462 BACTERIOPHAGES 



immunologic stimulus and to use enough animals to allow for 

 individual variation in immunologic response. 



Procedure. The rabbit is the most convenient laboratory 

 animal to use for production of antisera. At least 3 animals 

 should be immunized with each antigen because rabbits im- 

 munized in the same way with the same antigen may vary 10-fold 

 in the final titer of antibody produced. This also is insurance 

 against loss of animals through sickness or accidental death. 

 The phage stock used for immunization should have at least 

 10^" plaque-forming particles/ml. and may have been produced 

 in either broth or chemically defined media with equally good 

 results. The phages should not be treated with formalin or 

 heat since they are not infectious for the rabbit. However, it 

 is usual to filter the phage stocks to remove bacteria because 

 bacterial infections can occur. 



The route of injection seems to be of no significance if high 

 titer stocks are used, for we have obtained equally good results 

 from subcutaneous, intravenous, and intraperitoneal injections. 

 If the stock is of low titer, or the phage a poor antigen, the 

 subcutaneous route is probably preferable. 



The time schedule of the injections is not particularly impor- 

 tant, provided only that enough antigen is given over a long 

 enough period. We have had good results with 2 injections of 

 5 ml. each per week for 3 weeks with a test bleeding 1 week 

 after the last injection. Test bleeding is most conveniently 

 made by slitting the marginal ear vein and collecting about 

 5 ml. of blood. The blood is allowed to clot at 37 °C. in pet- 

 rolatum-lined centrifuge tubes and placed in the refrigerator 

 overnight. After centrifugation the serum is decanted, cen- 

 trifuged again if necessary to remove residual red cells, and 

 stored in sterile screw-capped vials in the refrigerator. 



The serum is now assayed for antiviral activity and if suffi- 

 ciently potent for use the rabbit is bled by cardiac puncture. 

 As much as 50 ml. of blood may be removed without damage to 

 the rabbit. After a week's rest the rabbit may be given a second 

 course of injections and bled again. 



