350 COLLECTED STUDIES IN IMMUNITY. 



collect the blood directly from the vessels of the neck and then tie 

 the vessel. It is an easy matter to obtain very small quantities 

 of sterile pigeon blood from the wing veins by first carefully removing 

 the feathers, disinfecting the skin with alcohol and then after incising, 

 touching the skin as little as possible. 



For purposes of collecting the serum, the blood is either allowed 

 to stand overnight (see the preceding chapter), or by means of a 

 sterile funnel is allowed to flow into a sterile bottle containing sterile 

 glass beads or steel shavings. The bottle is then stoppered with 

 a cork (previously burnt off), the blood defibrinated by shaking, 

 and then centrifuged. As a rule, centrifuging does not injure the 

 serum, especially if afterwards the upper layer of fluid is siphoned 

 off. For absolutely certain sterility the spontaneous separation 

 of the serum is to be preferred to defibrination and centrifuging. 



The active sera used for complementing are to be employed as 

 fresh as possible, in no case more than two or three days old (refriger- 

 ator). The immune sera, which are usually employed in the inactive 

 state, will keep in the refrigerator for a long time. Even in these, 

 however, a loss of power is observed. In the case of high-grade 

 immune sera the addition of 0.5% phenol is allowable for preserva- 

 tion. In the small quantities in which the serum is used in experi- 

 ments (about 0.01 cc.) this amount of phenol is without effect either 

 on the bacteria or on the complements. 



Before commencing the experiment proper it is necessary to 

 determine what amount sown gives the most favorable results. 

 Thus in many experiments it may be of advantage to always sow 

 Vsoo cc. of a one-day bouillon culture, whereas with another bac- 

 terium sowing Viooo or Vioooo l^op of a one-day agar culture will 

 give more uniform results. It is further necessary to repeatedly 

 convince one's self that the control plates regularly show a uniformly 

 good growth, for only when that is the case can uniform results be 

 expected. For example, although the bacillus of hog cholera grows 

 very well on ordinary slant agar, the control plates may result 

 most irregularly. In that case one can make use of glycerine agar. 

 Other bacteria again do not bear suspension in 0.85% salt solution 

 at all well; in that case one must use bouillon cultures and make 

 the dilutions with bouillon instead of with salt solution. The dilu- 

 tion should always be managed so that the amount finally sown is 

 about 5-10 drops, for in sowing only 1 or 2 drops considerable varia- 

 tions in the number of colonies may occur. In any case, however, the 



