Development and validation of the spiral Salmonella assay: an automated approach to bacterial mutagenicity testing

Since its development by Dr. Bruce Ames and his colleagues more than a decade ago, the Salmonella/mammalian microsome mutagenicity assay has become a widely accepted tool to assist in the identification of chemicals with mutagenic and carcinogenic potential. Several automated approaches to Salmonella testing have been proposed in recent years but have failed to gain acceptance in the scientific community due to poor performance or lack of demonstrated usefulness. In this paper we report on an automated system that successfully generates dose-response data and, moreover, reduces the labor, materials, and sample mass required to obtain such information. In the standard plate-incorporation assay, dose-response relationships are defined by testing discrete doses of the test agent on a series of agar plates. In contrast, the spiral Salmonella assay generates dose-response data from a continuous concentration gradient on a single agar plate. Upon analysis, each spiral plate yields a dose-response curve consisting of 13 data points that span a concentration range of about 15:1, which is equivalent to 5 two-fold serial dilutions. The performance of the spiral Salmonella assay was compared to that of the conventional plate-incorporation assay using 13 mutagens and 7 nonmutagens selected from a variety of chemical classes. Concordant qualitative responses were obtained for all compounds tested, and comparable dose-response relationships were generated by all mutagens with the exception of sodium azide and cyclophosphamide, which are highly water-soluble and, thus, are unable to maintain a well-defined concentration gradient on a spiral plate due to rapid diffusion. In general, toxicity was expressed at a lower dose in the spiral assay, and the mutagenic potencies (slopes of the dose-response curves) were greater in the spiral assay relative to the plate-incorporation assay. These differences will be discussed, as will the applicability of the spiral plating technique to routine screening and its relevancy to future mutagenesis testing.