464 BACTERIOPHAGES 



time, 5 min. or 30 min. It must be remembered that the 

 equation describes the course of the reaction only over a hmited 

 range of inactivation, usually between 90 and 99 per cent of the 

 phage, and cannot be used outside this range. Also, the value 

 of K is not absolutely independent of the concentration of serum 

 but usually increases somewhat as D increases. However, the 

 A' value is an extrem.ely valuable characteristic of the serum 

 within its limitations. 



Procedure. For use, the serum is diluted in the same medium 

 in which the phage is to be diluted, usually broth or some diluent 

 (p. 454), with the additional proviso that the diluent should be 

 approxim.ately isotonic so that the serum globulins will not 

 precipitate. The phage stock is diluted to a titer of lOVml. 

 and 0.1 ml. of phage is added to 0.9 ml. of diluted serum at 37 °C. 

 At 5 min. intervals 0.1 ml. samples of the phage-serum mixture 

 are added to 9.9 ml. of diluent to stop antibody reaction and 

 0.1 ml. samples of this dilution are plated by the agar layer 

 technique. If no inactivation of phage has occurred, about 

 1,000 plaques will be found after incubation of the plates; 

 after 90 per cent inactivation the count will be 100, etc. The 

 serum should be tested at dilutions of 1 :100 and 1 : 1,000. 



From such an experiment it should be possible to calculate the 

 K value of the serum, the range of inactivation over which the 

 equation holds and the effect of serum dilution on the K value. 

 For instance if the serum at a dilution of 1 : 1 00 inactivates 90 

 per cent of the phage in 5 min., the K value = 2.3 X 100/5 X 

 log 100/10 = 46. The higher the A' value of a serum, the 

 further it can be diluted and still give satisfactory neutralization 

 of phage. Various phages differ greatly with respect to their 

 rates of reaction with homologous antisera. The K values of 

 antisera against coli phages T3 and T7 usually range between 

 500 and 3000, and against T2, T4, and T6, between 200 and 

 1,000. The coliphages Tl and T5 appear to react quite 

 slowly with their antisera since the K values usually range be- 

 tween 20 and 100 and it is most unusual to obtain a serum with 

 a K value higher than 200 for these phages. 



