90 THE BACTERIOPHAGE AND ITS BEHAVIOR 



The following experiments permit of no doubt on this question. 



Bail/^ Doerr and Berger/^^ and Nakamura''^^ have observed that 

 plaques do not form on gelatin, and they have invoked the intervention 

 of colloidal reactions to explain this fact. The deduction of these 

 authors is absolutely correct, and the following experiments provide the 

 true explanation for the inhibition which they observed. 



Prepare three series of Petri dishes, as follows : (a) containing an agar 

 medium (bouillon with 2 per cent agar) ; (6) containing a gelatin medium 

 (bouillon with 15 per cent gelatin; (c) containing agar (the 2 per cent 

 agar of (a)) to a depth of 8 mm. Immediately after the introduction of 

 the agar the dishes of this series are placed in a horizontal position 

 (obtained by means of levelling screws) and the agar is allowed to solidify. 

 They are then placed in an incubator at 45°C. to warm them somewhat, 

 and over the surface of the agar is poured a quantity of gelatin (the 15 

 per cent gelatin mentioned above, in (6)), melted and cooled to about 

 60°C., in such a way that by tipping the dish in all directions the entire 

 surface is covered by a thin layer. Adjust the dishes with the levelling 

 device so that they are perfectly horizontal and allow the medium to 

 harden in the ice-box. Prepared in this way these dishes will con- 

 tain a layer of agar upon which is superimposed a thin layer of gela- 

 tin. When planted the material is distributed solely upon the gelatin, 

 and this medium will differ in no way from that present in the dishes 

 containing gelatin alone (series b, above). The only difference will be 

 that the c series will have a substratum of agar into which those pro- 

 ducts resulting from reactions taking place on the surface may diffuse. 



When these Petri dishes are thus ready, take 4 tubes, each containing 

 a suspension of Shiga bacilh, 250 million per cubic centimeter. Inocu- 

 late the first with 0.1 cc. of a Shiga-bacteriophage; the second with 0.1 

 cc. of the first: the third with 0.1 cc. of suspension 2; and the fourth 

 with 0.1 cc. of number 3. 



With these four suspensions the Petri dishes are implanted, 0.05 cc. 

 of each of the individual suspensions being spread uniformly over the 

 surface of a dish. When the results are read after 4 days, we find : 



I. Agar plates held at 37°C. 



Suspension 1 . . . Surface sterile 



Suspension 2. . . A few scattered traces of growth 



Suspension 3 . . . Fragments of growth 



Suspension 4. . .About 50 plaques, having a diameter of about 5 mm. 



