The general steps followed in the inoculation of embryonated chicken eggs with a virus

Written by MicroDok

The general steps followed in the inoculation of embryonated chicken eggs with a virus or virus-containing sample are highlighted in this section.  

  • Candle the eggs and place them gently on an egg rack making sure to place the eggs with the inoculation sites or routes facing up.
  • Mark the inoculation site of the embryonated eggs on the egg rack.
  • Disinfect the surface or inoculation site of the embryonated chicken eggs (of about 10-14 days old) to be inoculated with 70 % ethanol/alcohol or iodine.

Figure 1: Illustration of an embryonated egg for viral inoculation.

Figure 2: Illustration of egg shell punching device, an improvised punching tool used to create hole on an embryonated egg.

  • A cotton wool socked in the disinfectant should be used to undertake this disinfection process. It is important to allow the disinfectant to dry off from the site of inoculation before proceeding with the technique.
  • Pierce or make a hole in the end of the egg (i.e. at the inoculation site) using an egg punching device (Figure 2). Other tools used to punch the shell of an embryonated egg include dental drill. The egg shell punch is made from a copper wire.
  • After making the drill or hole on the correct inoculation site, inoculate the right concentration of the viral sample or particle into the inoculation site using a sterile syringe and needle.
  • Ensure the needle on the syringe penetrates well via the punctured site before dispensing its content especially for the inoculation of the allantoic cavity, yolk sac and the amniotic cavity which are deeper than the chorioallantoic membrane.
  • After injecting the viral inoculum into the appropriate site, withdraw the needle gently from the embryonated egg.
  • Seal the site of inoculation with paraffin or gelatin. A stationary tape can also be used for the sealing.
  • Incubate the inoculated embryonated eggs in the incubator while ensuring that the temperature condition and humidity of the incubator are balanced for the optimal growth of the virus being cultivated.
  • Inoculated embryonated eggs are usually incubated at 37oC for 2-3 days.
  • After incubation, the eggs are broken and the virus is isolated from the tissues of the embryonated eggs.
  • The formation of pocks or lesions on the membranes of the egg especially at the chorioallantoic membrane for some viruses indicates viral growth and replication. However, the death of the chicken embryo is also a strong indication of viral growth and multiplication.


Acheson N.H (2011). Fundamentals of Molecular Virology. Second edition. John Wiley and Sons Limited, West Sussex, United Kingdom.

Alan J. Cann (2005). Principles of Molecular Virology. 4th edition. Elsevier Academic Press,   Burlington, MA, USA.

Alberts B, Bray D, Johnson A, Lewis J, Raff M, Roberts K and Walter P (1998). Essential Cell Biology: An Introduction to the Molecular Biology of the Cell. Third edition. Garland Publishing Inc., New York.

Balows A, Hausler W, Herrmann K.L, Isenberg H.D and Shadomy H.J (1991). Manual of clinical microbiology. 5th ed. American Society of Microbiology Press, USA.

Barrett   J.T (1998).  Microbiology and Immunology Concepts.  Philadelphia,   PA: Lippincott-Raven Publishers. USA.

About the author


Leave a Comment