thymidine and hypoxanthine) not required exogenously by the cells for 
optimal growth. Mutants are detected by screening populations of cells 
exposed to test compounds for nutritionally deficient forms (auxotrophs) 
requiring one or more of the nine nutrilites omitted from FI 2D medium. 
Operationally, lO^ 7 cells are exposed in four parallel cultures to single or 
multiple doses of the test agent, following inoculation and cell attachment 
(point A, Figure 7-3). Because even induced mutation is a rare event, there will 
generally be, following expression, a small number of mutant cells growning 
among millions of nonmutants in enriched medium. To identify the mutants, it 
is necessary to introduce a procedure which will eliminate the prototrophic 
(wild-type) cells, while allowing auxotrophs to survive. This is accomplished by 
taking advantage of the fact that mutants auxotrophic for one or more of the 
nine nutrilites omitted from FI 2D medium will be unable to grow in this 
medium, whereas wild-types will. Thus, at point B, Figure 7-3, FI2 medium is 
replaced with FI 2D medium. This initiates the selective process by effectively 
terminating protein and nucleic acid synthesis. The thymidine analog, BrdU, is 
then added to the FI 2D medium from which it is incorporated into the DNA 
of wild-type cells (point C, Figure 7-3). Subsequent illumination of the cell 
population with white light is lethal to those cells having incorporated 
sufficient BrdU. Mutant cells do not incorporate BrdU, and survive the 
selective process (point D, Figure 7-3). Wild-type cells survive selection to an 
extent approaching 0.02 percent. In the presence of FI2 medium, mutant cells, 
along with some wild-types, grow into macroscopic colonies (point E, Figure 
7-3). These are picked and tested for mutant identification. It is important to 
note that the use of a known mutagenic agent (BrdU) in the selective process is 
of no consequence, as the only cells incorporating BrdU are wild-types destined 
for death. Mutants to be isolated are existent in the population at the end of 
the expression period prior to the application of selective conditions. 
The procedure illustrated in Figure 7-3 and described above was applied in 
collecting the mutagenesis data presented below. Figure 7-4 shows the lighting 
apparatus used to illuminate cell populations following exposure to BrdU. 
Figure 7-5 shows cell survival in four randomly selected dishes several days 
after illumination. Cells not killed in selection have grown into macroscopic 
colonies. Because mutant and wild-type cells differ only in their requirements 
tor exogenous nutrilites, they may be distinguished only by analysis of their 
growth properties in enriched and deficient media. 
Statistical Analysis of Data 
When testing compounds for mutagenic activity, we usually want to know if 
the number of mutants per unit number of viable cells screened is sufficiently 
larger in experimental, versus control situations, to support a conclusion of 
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