26 THE BACTERIOPHAGE 



material more or less fluid, pasty, or solid; or excreta admixed to 

 a greater or less degree with earth, organic debris, etc. In such 

 a case it is necessary to disintegrate as completely as possible 

 the material to be examined. 



To effect such a disintegration the most simple procedure consists in care- 

 fully suspending the material in bouillon, about 5 gm. to 50 cc. of the 

 medium, and incubating this suspension at 37°C . for from twelve to eighteen 

 hours. The bacterial fermentations which ensue, because of the diverse 

 organisms introduced into the medium, lead to a sufficient disintegration. 

 Upon removal from the incubator the material may be treated, as indicated 

 above, by filtration through infusorial earth and a bougie. 



If the material under examination contains the bacteriophage 

 and has been subjected to filtration, the ultramicrobe will be 

 found in the filtrate. The methods of purification outlined above 

 are applicable to two purposes, (A) the detection of a bacterio- 

 phage active toward a given bacterial type, and (B) to test the 

 activity of a bacteriophage, either upon diverse organisms or 

 against a single bacterial strain of indeterminate type. 



A. The first case is the more simple and will be considered 

 first, taking as an example the detection of a bacteriophage active 

 against B. dysenteriae Shiga. The day before the test is to be 

 made an agar slant is inoculated with the dysentery strain. From 

 this fresh culture, on the day of the test, four tubes of peptone 

 broth are inoculated. 2 To the first of these tubes is added one 

 drop of the filtrate, to the second, ten drops, and to the third, 

 two cubic centimeters. One tube, simply inoculated with the dys- 

 entery organism, serves as a control. The tubes are incubated 

 at 37°C. After twelve to eighteen hours one of several results 

 may be observed: the three tubes (in addition to the control tube) 

 may all show a turbidity due to the growth of the dysentery 

 organism, only one or two of the tubes may be turbid, or the 

 three tubes may be clear. 



1 As will be shown later this bouillon should be alkaline in reaction. 

 The ordinary neutral bouillon (I have always used by preference the 

 bouillon of Martin) to which is added 6 cc. of N/1 NaOH per liter is per- 

 fectly satisfactory. This is, moreover, the degree of alkalinity most 

 frequently employed in bacteriological work. 



