embryo.
 2. Turn the egg a quarter
turn away from the head.
 3. Draw a line on the shell
marking the edge of the air
 4. Draw an X approximately
2 mm above this line.
 5. The X marks the
inoculation site.
Dr.T.V.Rao MD
Materials Needed for Egg
 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
 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.
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Inoculation of the
Allantoic cavity
 1.
Use cotton wool and 70 percent alcohol to
swab the end of the eggs to be inoculated.
Allow the alcohol to evaporate.
2. Swab the eggshell punch with 70 percent
alcohol solution. Place used cotton wool in
discard tray.
3. Pierce a hole in the end of the egg at the
marked inoculation site.
4. Attach needle to 1 mL syringe.
5. Draw inoculum into 1 mL syringe.
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Inoculation of the
Allantoic cavity
 6 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.
 7. Inject 0.1 mL of inoculum into the egg.
 8. Withdraw the needle from the egg.
 9. Seal the hole in the shell with stationery tape or melted
 10. Discard the used needles and syringes.
 11. Place the inoculated eggs into a second incubator.
Check the temperature and humidity of incubate
Dr.T.V.Rao MD
Piercing a hole in the egg shell
 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
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Routes of Egg Inoculation
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Inoculating the Specimens
 The rest of the embryo then
gets exposed and ready for
use. Virus suspension to be
cultivated is taken in dropper
and gently spread over the
exposed embryo. After
inoculation is thus completed,
the open area of the shell is
sealed eggs are incubated for
one week as in hatching. The
virus particles infect the
membrane at random and
create pock marked
appearance against the
transparent background. This
indicate viral basis.
Dr.T.V.Rao MD
Chorioallantoic membrane
 CAM is inoculated mainly for
growing poxvirus. Herpes
simplex virus is also grown.
Virus replication produces
visible lesions, grey white area
in transparent Cam. Each pock
is derived from a single virion.
Pocks produced by different
virus have different
morphology. Under optimal
conditions, each infectious
virus particle can form one
pock. Pock counting, therefore
can be used for the assay of
pock forming virus such as
Dr.T.V.Rao MD
Piercing the Chorioallantoic Membrane
Little holes are
drilled through
the egg shell
for infection of
the chorioallantoic
Dr.T.V.Rao MD
Can be used in few Fungal
 They provide a complex
environment, including
phagocytic cells, to study
fungal host-pathogen
interaction, but are of a
lower developmental
stage than adult mice.
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Dr.T.V.Rao MD
Piercing the Shell with Needle
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Injecting Infective Material with
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Overview of
Inoculating Sites
Dr.T.V.Rao MD
Allantoic cavity:
 Inoculation into the allantoic cavity provides a rich
yield of influenza and some paramyxoviruses.
Allantoic inoculation is employed for growing the
influenza virus for vaccine production. Other
allantoic vaccines include Yellow fever (17D strain),
and rabies vaccines. Duck eggs are bigger and have a
longer incubation period then hen’s egg. They
therefore provide a better yield of rabies virus and
were used for the preparation of the inactivated nonneural rabies vaccines.
Dr.T.V.Rao MD
Dr.T.V.Rao MD
Amniotic cavity:
The amniotic
sac is mainly
inoculated for
isolation of
influenza a
virus and the
mumps virus.
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Amniotic Route of
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Yolk sac:
It is inoculated
for the
cultivation of
some viruses as
well as for
some bacteria
like Chlamydia
and Rickettsia.
Dr.T.V.Rao MD
Dr.T.V.Rao MD
Influenza Vaccine Development
in Fertilized Eggs
Dr.T.V.Rao MD
Influenza Vaccine Traditional MethodsInfluenza Examining the infected eggs
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How Vaccines are Produced
in Eggs
 In egg culture, flu viruses are
injected into chicken egg
embryos, where they
multiply. After several days
of incubation a machine opens
the egg and harvests the
virus, which is then purified
and chemically killed.
On average it takes one or
two eggs to produce a single
dose of annual flu vaccine.
In cell culture, the virus is
grown in animal or human
cells, which are available in
unlimited supply.
Dr.T.V.Rao MD
How the Reassortant Vaccines
for Influenza Produced in Eggs
 The egg is inoculated
with a mixture of the
epidemic influenza virus
strain (red) and a
standard strain (green)
that can replicate in
chicken eggs. Both strains
replicate themselves, but
as they do so their genetic
material becomes mixed,
producing hybrid viruses
known as reassortants
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Eggs as Tools for Developing
Influenza Vaccines
 Influenza vaccine
manufacture in eggs,
computer artwork. Fertilized
chicken eggs can be used to
produce vaccines against
influenza viruses. The
reassortants are analyzed,
and those which have the
epidemic strain surface
proteins but other genes of
the standard strain will be
selected. These are injected
into different eggs to
replicate before harvesting.
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Eggs are Used in Mass Scale
Development of Vaccines
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Egg Allergies and Vaccines
 No suitable cell culture
system exists and egg
inoculation is the
method of
choice. Influenza virus
vaccines are still
cultivated in eggs, and
hence people with egg
allergies cannot tolerate
the influenza vaccines.
Dr.T.V.Rao MD
Follow all the Biosafety
 All procedures involving
the manipulation of
infectious materials are
conducted within
biological safety cabinets,
specially designed hoods,
or other physical
containment devices, or
by personnel wearing
appropriate personal
protective clothing and
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Created by Dr.T.V.Rao MD for
‘e’ learning for Medical and
Paramedical students in the
Developing world
[email protected]
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