Chicken embryo culture experiments of animal viruses

Summary

Chicken embryo culture is a culture method used to cultivate certain animal viruses that are sensitive to chicken embryo, and this method can be used for the isolation and cultivation of many kinds of viruses, titration of virulence, neutralization test, and preparation of antigens and vaccines. The technique of chicken embryo culture is easier to succeed than tissue culture, and also easier than the animal source of inoculated animals, without special requirements of feeding management and isolation, etc., and the chicken embryo is generally free of viral latent infections, and at the same time it has a wide range of sensitivity to a wide variety of viruses, so it is a commonly used method of culturing animal viruses. Source: Experiments in Microbiology (Third Edition)

Operation method

basic program

Principle

In this experiment, chicken embryos were inoculated with poxvirus and chicken Newcastle disease virus. Poxvirus is suitable for growth on the chorionic allantoic membrane, and after incubation, it produced white pox blister-like lesions visible to the naked eye, resembling small nodules or small white pieces of cataract. Chicken Newcastle disease virus is suitable for inoculation in the allantoic cavity and amniotic cavity, after growth, the chicken embryo all over the body skin hemorrhagic spots, most notable in the back of the brain.

Materials and Instruments

Poxvirus (Vavvinia virus) Chicken Newcastle disease virus (Newcaatle distase virus) Fertilized eggs with white shells
2.5% iodine 70% ethanol
Egg incubator Egg inspection lamp Toothed drill Shell grinder Steel needle Egg stand Syringe Tweezers Scissors Sealing wax Sterilized Petri dish Sterilized coverslip

Move

1. Preparation of egg embryos

Before incubation, the chicken eggs should be washed with water and dried with a dry cloth, and then put into the incubator for incubation (37℃, relative humidity is 45～60%), after incubation for 3 d, the eggs should be turned over 1～2 times a day. Incubated to the 4th d, with the egg detection lamp to observe the development of chicken embryo, unfertilized eggs, only see the vague yolk black shadow, do not see the shape of the chicken embryo, this chicken eggs should be eliminated. Live embryos can see clear blood vessels and the dark shadow of the chicken embryo, larger ones can see the embryonic movement, and then observed once a day, the embryonic movement is stagnant or no movement, blood vessels are dark and fuzzy, that is, it may be dead or dying chicken embryo, should be eliminated at any time. The well-grown embryos should be incubated until the time of inoculation, and the specific age of the embryos depends on the type of virus to be cultivated and the route of inoculation.

2. Inoculation

2.1 Chorioallantoic membrane inoculation

2.1.1 Place the egg embryos incubated for 10~12 d on the egg detection lamp, and outline with a pencil where the chorionic allantoic membrane is well developed at the end of the gas chamber and the embryo slightly near the gas chamber.

2.1.2 Disinfect the top of the gas chamber and the chorionic allantoic membrane with iodine, and use a shell grinder or a tooth drill to grind a triangular or square (about 5-6 mm on each side) window in the eggshell at the marked area, without breaking the shell membrane below. Drill a small hole in the top of the air chamber.

2.1.3 Use small forceps to gently remove the eggshell at the small window opened, revealing the shell membrane under the shell, put a drop of saline on the shell membrane, and use the tip of the needle to carefully scratch the shell membrane, but be careful not to hurt the chorionic urothelium that is tightly adhered to the underside of the shell membrane, and at this time, the saline flowed from the breach to the chorionic crumb membrane, so as to facilitate the separation of the two membranes.

2.1.4 Use a needle to puncture the shell membrane at the small hole of the air chamber, and then use a rubber nipple to suck out the air inside the air chamber, so that the chorionic villous allantoic membrane is subsided to form an artificial air chamber (Figure 1).

Figure 1 Chorionic allantoic membrane inoculation method
A. Preparation before inoculation; B. State of chicken eggs after inoculation

2.1.5 Use a syringe to drop 0.05~0.1 ml of smallpox virus onto the chorioallantoic membrane through the hole in the shell membrane of the window.

2.1.6 Apply semi-solidified paraffin wax around the window of the eggshell to form a dike, and immediately cover with a sterilized coverslip. Alternatively, the eggshells can be sealed with the uncovered eggshells, then the eggshells are covered and the seams are coated with paraffin wax, but the paraffin wax must not be too hot so as not to flow into the eggs. The eggs were incubated at 37 ℃ with the artificial air chamber in the upper position at all times, and the results were observed for 48-96 h. The results were summarized as follows.

Temperature has a significant effect on the formation of foci such as poxvirus, and the incubation temperature should be strictly controlled at 37℃, and typical foci cannot be produced in chick embryos at incubation temperatures higher than 40℃.

2.2 Urocystic cavity inoculation

Use egg embryos incubated for 10~12 d, because this is the time when the allantoic fluid accumulates the most.

2.2.1 Illuminate the egg embryo on the egg inspection lamp, draw the position of the gas chamber and embryo with a pencil, and mark the place where the chorionic allantoic membrane is less vascularized.

2.2.2 Place the egg embryo vertically on the wooden frame of the egg holder with the blunt end upwards. The eggshell was sterilized with iodine in the gas chamber and a small hole was drilled at the mark with a steel needle.

2.2.3 A 1 ml syringe with an 18 mm long needle was used to draw up chicken Newcastle disease virus solution. The needle was pierced into the hole and 0.1 ml of virus solution was injected into the allantoic cavity through the chorioallantoic membrane (Fig. 2).

Fig. 2 Inoculation in the urinary bladder cavity

2.2.4 Seal the holes with paraffin wax and incubate at 37℃ for 72 hours.

2.3 Amniotic cavity inoculation

2.3.1 Visualize the egg embryos incubated for 10 to 11 d. Draw the range of air chambers and mark the side of the embryo closest to the egg shell.

2.3.2 Disinfect the eggshell with iodine at the site of the gas chamber. A tooth drill was used to make a triangular shaped fissure of about 1 cm on each side at the top of the gas chamber.

2.3.3 Remove the eggshell and shell membrane with sterilized forceps and add one drop of sterilized liquid paraffin to the lower shell membrane to make it transparent for observation, or more clearly if the embryo is placed on the egg detection lamp.

2.3.4 Use sterilized pointed forceps, with both pages well together, to pierce the lower shell membrane and chorionic villous urothelium where there are no blood vessels, and clamp the amniotic membrane and pull it out from the place where it has just been perforated (Fig. 3).

Figure 3 Amniotic cavity inoculation

23.5 Hold the amniotic membrane in the left hand with another pair of non-toothed forceps. In the right hand, hold a syringe with a 26-gauge needle and insert it into the amniotic cavity to inject 0.1 ml of Newcastle Disease Virus into the embryo, preferably with a blunt needle without a sharpened tip to avoid injury to the embryo.

2.3.6 Seal the small window of the eggshell with the closure method of the chorionic allantoic membrane inoculation method, incubate the eggs in an incubator at 37℃ for 48-72 hours, and keep the blunt end of the egg embryo facing up.

The process of virus inoculation of chicken embryos and the use of instruments should be strictly aseptic and carried out on a sterile bench as far as possible.

3. Harvesting

3.1 Chorionic allantoic membrane

3.1.1 Sterilize the eggshells on the artificial air chambers with iodine and remove the caps on the window holes.

3.1.2 Insert sterilized scissors into the window and cut off the shell membrane along the boundary of the artificial air chamber, exposing the chorionic allantoic membrane, then use sterilized ophthalmic forceps to pinch the membrane right in the middle, use scissors to cut off the membrane along the edge of the artificial air chamber, put it into a petri dish with sterilized saline, and observe the shape of the lesion. The membrane is then either used for passaging or preserved with 50% glycerol.

3.2 Urocystic fluid harvesting by inoculation of the urinary bladder cavity

3.2.1 Place the egg embryo in the refrigerator for half a day or overnight to allow vasoconstriction in order to obtain pure allantoic fluid without fetal blood.

3.2.2 The eggshell at the air chamber is disinfected with iodine and removed with sterilized scissors. The shell membrane and the chorionic allantoic membrane beneath it were incised and turned over to the side of the eggshell.

3.2.3 Tilt the egg to one side and aspirate the allantoic fluid with a sterilized pipette. About 6 ml of allantoic fluid can be harvested from one egg embryo. The harvested allantoic fluid should be temporarily stored in the refrigerator at 4℃, and then stored at -30℃ for a long period of time after passing the sterility test.

3.2.4 Observe the chicken embryo to see if there are typical symptoms.

3.3 Amniotic fluid harvesting by amniotic cavity inoculation method

3.3.1 Sterilize and remove the shells according to the method of harvesting allantoic fluid, and turn over the shell membrane and allantoic membrane.

3.3.2 Aspirate the allantoic fluid first.

3.3.3 Use forceps to clip out the amniotic membrane, insert a pointed capillary pipette into the amniotic cavity, aspirate the amniotic fluid and put it into a sterilized test tube, 0.5-1.0 ml for each egg embryo. after aseptic test, store it in low temperature.

3.3.4 Observe the symptoms of chicken embryos.

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