VIROLOGICAL METHODS 253 



desirable for maximum production of viable virus. Not uncommonly 

 one may sacrifice high-infectivity titers in his harvests if a high concen- 

 tration of seed virus is used. This is particularly true if strictly fresh 

 seed virus is not used. It is said to be the result of inactive virus inter- 

 fering with the growth of active virus. Inocula of high viral concentra- 

 tion are more likely to produce the course of disease and pathology of 

 toxicity rather than that of infection. It should be remembered that high 

 dilutions of virus are prone to become inactive more readily than low 

 dilutions. Obviously, the investigator must vary the concentration of 

 the inoculum to suit his special purposes. 



Inoculation, A few additional suggestions might be in order concern- 

 ing the actual inoculation of animals. The mortality rate of intranasal 

 inoculation attributable to the inoculation procedure, per se, increases 

 under certain conditions. Hot humid w^eather, deep instead of light 

 ether anesthesia, and an inoculum of high viscosity may all increase the 

 mortality rate. It is especially desirable to anesthetize the animals 

 lightly for intracranial inoculation and to inoculate as quickly as possible. 



Intracranial. Before starting intracranial inoculations it is advantage- 

 ous to open the skull of a deceased animal and study the dimensions and 

 relationships of the brain to the cranial vault. Placing the inoculum in 

 the center of the brain substance is usually desirable. The mouse is 

 probably used for intracranial inoculation in virology more often than 

 other animals. A 0.25-ml syringe fitted with a sharp 27-gauge needle 

 aids in injecting and controlling the small volumes required, 0.01-0.03 ml. 



While the operator is seated at a desk, the mouse is grasped by the 

 dorsal skin behind the head, using the thumb and forefinger, and pressed 

 gently against a firm surface. The hand holding the syringe is steadied on 

 the desk and rocked slowly forward to make the inoculation. It is wise 

 to hold the needle in situ for a few seconds. Usually less inoculum follows 

 the needle when you withdraw it if this procedure is followed. Larger 

 animals may require trephining as described in Chap. X. 



Intravenous. Several factors aid in making successful intravenous 

 inoculations in mice. Three- to four-week-old mice are easier to inoculate 

 than older mice. If the mice are placed in a 37°C incubator J^ hr before 

 inoculation, the tail-vein dilation makes the task easier. A 0.25-ml 

 syringe fitted w^ith a sharp 27-gauge needle is ideally suited for the pro- 

 cedure. The distal one-half of the tail vein is more readily entered than 

 the proximal one-half. The tail, needle, and syringe should be held so 

 the approach is one of putting a stilette in a needle. Trying to enter the 

 vein at an angle will usually fail. If the distal end of the tail is bent over 

 the forefinger and secured with the thumb, the remainder of the tail vein 

 can be approached as described above. 



Intratesticular. Intratesticular inoculation is used to propagate some 



