496 BACTERIOPHAGES 



T7 is made and the mixture diluted so that a 0.1 ml. sample will 

 contain about 100 particles of each kind of virus. This mixture 

 of viruses is to be plated by the agar layer technique on strain B, 

 on each indicator strain separately and on a mixture of the 2 in- 

 dicator strains. Both phage Tl and phage T7 produce large 

 plaques, but the plaque morphologies are distinctive enough so 

 that an experienced observer can usually distinguish them. In 

 the case of other pairs, such as T2 and T6, this is usually not pos- 

 sible. The appropriate indicator strains to use are B/1,5 and 

 B/3,4,7. The efficiency of plating of each phage on the sensitive 

 indicator strain is better than 90 per cent as compared with 

 strain B, even when an excess of the other phage is present. 

 Slants of 2 per cent agar heavily inoculated with B, with B/1,5 

 or with B/3,4,7 have been incubated overnight. Two ml. of 

 sterile broth is added to each slant, the bacterial growth is rubbed 

 off with the pipet and dispersed by filling the pipet and forcibly 

 blowing out the contents several times. Strains B and B/1,5 are 

 readily suspended, but B/3,4,7 is difficult to disperse and the 

 broth suspension may have to be aerated for 20 min. at 37 °C. 

 The suspension is allowed to stand for 10 min. so that undis- 

 persed clumps will settle out. The bacterial suspensions are 

 then removed with pipets and placed in test tubes. Usually the 

 suspensions of strains B and B/1,5 will be much more turbid 

 than the suspension of B/3,4,7. Broth is added to dilute the 

 more concentrated suspensions until the turbidities of the 3 sus- 

 pensions appear to be about the same. Then 1 part of the sus- 

 pension of B/1,5 is added to 4 parts of the suspension of B/3,4,7 

 to give the mixed indicator, and the suspensions of B and B/1,5 are 

 diluted 1/5 with broth. The suspension of B/3,4,7 is not di- 

 luted. These relative concentrations have been chosen by expe- 

 rience so that the larger inoculum of B/3,4,7 will compensate for 

 its slower growth rate and result in an equal development of tur- 

 bidity on the plate in 5-6 hr. when the plaques are well devel- 

 oped. With other combinations of indicator strains the propor- 

 tions to use in mixtures must be found by trial. A flask of 0.7 per 

 cent agar is melted, cooled to 45 °C. and dispensed in 2.5 ml. 



