Cultivation of Viruses in Chicken Embryonated Egg (CEE)

Introduction

This tutorial provides a step-by-step guide on cultivating viruses in chicken embryonated eggs (CEE). This method is commonly used in virology for vaccine production and research. Understanding the process and techniques involved can help in various applications, including vaccine development and studying viral pathogenesis.

Step 1: Selecting the Right Eggs

• Choose fertilized chicken eggs that are approximately 9 to 12 days old.
• Ensure that the eggs are clean and free from any cracks or contamination to avoid introducing unwanted bacteria during the cultivation process.

Step 2: Preparing the Working Environment

• Maintain a sterile working environment to prevent contamination.
• Use a laminar flow hood or a clean bench with disinfectants to sanitize all surfaces and equipment.
• Equip yourself with personal protective equipment (PPE), including gloves, masks, and lab coats.

Step 3: Inoculating the Eggs

• Carefully disinfect the surface of the eggs with 70% ethanol.
• Using a sterile needle or syringe, create a small hole in the egg shell to introduce the virus.
• Inject the viral sample into the allantoic cavity of the egg. Use the following guidelines:
  • For most viruses, a volume of 0.1 to 0.5 ml is sufficient.
  • Ensure that the needle does not penetrate too deeply to avoid damaging the embryo.

Step 4: Incubation

• Place the inoculated eggs in an incubator set at 37-38°C (98.6-100.4°F).
• Maintain proper humidity levels (around 60-70%) during the incubation period.
• Monitor the eggs regularly for signs of development and contamination.

Step 5: Harvesting the Virus

• After an incubation period of 2 to 7 days, check the eggs for signs of viral infection, such as embryo lethargy or death.
• To harvest the virus, re-disinfect the surface of the egg and carefully open it under sterile conditions.
• Collect the allantoic fluid using a sterile syringe, which contains the cultivated virus.

Step 6: Storing the Virus Sample

• Store the harvested virus in appropriate vials or tubes.
• Label the samples clearly with details such as the virus type, date of collection, and any other relevant information.
• Freeze the samples at -80°C for long-term storage or store them at -20°C for short-term use.

Conclusion

Cultivating viruses in chicken embryonated eggs is a vital technique in virology that allows for the study and production of vaccines. Key steps include selecting the right eggs, maintaining a sterile environment, careful inoculation, proper incubation, and virus harvesting. Following these guidelines will help ensure successful cultivation. For further applications, consider exploring different viral strains and their effects on the embryonated eggs.