224 



produced a filtrable agent (FA), under 

 stimulation from LA-22, that could 

 elicit prototrophs from LA-22. Fil- 

 trates of LA-22 cultures, containing 

 substantial amounts of phage (PLT- 

 22) active on LA-2, also stimulated FA 

 production from LA-2. The role of 

 this phage will be discussed later. 



To help the further exposition of 

 our experiments, we shall use the term 

 transduction for genetically unilateral 

 transfer in contrast to the union of 

 equivalent elements in fertilization. 

 The working hypothesis that 5^;/- 

 monella FA is an agent of genetic 

 transduction provides a useful frame 

 of reference for our discussion. 



Assay of FA. Stock FA was pre- 

 pared by growing LA-22 and LA-2 in 

 mixed culture in broth. After 48 hours, 

 the cells were sedimented and the 

 supernatant passed through a sintered 

 pyrex filter. The sterility of a filtrate 

 was verified by inoculating samples 

 into broth at the time of preparation 

 and by platings in agar as controls for 

 particular experiments. This precau- 

 tion was taken although complete 

 sterility is not critical to most experi- 

 ments since more than a million cells 

 of LA-2 per plate are needed to inter- 

 act with LA-22 to give prototrophs 

 in the "direct crosses". These prepara- 

 tions have been stored in the refrigera- 

 tor for several months without loss of 

 activity. 



A standard procedure for assay of 

 FA was developed for further work. 

 LA-22 was grown on nutrient agar 

 plates and harvested in dense saline 

 suspension. The viable count was ob- 

 tained by plating suitable dilutions on 

 nutrient agar. Various dilutions of cells 

 were plated with a constant volume 

 of an FA preparation on minimal agar. 

 Prototrophs appeared at 24 hours and 

 were counted after 48 hours. Figure 1 

 shows that a constant response was 

 found with about 10^ to lO^*' cells per 



ZINDER AND LEDERBERG 



plate. The decline at high cell den- 

 sities was probably due to overcrowd- 

 ing and inhibition of colony forma- 

 tion, and at lower densities to physical 

 separation of cells and agent or to the 

 saturation of susceptible cells. 



lO'* cells of LA-22 were plated with 

 serial dilutions of FA. Over a consider- 

 able range a linear relationship was 

 found between the yield of proto- 

 trophs and amount of FA (figure 1). 

 The efi^ect of higher concentrations of 

 FA will be discussed in a later section. 



A unit of FA may be defined as the 

 content of a filtrate that will elicit a 

 single prototroph from an optimum 

 concentration of LA-22 cells. Filtrates 

 from mixed cell preparations usually 

 contain about 2,500 such units per ml. 



Cheffiical reactivity of FA. With 

 the development of a standardized 

 assay it was possible to compare the 

 effects of various treatments on FA 

 and bacterial cells. The latter are steri- 

 lized by shaking with such agents as 

 chloroform, toluene, alcohol, and 

 formalin. Of these only formalin in- 

 activates FA. The bacteria are steri- 

 lized by heating at 56 C for 30 minutes. 

 Temperatures of 70 C are necessary 

 for detectable effects on FA. It is 

 rapidly inactivated only when 100 C 

 is approached. 



FA is quantitatively precipitated 

 from broth by one to two volumes of 

 cold alcohol or half saturated ammo- 

 nium sulfate. A heavy floe appears in 

 both cases which, for the most part, 

 remains water insoluble; FA, however, 

 redisperses. 



None of several enzymes tested 

 affected FA. They were added di- 

 rectly to the active filtrates and in- 

 cubated for two hours. The tests in- 

 cluded pancreatin (100 mg/ml), tryp- 

 sin (100 jLtg/ml), Taka-diastase (100 

 mg/ml), ribonuclease (10 fig/ml), and 

 desoxyribonuclease (20 jxg/ml). The 

 failure of desoxyribonuclease to inac- 



