RESISTANCE OF BACTERIA 237 



on ordinary agar may not suffice, for all of the colonies may then be 

 contaminated. I would recommend in such a case''-^ the use of an agar 

 containing sugar, that is to say, a medium containing some sugar fer- 

 mented by the bacterium which is undergoing purification. Here it is 

 necessary to resort to several successive purifications, each time taking 

 the material for the new isolation from the margin of a colony as close 

 as possible to the edge. 



If the mixed culture can not be purified by this method it may be 

 necessary to carry out a series of cultures in liquid media, acid in reac- 

 tion and at the limiting temperature of the culture. In other words, 

 it may be necessary to make a series of 8 to 10 cultures at the highest 

 temperature in media containing the greatest amount of acid compatible 

 with the development of the bacterium involved. After this ultrapure 

 colonies may be obtained by cultivation upon a sugar-containing agar. 

 These methods are based upon the fact that the bacteriophage may not 

 develop in a medium whose acidity still allows the development of 

 bacteria, especially at the temperature limit for the development of the 

 bacterium. 



An additional method for isolation, proposed by Prausnitz,^^^ depends 

 upon the use of gelatin as a culture medium. 



Still another method, appHcable to certam cases, consists in the use 

 of the antiserum. Such an antiserum is prepared by giving a rabbit a 

 series of injections of the natural mixed culture. It has been shown by 

 Bordet that the serum of an animal prepared by injections of mixed 

 cultures possesses antibacteriophage properties. Isolations can then 

 be made upon an agar medium in which this antiserum is incorporated. 

 From the point of view of efficiency this procedure is inferior to the 

 other methods described. 



It should be stated that in those very rare cases where isolation on 

 ordinary agar does not permit the selection of ultrapure colonies, 

 isolation upon sugar-containing agar has always permitted me to 

 isolate such a strain from naturally mixed cultures, except in one in- 

 stance (among about 100 experiments) , where it was necessary to employ 

 passages in an acid medium at the temperature limit. 



14. ISOLATION OF THE BACTERIOPHAGE FROM NATURALLY MIXED 

 CULTURES 



When dealing with bacteria belonging to homogeneous species it is 

 usually only necessary to filter a bouillon preparation of the mixed 

 culture and to combuae the filtrate with an ultrapure suspension of 



