280 MANUAL OF MICROBIOLOGICAL METHODS 



24-48 hr should be allowed to incubate an extra day. End points may 

 be reported on pooled specimens of like dilutions, or the 50 per cent end 

 points may be calculated as discussed elsewhere. 



Route of inoculation. The route of inoculation will vary according 

 to the potentiality of the underneutralized virus to produce a recognizable 

 end point. Dermatropic viruses ordinarily would be inoculated intra- 

 cutaneously in a susceptible animal, although other methods, such as 

 corneal scarification, might be used. Neutralization end points are 

 known to vary, even with the same virus and antibody preparations, 

 when different routes of inoculation are used even in the same species of 

 animal. 



Bleeding mice for serum. Mice are not uncommonly used to produce 

 antibody where small amounts of several different kinds are desired. 

 Eight-week-old mice will yield about 0.25 ml of serum each. Since mice 

 are susceptible to several virus diseases, convalescent as well as hyper- 

 immune serum may be obtained. Mice can be bled quickly if a suitable 

 method is used. The mice are anesthetized in an ether jar. They are 

 then held in one hand by the scruff of the neck ventral side up. The 

 thorax is punctured at the xiphoid process with the sharp blade of a 

 pair of scissors. The blades are closed to make a diagonal cut toward 

 one shoulder and withdrawn. The incision is inverted over a test tube 

 having an 18-mm opening. The blood that will flow out quickly is 

 collected, and the mouse is placed on the table, cut side up. A second 

 mouse is treated in the same fashion. In returning to the first mouse 

 more blood can be collected. With a little practice and forethought one 

 operator learns to have three to four mice under the proper depth of 

 anesthesia at all times as he bleeds others more or less continuously. A 

 practiced operator can bleed 100 mice in an hour. While this method of 

 bleeding will cause some hemolysis, the hemolysis can be minimized by 

 centrifuging the blood immediately after it is collected. 



To obtain clear serum or serum specimens from individual mice requires 

 more time and patience. The anesthetized mouse is secured, ventral side 

 up, on a mouse board. Spring clamps, which can be easily and quickly 

 released, are used to immobilize his extremities. At least 0.1 ml of blood 

 may be obtained by cardiac puncture, using a 27-gauge needle and 

 0.25-ml tuberculin syringe. It is advisable for two people to cooperate, 

 one to handle the mice and the other to bleed. Depending on the 

 amount of blood removed, the animals may be allowed to recover from 

 the anesthetic or sacrificed by returning them to the ether jar. 



CONCENTRATING VIRUSES 



Gravitation. For various reasons the virologist may wish to con- 

 centrate viruses or their products or both. To concentrate particles of 



