Primary Isolation Using Guinea Pigs and Embryonated Eggs 



tubes and store as described above. Save one aliquot to be inoculated into embryonated 

 eggs.* Any remaining guinea pigs should be allowed to convalesce and then should be bled 

 by cardiac puncture approximately 4 weeks after they were inoculated with the tissue 

 suspension in order to obtain sera for serologic tests. 



D. Inoculating Embryonated Eggs 



1. Select twelve, 6- to 7-day-old embryonated hens' eggs from antibiotic-free tlocks. Candle 

 the eggs just before inoculating them to confinn that they are healthy. Label the eggs from 

 1 through 12 with a soft-lead pencil. 



2. Working in a biological safety cabinet, dilute a 1-ml aliquot of the lO'r guinea pig spleen 

 suspension with 9 ml of PBS to form a 1% suspension. Draw up 6 ml of the 1% suspension 

 into a 6-ml syringe fitted with a 20-gauge, ll/z" needle. Cover the needle with the shield. 



3. Disinfect the top of each egg with 70% ethanol (or tincture of Merthiolate). Allow the 

 disinfectant to air dry. Disinfect an egg punch by dipping it in 70% alcohol and passing it 

 through a tlame. Use it to puncture the top of each egg. Inoculate each egg with 0.5 ml of 

 the 1% guinea pig spleen suspension. (Be sure to insert the needle completely into the egg.) 

 Seal the eggs with Duco Cement or its equivalent, and then incubate them at 35°-37° C in a 

 humid incubator for 10 days. 



4. Candle the eggs daily and discard all that die within the first 3 days after inoculation. 

 Harvest eggs that die between the 4th and 10th days postinoculation on the day they die to 

 obtain infected yolk sacs. 



Harvesting Infected Yolk Sacs 



1. Disinfect the top of an egg with 70%' ethanol. 



2. Disinfect a pair of blunt forceps by dipping them in 70% ethanol and passing them 

 through a tlame. Crack the shell of the egg with one prong of the forceps, and remove the 

 uppermost quarter of the shell with the forceps. 



3. Disinfect a second pair of blunt forceps as above. Use them to separate the yolk sac 

 membrane from the other membranes; remove the yolk sac membrane, and place it in a 

 sterile petri dish. Aseptically tear off a small piece of the membrane and drop it onto a 2" x 

 2" sterile gauze pad. Place the remainder of the yolk sac into a sterile 13- X 100-mm 

 screw-capped test tube labelled with the appropriate egg number and date. 



4. Blot the excess yolk from the small piece of yolk sac membrane on the gauze pad with 

 another pad. Hold the tissue with sterile forceps, and use it to make smears on glass slides. 

 Label each shde with the appropriate egg number and date. (Be sure that a particular egg, 

 the smears of its yolk sac tissue, and the test tube used to store that yolk sac are assigned 

 the same numbers.) 



5. Wipe the excess tissue from the forceps with a clean 2" x 2" gauze pad, disinfect the 

 forceps as above, and continue to harvest any remaining infected yolk sacs as instructed. 



6. Flash-freeze the yolk sacs in a dry ice-alcohol bath, and store at -70° C. 



*lf embryonated eggs of the proper age are not available at the time the guinea pigs are sacrificed, guinea pig spleen suspensions 

 can be quick-frozen and stored at -70° C until such eggs are available. 



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