366 GROWTH OF BACTERIOPHAGE 



EXPERIMENTAL 



Bacteria Culture. — Our host organism was a strain of Escherichia coli, which 

 was kindly provided by Dr. C. C. Lindegren. Difco nutrient broth (pH 6.6- 

 6.8) and nutrient agar were selected as culture media. These media were selected 

 for the present work because of the complications which arise when synthetic 

 media are used. We thus avoided the difficulties arising from the need for 

 accessory growth factors. 



Isolation, Culture, and Storage of Phage. — A bacteriophage active against this 

 strain of coli was isolated in the usual way from fresh sewage filtrates. Its homo- 

 geneity was assured by five successive single plaque isolations. The properties of 

 this phage remained constant throughout the work. The average plaque size on 

 1.5 per cent agar medium was 0.5 to 1.0 mm. 



Phage was prepared by adding to 25 cc. of broth, 0.1 cc. of a 20 hour culture 

 of bacteria, and 0.1 cc. of a previous phage preparation. After 3^ hours at 37° 

 the culture had become clear, and contained about 10^ phage particles. 



Such lysates even though stored in the ice box, decreased in phage concentra- 

 tion to about 20 per cent of their initial value in 1 day, and to about 2 per cent 

 in a week, after which they remained constant. Part of this lost phage activity 

 was found to be present in a small quantity of a precipitate which had sedimented 

 during this storage period. 



Therefore, lysates were always filtered through Jena sintered glass filters (5 on 

 3 grade) immediately after preparation. The phage concentration of these fil- 

 trates also decreased on storage, though more slowly, falling to 20 per cent in a 

 week. However, 1:100 dilutions in distilled water of the fresh filtered lysates 

 retained a constant assay value for several months, and these diluted preparations 

 were used in the work reported here, e.xcept where otherwise specified. 



This inactivation of our undiluted filtered phage suspensions on standing is 

 probably a result of a combination of phage and specific phage inhibiting sub- 

 stances from the bacteria, as suggested by Burnett (4, 5). To test this hypothesis 

 we prepared a polysaccharide fraction from agar cultures of these bacteria, ac- 

 cording to a method reported by Heidelberger et al. (6). Aqueous solutions of 

 this material, when mixed with phage suspensions, rapidly inactivated the phage. 



Method of Assay. — We have used a modification of the plaque counting method 

 of d'Herelle (7) throughout this work for the determination of phage concentra- 

 tions. Although the plaque counting method has been reported unsatisfactory 

 by various investigators, under our conditions it has proven to be entirely satis- 

 factory. 



Phage preparations suitably diluted in 18 hour broth cultures of bacteria to 

 give a readily countable number of plaques (100 to 1000) were spread with a bent 

 glass capillary over the surface of nutrient agar plates which had been dried by 

 inverting on sterile filter paper overnight. The plates were then incubated 6 to 

 24 hours at 37°C. at which time the plaques were readily distinguishable. The 

 0.1 cc. used for spreading was completely soaked into the agar thus prepared in 



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