HAAS, ET AL.: MUTATION INDUCTION IN BACTERIA 147 



2. For prototrophic reversion. — M medium supplemented with 

 2.5 per cent Difco nutrient broth and solidified with 3 per cent Bat to 

 agar was used for final platings. The low amino acid concentrations 

 in this medium allows unreverted cells to make sufficient divisions to 

 develop small visible colonies (26). It also enables reverted cells to 

 become phenotypically expressed. Unreverted survivors are counted 

 as small colonies on high-dilution platings, while revertants appear 

 as large colonies against a background of auxotrophic growth at low 

 dilutions. 



3. For mutation expression. — M medium solidified with 3 per 

 Baci 

 medium. 



cent Bacto agar was used. No nutrient broth was added to this 



Radiation sources 



Ultraviolet irradiation (UV). — A model 30600 Hanovia mercury- 

 vapor lamp. The UV output at the position of the cells was 92.5 

 ergs /mm 2 /sec at wave lengths below 2800 A (determined by a Han- 

 ovia model AV-971 ultraviolet meter). 



X-ray.— A General Electric "Maxitron 250" unit set at 200 KVP 

 and 30 MA, with 1-mm aluminum filtration added. X-ray output at 

 the locus of cell suspensions was approximately 2860 r/min. 



Preirradiation and postirradiation treatments 



The techniques employed have been previously described (3, 4, 

 7, 9). Briefly they are as follows: A 24-hour-old slant culture was used 

 to inoculate 50 ml sterile M medium (plus growth factors in the case 

 of auxotrophic strains). This culture was grown for 15.5 hours at 

 37° C with aeration, and then held at 6 C for 1 hour to synchronize 

 cell division. The cells were centrifuged down in the cold and 

 resuspended in 50-ml fresh M medium (plus supplements if neces- 

 sary). In the preirradiation experiments supplements under test were 

 added to this medium. The culture was grown for an additional 

 period, aliquots taken for test at various intervals, and chilled to halt 

 further growth. For postirradiation experiments the cells were incu- 

 bated for 50 minutes before chilling. Chilled cells were washed with 

 cold 0.9 per cent saline and M medium, then resuspended in cold 

 M medium so as to titer approximately 6 X 10 8 colony-forming organ- 

 isms per ml in the UV studies and 2 X 10 10 organisms per ml in the 

 X-ray studies. Aliquots of this suspension at proper dilution were 



