| | 2.0, and 4.0 ml. | | | | Series 3: Subcutaneous, 0.5, | | 1.0, 2.0, and 4.0 ml. --------------------------+----------+--------------------------------- _Pipilo erythrophthalmus_ | 0.92 | Antiserum not prepared. --------------------------+----------+--------------------------------- _Junco hyemalis_ | 0.56 | Same as for _Spinus_. --------------------------+----------+--------------------------------- _Spizella arborea_ | 0.48 | Same as for _Spinus_. --------------------------+----------+--------------------------------- _Zonotrichia querula_ | 0.48 | Same as for _Spinus_. --------------------------+----------+--------------------------------- _Zonotrichia albicollis_ | 0.92 | Antiserum not prepared. (Gmelin) | | --------------------------+----------+--------------------------------- [A] Antiserum prepared against formolized antigen. Preparation of Antisera All antisera were produced in rabbits (laboratory stock of _Oryctolagus cuniculus_). Three methods of injection of antigen were used in various combinations: intravenous, subcutaneous, and intraperitoneal. Injection schedules used in the production of each antiserum are listed in Table 1. Both formolized and "native" antigens were used. Each rabbit received one or more series of four injections, each injection being administered on alternate days and doubling in amount: 0.5 ml., 1.0 ml., 2.0 ml., and 4.0 ml. In all but two instances more than one series of injections was necessary to produce a useful antiserum. More than two series, however, resulted in little or no improvement of the reactivity of the antiserum. The injection-series were separated by intervals of eight days. On the eighth day after the last injection of each series, 10 ml. of blood were withdrawn from the main artery of the ear of the rabbit, and the antiserum was used in a homologous precipitin test to determine its usefulness. If the antiserum contained sufficient amounts of antibodies to conduct the projected tests, the rabbit was completely exsanguinated by cardiac puncture, by using an 18-gauge needle and a 50 ml. syringe. The whole blood was placed in clean test tubes and allowed to clot. It was allowed to stand at 2°C. for 12 to 18 hours so that most of the serum would be expressed from the clot. The serum was then decanted, centrifuged to remove all blood cells, sterilized in a Seitz filter, bottled in sterile vials, and stored at 2°C. until used. Methods of Serological Testing