Chicken embryo inoculation technology
2020-12-04
Chicken embryo vaccination technology has a wide range of uses. In addition to isolating and cultivating pathogens such as viruses, mycoplasma, and chlamydia to diagnose infectious diseases, it can also be used for virus identification, titer determination, pathogenicity determination, and the manufacture of vaccines and antigens. Therefore, mastering the technique of chicken embryo inoculation is very important for the research and prevention of infectious diseases.
1 Pick embryo
Choose chicken embryos of suitable age according to your needs. Most viruses are suitable for chicken embryos of 9-12 days old. Chicken embryos should be white eggshells for easy observation. Chicken embryos should be developed normally, healthy and lively, and should not hatch embryos that are too large, too small or deformed. Chicken embryos should come from healthy and disease-free chickens, and they should not contain maternal antibodies that can inhibit the inoculated virus. It is best to use SPF chicken embryos or non-immune chicken embryos.
2 Positioning according to the egg
Before inoculation, the chicken embryos should be inspected under the egg-shaping device to pick out dead and weak embryos. For all healthy embryos, first draw the air chamber position with a red pencil, and then draw the inoculation site (needle entry point) on the side of the embryo. The inoculation site should avoid large blood vessels, close to the edge of the air chamber, and about 1 cm away from the embryo head.
3 Vaccination
After positioning the eggs, you can start inoculation. First use tincture of iodine to disinfect the inoculation site and air chamber, then use 70% alcohol to deiodize, and then use a triangular needle or a 20-gauge needle to puncture the eggshell as needed. Allantoic cavity vaccination: The most commonly used vaccination method, Newcastle disease, egg drop syndrome virus, infectious bronchitis virus, bursal bursal virus, etc. can be used in this method. After the eggshell is disinfected, first make a small hole on the air chamber, and then make a small hole at the position on the embryo side near the air chamber, and insert a syringe with a 5-7 gauge needle into the hole about 1.5 cm. Inoculate 0.1～0.2mL of inoculation material. When inserting the needle, the needle and the egg shell should be kept at a 30°C angle, and the needle should not be inserted vertically. After the injection is completed, immediately seal the injection port and the small hole on the air chamber with melted hot paraffin, and then return it to the 37°C incubator for 72～120h incubation .
Chorioallantoic membrane inoculation: mainly used for the isolation and culture of pox virus, laryngotracheitis virus and Marek's virus. After sterilizing the egg shell, first make a small hole in the air chamber, and then avoid the large blood vessels at the embryo side near the air chamber. Use an electric soldering iron or a hacksaw blade to open the egg shell into a 4mm square opening, and carefully pry it with the tip of a knife. Open the eggshell without damage to the eggshell membrane and form an egg window. Use a needle to gently break the eggshell membrane in the center of the egg window, but not damage the chorioallantoic membrane under the eggshell membrane. Put 1 drop of sterile saline or glycerin on the needle tip, and then use the suction tip to inhale on the air chamber pores to cause negative pressure in the embryo. At this time, the chorioallantoic membrane at the egg window sinks to form an artificial air chamber , At this time, physiological saline or glycerin can be seen to penetrate rapidly. Use a syringe to add 2 to 3 drops of inoculum to the chorioallantoic membrane of the egg window, and seal the egg window with transparent tape. The small holes of the air chamber are sealed with paraffin, and the inoculated chicken embryos are placed flat in the incubator with the egg window upwards. Do not transfer.
Yolk sac inoculation: suitable for separation and culture of Marek's virus, Toga virus, Chlamydia and Rickettsia. Inoculation method: After the eggshell at the end of the air chamber is disinfected, a small hole is made in the center of the air chamber, and a 7-gauge needle syringe is used to vertically pierce the person for about 3 cm, and the inoculum is injected about 0.1 to 0.2 mL. Seal with paraffin and continue to incubate at 37°C, turning the eggs twice a day.
4 Incubation and observation
After inoculation, continue to incubate at 37°C, and take out the eggs twice a day. Generally, embryos that die within 24 hours are mostly caused by trauma or bacterial contamination, so they are discarded. Embryos that died after 24h were taken out and placed in a refrigerator at 4°C and marked. The length of incubation time after inoculation varies with the type of virus. Generally, Newcastle disease virus, bronchitis virus, egg drop syndrome virus and bursal disease virus need to incubate for 48 to 120 hours, and laryngotracheitis virus and Marek's virus need to incubate for 48 to. At 96h, the poxvirus must be incubated for 4 to 5 days, and the encephalomyelitis virus must be incubated until the chicks emerge.
5 Collect poison
After the inoculated chicken embryo hatches to the specified time, it should be taken out of the incubator and frozen at 4°C for about 36 hours, or quick-frozen below 10°C for about 4 hours, but the chicken embryo should not be frozen, otherwise hemolysis will easily occur. After freezing, follow the steps below to collect poison. Place the chicken embryo on the egg tray or egg rack with the air chamber upward. After disinfection with iodine tincture and alcohol, use sterile forceps to knock open the air chamber eggshell or remove the transparent adhesive paper of the artificial egg window to expose the egg membrane. Be careful not to let the egg shell fragments fall into the chicken embryo. Allantoic fluid: After the air chamber is opened, use sterile ophthalmic forceps to slowly tear off the egg shell membrane, then clamp the chorioallantoic membrane and gently lift it up, puncture the choriouric membrane with a Pasteur pipette to directly suck the allantoic fluid. To prevent the amniotic membrane from blocking the straw opening, use tweezers to gently press down the amniotic membrane and fetal membrane, and then suck again. Each chicken embryo can generally receive about 8mL allantoic fluid. Amniotic fluid: After sucking the allantoic fluid, use forceps to clamp the amniotic membrane, insert the Pasteur pipette directly into the amniotic cavity to absorb the amniotic fluid, and then lift the amniotic membrane with forceps or press the fetus until the amniotic fluid is harvested. A considerable part of the viral amniotic fluid and allantoic fluid can be mixed. Some can be mixed, and the two should be received and released separately. Chorioallantoic membrane: If you want to harvest the entire allantoic membrane, you can disinfect, open the egg shell, tear off the shell membrane, and pour out the entire egg content. At this time, the chorioallantoic membrane is attached to the inner wall of the egg shell and used Clamp it with tweezers and tear it off. Embryos: After disinfecting and opening the egg shell of the air chamber, tear the egg shell membrane and chorioallantoic membrane with tweezers, clamp the embryo, cut the yolk band with scissors, and put the fetus in the sterilization container.
