The amount of mutagenesis caused by a mutagen is determined by three factors: (1) the chemical reactivity of the mutagen with DNA, (2) the concentration of the mutagen and the DNA target, and (3) the amount of time the DNA is exposed to the mutagen. It is typically difficult to control the chemical reactivity of the mutagen, so the dose of a mutagen is usually controlled by varying the concentration of the mutagen or the time of exposure. A few practical considerations to think about when mutagenizing cells are listed below.

• When a population of cells are heavily mutagenized, many cells may accumulate multiple mutations. The probability that a mutagen will cause multiple mutations depends upon the dose of the mutagen, so it is possible to limit this problem by using a low dose of mutagen. (Of course, this will also decrease the mutant frequency.) The probability that a cell will recieve multiple mutations can be calculated from the Poisson distribution.

• Some mutations are lethal -- that is, they prevent the further reproduction of the cells. This may be because the mutation blocks DNA replication (for example, cross-linking of the DNA strands by mitomycin C) or because the mutation prevents the synthesis of some essential gene product (for example, a subunit of RNA polymerase). If the dose of the mutagen is too high, multiple mutations will occur in every cell but lethal mutations will decrease the recovery of desired mutants (because you can only isolate a mutant if the cell survives). A plot of the number of mutants obtained at a given dose of mutagen is called a "dose-response curve". An example of hydroxylamine mutagenesis of phage is shown below (total plaques indicates phage survival and clear plaques indicates phage mutants).
  

  Cells exposed to a mutagen die at a constant rate. At lower doses of mutagen, both the absolute number of mutants in the surviving population and the ratio of mutants to nonmutants in the population increase in direct proportion to the dose of mutagen. However, beyond a certain dose of mutagen the probability that the cell will recieve a lethal mutation exceeds the probability of obtaining the desired mutation, so even though the ratio of mutants to non-mutants in the population increases, the actual number of mutants in the population decreases.

• Some mutant phenotypes are not observed until after the cells have undergone one or two cell divisions -- a process called "phenotypic lag". For example, if you obtain a mutation in a thi gene required for the biosynthesis of thiamine (vitamin B1),the cells will not show an auxotrophic requirement for thiamine until the concentration of thiamine made previously and the wild-type enzymes are diluted out by cell division. To avoid this problem, cells are often grown for several generations after mutagenesis before plating on selective growth conditions. However, if the cells are allowed to go through multiple generations, every mutant will yield siblings that carry the same mutation, so the outgrowth is usually limited to less than or equal to one generation time under the conditions used.