228 IMMUNOLOGY 



tube added 0.05 c.c. of immune serum. The tubes were incubated 

 at room temperature, apparently witliout shaking, and observed 

 closely during the first tive minutes and at intervals for 24 hours. 

 Adequate controls were also included. 



Quantitative Technique of Nuttall.— In his quantitative 

 technique he employed as a standard, representing 100 per cent 

 precipitate, the amount formed when 0.1 c.c. of antiserum is 

 added to 0.5 c.c. of a 1 :100 or 1 :200 dilution of homologous antigen. 

 He considered it necessary that the ratio of antiserum to antigen 

 be 20 :1 to 200 :1 or more. After the antiserum is added the tubes 

 are shaken. 



Time of Incubation. — The tubes are then incubated for twenty- 

 four hours, the supernatant fluid decanted, and the precipitate 

 drawn up into capillary tubes 12 cm. long and having a lumen 

 of 1 mm. The dry end of each capillary tube is sealed and the 

 tubes are allowed to stand vertically for 24, 48 and 72 hours and 

 the volumes (height) are measured. 



The Ring Test.— He says that Ascoli (1903) suggested stratify- 

 ing immune serum under varying dilutions of antigen and deter- 

 mining the highest dilution of antigen that produces a ring of 

 precipitate at its junction with immune serum. This is essentially 

 the ring technique of Fornet and IMiiller (1910) and the one Avhich 

 Hektoen (1928) seems to prefer. In this test small clean vials or 

 test tubes having a diameter of approximately 0.5 cm. are placed 

 in a special rack. A series of dilutions of antigen varying from 

 1:1,000 to 1:10,000 or 1:20,000 are prepared. By means of a 

 capillary pipette a measured amount of saline (0.85 per cent) 

 is put into the last vial, the same amount of the highest dilution 

 of antigen is put into the next vial and so on until vial I receives 

 the same amount of the 1 :1,000 dilution of antigen. With an- 

 other capillary pipette a measured amount of clear immune serum, 

 to be titrated, is stratified in the bottom of the saline control 

 and a corresponding amount stratified under each antigen dilu- 

 tion, beginning with the highest dilution and proceeding to the 

 lowest. Instead of stratifying the immune serum under the anti- 

 gen dilutions as recommended by Hektoen (1928) some prefer 

 to layer the antigen dilutions over 0.1 c.c. of immune serum that 

 has previously been placed in each vial. The tubes are incubated 

 at room temperature and the titer is recorded as the highest 



