VIROLOGICAL METHODS 263 



results should be viewed with suspicion. Sometimes only a few eggs out 

 of several inoculated will show the undesirable signs and can be dis- 

 carded, thus saving the bulk of the harvest. A 20-gauge needle fitted to 

 a 5- to 10-ml syringe works well for removing the fluid. The needle is 

 inserted into one of the larger pockets of fluid close to the shell, and with a 

 gentle suction the fluid is removed. If suction is applied too rapidly, the 

 membranes are pulled to the needle opening and block the aspiration 

 procedure. Depressing the membranes with a sterile spatula helps 

 obviate the latter problem. 



Allantoic fluid can be aspirated with ease from the windowed eggs 

 because a considerable area of the membranes and embryos is under 

 direct vision. A good light source appropriately directed and close to 

 one's work is invaluable for any type of harvest. From 5 to 10 ml of 

 allantoic fluid can be drawn from the allantoic sac, depending on the age 

 of the embryos and the agent under propagation. 



Amniotic fluid. Withdrawing amniotic fluid requires more technical 

 skill than other methods of harvest, especially if it is desired to keep it 

 free from the other fluids. Perhaps one of the more certain methods 

 involves preliminary removal of as much allantoic fluid as possible, 

 followed by careful evacuation of the eggs' contents into a sterile petri 

 dish. Care is exercised not to break the allantoic and yolk sacs and to 

 have the embryo end up on the top side. While one is seated at a table 

 to steady one's self, a 22-gauge l3^^-in. needle, fitted to a 1.0-ml tuberculin 

 syringe, is used to withdraw the fluid. By careful observation a pocket 

 of fluid somewhere around the embryo is located. With the hand holding 

 the syringe steadied on the table, the needle is carefully inserted into the 

 pool of fluid. The other hand is held in readiness to draw back on the 

 plunger of the syringe. Success comes to those with sharp eyes and 

 steady hands. About 0.1 ml of air can be injected to verify the position 

 if desired once the operator feels he has the needle in the amniotic sac. 

 The bubble will be trapped in the sac surrounding the embryo and not 

 move out into the large allantoic sac. From 0.1 to 2.0 ml may be drawn 

 from the amniotic sac, depending on many variables. 



Yolk fluid. Yolk fluid is sometimes harvested, although the mem- 

 brane is probably more commonly collected. Fluid can be withdrawn if 

 a large-bore needle, e.g., 18-20-gauge, is used. Following some infec- 

 tions the yolk granules are quite fluid. Other infections do not produce 

 this effect, and the yolk, particularly in older embryos, is thick and 

 viscid and therefore difficult to withdraw. 



Membranes. If viruses are propagated on the membrane, the egg 

 has probably been windowed. When the chorioallantoic membrane is 

 harvested, the gummed tape is removed and the shell broken away down 

 to the membrane. Care is used to avoid shell fragments falling on the 



