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EMBRYONATED EGG INOCULATION

By : Chinithung ngullieFinal year

M.Sc medical microbiology

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virus cultivation

– isolate and identify viruses

– viral structure, replication, genetics & pathogenesis

– vaccine production

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TECHNIQUES OF VIRUS CULTIVATION EMBRYONATED EGG INOCULATION

ANIMAL INOCULATION

TISSUE CULTURE

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Introduction

• Cultivation of viruses in chick embryo • different type of approach.• For all practical purposes they behave as

tissue cultures

History

Burnet

First used for cultivation of viruses by Ernest GoodPasteur and Burnet (1931) F.M. Burnet in thelaboratory in theearly 1950's, wasexperimentingon influenzavirus genetics,using thedeveloping hen'segg.

goodpasture

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EMBRYONATED EGG

state of a fertilized egg containing an embryo foetus in its early stages of developments especially before it has reached a distinctively recognizable form).

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EGGS USED IN VIROLOGY

• HEN EGG• DUCK EGG• TURKEY’S EGG

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Selection of egg

• must be sterile • shell should be intact and healthy.• should be obtained from non-vaccinated,

disease-free flocks

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Process of artificial incubation

• incubation - 38 – 39°C and 60 – 70% humidity.• need to be turned at least twice a day or• rolled continually in a specially designed egg

incubator.

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EGG INCUBATOR

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Egg incubator• artificially hatched - controlled and favourable conditions• maintain favourable incubation/ environment - constant temperature

over a specified period.• electrically heated – thermostat• intelligent control system - correct measurement of heat quantity ,• - adjusting hatching control

temperature constantly • variation of temperature - ambient to 70° C • controlled by “JUMO”/ EGO” German

Capillary thermostat having accuracy of + 0.5° c.• Capacity:• 50 to 2000 eggs•

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ADVANTAGES OF EMBRYONATED EGG

Ideal for viruses to grow, offers several sites for virus cultivation Isolation and cultivation of many avian viruses and few mammalian viruses Sterile and wide range of tissues and fluids Economical and Readily available Maintenance easier Less labour (not need feeding and caging) They do not have immune mechanism like animals to counteract virus

infection.

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DISADVANTAGES

Some viruses do not show growth on primary inoculation into the egg.

Slight amount of bacterial contamination in the inoculum may kill the embryo.

Eggs may be contaminated with mycoplasma and latent fowl viruses which may interfere with the growth of other virus.

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CANDLING OF EGG:

process of holding a strong beam of light

above or below the egg

to observe the embryo.

done in a darkened room

or area shielded by curtains

Use candling box

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CANDLING BOX

consists of A candling lamp a strong electric bulb covered by a plastic or aluminium container

with handle and aperture.

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PROCEDURE OF CANDLINGThe egg is placed against the aperture and illuminated by light.

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• Under the candling lamp, the embryo appears as a dark shadow with the head as a dark spot (eye).

• Incubated eggs are candled daily to see the chicken embryos inside

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Live Embryonated Egg Healthy embryos will respond to the light by moving.

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Dead embryo-Candling will reveal a small dark area and disrupted blood vessels.

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MARKING OF AIRSAC 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.Draw a line on the shell marking the

edge of the air sac. 3.Draw an x approximately 2mm above

this line.

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ROUTES OF INOCULATION

1. Allantoic cavity2. Yolk sac 3. Chorio- allantoic membrane (CAM)4.Amniotic cavity

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Materials required for egg inoculation• Egg• Egg holders• Egg shell punch• Cotton wool• 70% alcohol• Syringe 1ml• Needles preferably 23-25 gauge• Stationery tape or melted wax to seal the inoculation site• Inoculum• Discard tray

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Egg shell puncturing tools

CARBORANDUM DISCENGROTOOL

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ALLANTOIC INOCULATION1. 9-12 days old egg required

VIRUS WHICH CAN BE HARVESTED

INFLEUNZA

MUMPS

AVIAN ADENOVIRUS

NEWCASTLE DISEASE VIRUS

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STEPSCandle the eggs

mark the airsac.

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Puncture the shell over the centre of the air sac.

Insert a 23- gauge needle, 1-1/2 inches in length on a 1 ml syringe, into the egg through the puncture in the shell at a 45 angle to the long axis of the egg and away from the embryo.

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• Inject O.2ml of fluid into the egg

• Seal the puncture with

- Nail polish/cellophone tape

• Position the eggs and incubate at 37o C.

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ALLANTOIC ROUTE – INOCULATION SITE DETERMINATION

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Harvesting of Allantoic Fluid• Eggs must be chilled to obtain allantoic fluid

free of RBCs.• Clean the upper half of the shell with 70%

alcohol.• Cut away the shell above the air space.• Peel away the white opaque shell membrane

lining the air space, exposing the transparent allantoic membrane directly beneath.

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• Tear the allantoic membrane with sterile forceps.

• Attach a ballpoint needle to a syringe and insert the needle into the cavity.

• Remove the fluid by suction.

• Culture the harvested fluid in a suitable medium for a sterility check.

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YOLK SAC INOCULATION

• 6-8 days old eggs required.

Virus and bacteria which can be harvested

Rickettsiae

Chlamydia trachomatis

C. psittaci

HERPES SIMPLEX

VIRUS

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STEPS1. Candle

the egg and mark the position of the airsac

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2.Puncture the shell over the centre of the air cell

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3. Insert a 22 gauge needle,2 inches in length on a syringe, into the egg via the puncture

4. Point the needle straight down for depth of about 1-1/2 inches.

5. Express 0.5 ml of inoculum into the yolk sac.

AIRSAC PUNCTURED hole

Yolk sac

needle

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6. Seal the puncture with nail polish or cellophane tape.

7. Incubate the eggs.

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HARVESTING OF YOLK SAC

• Disinfect the upper half of the shell.• Remove the shell, shell membrane and

underlying chorio-allantoic membrane.• Lift the embryo up with sterile forceps to

expose the attached yolk sac.• Pull the yolk sac free with another pair of

forceps and place it in a sterile petridish.

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Chorio- Allantoic Membrane (CAM) Inoculation

• 11- 14 days old eggs required

Viruses inoculated

HERPES SIMPLEX VIRUS

POX VIRUS

ROUS SARCOMA VIRUS

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Candle the egg and locate an area on the side of the egg that is free of large blood vessels. Mark this area with a pencil.

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grind a hole through the shell, but not the shell membrane , at the site marked.

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Puncture a second hole at the air sac end, this time penetrating the outer shell membrane.

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• Place a drop of sterile physiological saline on the side hole and gently tease apart the fibers of the shell membrane with a 27- gauge needle.

• When the shell membrane has been penetrated, the drop of saline will be drawn into the egg as a result of separation of the CAM and shell membrane.

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Apply negative pressure to the air space opening by means of mouth suction with a rubber tube.

As the air is removed the CAM will drop from the shell around the side hole, creating an artificial airspace , outline the limits of artificial airspace.

Express 0.2 ml of inoculum through the side opening onto the CAM.

Negative pressure

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• Seal openings with cellophane tape.

• Gently rotate the egg to spread the inoculum over the entire CAM under the false air space.

• Incubate the eggs on side with false air space upward.

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Harvesting the CAM Membrane

• Disinfect the shell.• With sterile scissors, cut through the shell

along the longitudinal axis, about 1/3 down from the upper surface.

• Gently remove the shell to a discard pan.• With sterile forceps,lift the CAM, cut free.• Place the CAM in sterile saline and float free

for examination.

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AMNIOTIC CAVITY INOCULATION

• 9-10 days old egg required.

VIRUS INOCULATED

INFLEUNZA VIRUS

MUMPS VIRUS

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STEPS

• Candle the egg and mark the position of the embryo and the outline of the airspace on the shell.

• Punch a hole through the shell at the edge of the airspace directly above the embryo.

• Using a 23- gauge, 1 inch needle on a syringe make a short jab through the punched hole, towards the embryo.

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• When the amniotic membrane is penetrated, the embryo will be seen to follow the movements of the needle.

• Express upto 0.2 ml of inoculum.

• Seal the puncture with nail polish or cellophane tape and incubate at 37o C.

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HARVESTING OF AMNIOTIC FLUID

• Remove the shell and shell membrane below the air space.

• Remove the fluid from the allantoic cavity , the amnion should then be clearly visible.

• Remove the amniotic fluid with a 20- gauge needle and syringe.

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death of the embryo embryo cell damage

• formation of typical pocks or lesions • on the egg membranes

oedema of the developing membranes inclusion bodies

Presence of viral antigen in egg fluids

Viral growth and

multiplication in the egg embryo is

indicated by

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CYTOPATHOGENIC EFFECTS

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GROWTH OF VIRUS ON THE CAM• Formation of characteristic pocks.• Variola produces small circular pocks, dome shaped, no

surrounding necrosis or haemmorrhage whereas• Vaccinia virus larger lesions , flattened with necrosis and

haemmorrhage.

• Herpes simplex virus- -small , oval shaped with- no evidence of necrosis

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GROWTH OF A VIRUS IN THE YOLK SAC

PRESENCE OF BASOPHILIC INCLUSION BODIES

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Viruses which can be harvested by various routes:An Overview

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PURPOSE OF INOCULATION

• Diagnostic- Poxvirus Herpes simplex virus Mumps virus

Vaccine Production- Influenza virus

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REFERENCES

• Principles of virology: Molecular biology, pathogenesis, and control; S.J Flint, L.W. Enquist, R.M. Krug, V.R. Racaniello, A.M. Skalka.

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THANK YOU