PRECIPITINS 227 



that 1.8 per cent saline be used in preparing dilutions of rooster 

 serum to avoid nonspecific reactions. 



When to Bleed Anbials. — In collecting blood from an im- 

 munized animal to be used in precipitin work, it is better to bleed 

 the animal just before feeding or after moderate fasting to avoid 

 cloudy or opalescent sera. Undoubtedly much of the difficulty 

 reported in the early literature was due to failure to observe this 

 precaution. 



Obtaining Immune Serum. — After it is determined that the 

 precipitin content is sufficiently high to react by the ring technique 

 to be described later, wdth dilutions of antigen of 1 :1,000 or 

 higher, the animals are bled to death u]ider aseptic conditions. It 

 is desirable to obtain the serum in a sterile condition, but if con- 

 tamination occurs the antisenun may be sterilized by filtration, 

 placed in sterile containers, sealed and kept in the refrigerator. 

 Preservatives should not he added, the serum should not be heated 

 or frozen. All cloudy or opalescent sera should be discarded. 



Titration After Uhlexhutii. — The next step would be the titra- 

 tion of the antiserum. According to Uhlenhuth (1909, p. 815) 

 he prepared three dilutions of antigen 1 :1,000, 1 :10,000 and 

 1:20,000 using physiological (0.85 per cent) saline as a diluent. 

 He then placed 4 small, clean test tubes in a rack and with a 

 sterile pipette added one cubic centimeter of sterile saline to tube 

 IV and one cubic <^entimeter of the 1 :20,000 dilution of antigen 

 to tube III, a corresponding amount of the 1 :10,000 dilution to 

 tube II and of the 1 :1,000 to tube I. He next took a sterile one 

 cubic centimeter pipette graduated to 1/100 of a cubic centimeter 

 and added 1/10 of a cubic centimeter of the clear immune serum 

 to each tube. The tubes were not shaken and were incubated at 

 room temperature and observed for five minutes. Tube IV re- 

 mained clear, but he says that a good serum should give a positive 

 reaction in tube I within two minutes and that within 3 to 5 

 minutes tubes II and III may become cloudy and a precipitate 

 form and settle out. The titer is recorded as the highest dilution 

 giving a definite reaction. 



Technique of Nuttall. — Nuttall (1904) in his routine qualita- 

 tive work employed a similar technique. As a rule he used one 

 cubic centimeter of a 1 :100 to 1 :200 dilution of antigen and to each 



