Dr.T.V.

Rao MD

Dr.T.V.Rao MD 1
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 2
 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 in the laboratory itself.
Dr.T.V.Rao MD 3
 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 4
 Techniques used
Microscopy

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 5
 Microscopy
Electron Microscope /

Immune Electron
Microscopy
Light microscope –
Inclusion bodies eg
Negri Body in
Rabies
Fluorescent
Microscope -
Fluorescent antibody
technique Dr.T.V.Rao MD 6
Demonstration of Viral Antigens


Precipitation on gel eg HBsAg
Immunofluorescence
Counter Immuno Electro Phoresis
(CIEP)
Enzyme Linkes Immuno Sorbant
Assay (ELISA)
Dr.T.V.Rao MD 7
 Isolation of Virus


 Laboratory animals
 Fertilized Hen’s Egg

Chorioallantoic
membrane

Allantoic cavity

Amniotic cavity

Yolk sac
 Organ/Tissue/Cell
Culture
 Growth identified by
serological method like Dr.T.V.Rao MD 8
neutralization.
 Virus Culture
Embryonated Egg Chorioallantioc membrane (CAM)
Allantoic cavity
Amniotic cavity
Yolk Sac

Cell Lines/ Tissue Primary

cultures Diploid/ Secondary
Continuous

Animal inoculation Suckling mice

Dr.T.V.Rao MD 9
 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 virus

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 10
 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 11
 Burnet as Director of the Hall
Institute, 1944-1965
F.M. Burnet in the

laboratory in the
early 1950's, was
experimenting on
influenza virus
genetics, using the
developing hen's
egg.

Dr.T.V.Rao MD 12
 Burnet
Wins Nobel Prize


 Burnet was confirmed by
the award of the 1960
Nobel Prize to him and
Peter Medawar for the
discovery of
immunological tolerance,
a discovery in
immunology of minor
importance compared
with the clonal selection
theory.
Dr.T.V.Rao MD 13
Only Embryonated Eggs Are
Suitable for Growing Virus


Inoculated eggs
are candled
daily to see the
chicken embryos
inside.

Dr.T.V.Rao MD 14
 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 15
 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 16
 Advantages of Using
Embryonated Eggs


 Isolation and cultivation of
many avian and few mammalian
viruses
 Ideal receptacle for virus to
grow
 Sterile & wide range of tissues
and fluids
 Cost- much less
 Maintenance-easier
 Less labor
 Readily available

Dr.T.V.Rao MD 17
Advantages of Fertilized Eggs are


 Free from bacteria
and many latent
viruses.
 Free from specific
and non specific
factors of defense.

Dr.T.V.Rao MD 18
Structure and Utility of
Fertilized Egg

Dr.T.V.Rao MD 19
Routes of Injecting the Fertilized
Eggs

Dr.T.V.Rao MD 20


Dr.T.V.Rao MD 21
 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 22
 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.

Dr.T.V.Rao MD 23
 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 sac.
 4. Draw an X approximately 2
mm above this line.
 5. The X marks the inoculation
site.

Dr.T.V.Rao MD 24
 Materials Needed for Egg
Inoculation


 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.

Dr.T.V.Rao MD 25
 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.

Dr.T.V.Rao MD 26
 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 wax.
 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 27
 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 procure.

Dr.T.V.Rao MD 28
Routes of Egg Inoculation

Dr.T.V.Rao MD 29
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 30
 Chorioallantoic membrane
(CAM):
 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 vaccinia.

Dr.T.V.Rao MD 31
Piercing the Chorioallantoic Membrane

Little holes are
drilled through
the egg shell for
infection of the
chorio-allantoic
membrane.

Dr.T.V.Rao MD 32
 Can be used in few Fungal
Infection


 They provide a complex
environment, including
phagocytic cells, to study
fungal host-pathogen
interaction, but are of a lower
developmental stage than
adult mice.

Dr.T.V.Rao MD 33
Dr.T.V.Rao MD 34
Piercing the Shell with Needle

Dr.T.V.Rao MD 35
Injecting Infective Material with
Needle

Dr.T.V.Rao MD 36
Overview of Inoculating
Sites

Dr.T.V.Rao MD 37
 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 non-
neural rabies vaccines.

Dr.T.V.Rao MD 38
 ALLANTOIC ROUTE –
INOCULATION SITE DETERMINATION

Dr.T.V.Rao MD 39
 Amniotic cavity:


The amniotic sac
is mainly
inoculated for
primary isolation
of influenza a
virus and the
mumps virus.
Dr.T.V.Rao MD 40
Amniotic Route of Inoculation

Dr.T.V.Rao MD 41
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 42
YOLK SAC ROUTE

Dr.T.V.Rao MD 43
Influenza Vaccine Development in
Fertilized Eggs

Dr.T.V.Rao MD 44
Influenza Vaccine Traditional Methods- Influenza
Examining the infected eggs Vaccine

Dr.T.V.Rao MD 45
 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 46
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

Dr.T.V.Rao MD 47
 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.
Dr.T.V.Rao MD 48
Eggs are Used in Mass Scale
Development of Vaccines

Dr.T.V.Rao MD 49
 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 50
 Follow all the Biosafety
Considerations


 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
equipment.

Dr.T.V.Rao MD 51


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Dr.T.V.Rao MD 52