Preparation of Test Agents

The chemicals to be tested are dissolved in water or dimethylsulfoxide. The test agent, or dilutions thereof, are placed onto filter disks by means of Eppendorf pipettes with disposable tips. Every agent is tested in duplicate on at least three different occasions. 

Because the assay depends on the differential inhibition of tester strains, the use of positive as well as negative controls is mandatory. For practical purposes, it is best to use commercial disks containing 30 fig chloramphenicol and freshly prepared discs containing 0 fi methyl methanesulfonate. Typical diameters of zones of growth inhibition are given in Table 1. If these controls do not give the expected results, the data are discarded. 

The standard E. coli pol Ai assay depends on the rate of diffusion of the test agent from a disk placed at the center of the plate. Results and their interpretation are then dependent on the differential inhibition of the pol A and pol Ai strains by DNA-modifying agents. This effect is expressed as dif- 

TABLE 1. Effects of Various Agents on the Growth of a DNA-Polymerase-Deficient Strain (Standard Assay) 

Ethyl methanesulfonate V-Methyl-V-nitrosourea N Ahy -N-nitrosourea V-Methyl-V-nitrosourethane V-Ethyl-V-nitrosourethane Formaldehyde V-Hydroxyurethane 2-Nitrosofluorene V-Hydroxylaminofluorene 1,2-Dimethylhydrazine NFTF  

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