Development of a microplate assay for the detection of single plaque-forming units of bacteriophage PhiX174 in crude lysates

Mice containing the PhiX174 am3 transgene can be used for measuring in vivo mutation; however, the single burst analysis method used for distinguishing in vivo mutations from mutations generated during sample processing is labor-intensive. A liquid microplate assay was developed that detects a single mutant plaque-forming unit (PFU) of PhiX174 bacterial virus in the presence of excess nonmutant virus. The assay is based on inhibiting reduction of the tetrazolium dye, MTS, by bacterial cells selective for mutant virus. The assay is performed with crude lysates of infected bacteria and is as accurate as scoring viral plaques on a bacterial lawn. This microplate assay may have application in increasing throughput of the single burst analysis of PhiX174 in transgenic mouse mutation assays.