Rapid detection of temperate bacteriophage using a simple motility assay

Summary Phage contamination is a common complication for the fermentation and pharmaceutical industries. The risk of bacteriophage contamination in laboratory processes increases with multiple rounds of genetic manipulation such as deletion and complementation. The contamination of temperate phages does not lead to immediate host cell lysis but could become a serious issue when the lytic cycle is activated under specific conditions. Our objective was to develop a quick and reliable detection method for checking possible temperate phage contamination. Here, using motility plates, we found that when the strain carries a newly acquired temperate phage, its presence can be easily detected by the formation of a clear ‘lysis zone’ when swimming against the original strain on the same swimming plates. Compared to the traditional double agar layer method and genomic sequencing‐based methods, the duration of the motility‐based assay is shorter and the procedure is simplified. More importantly, for the bacterial strains that already contain active prophages, this method can still easily detect the newly acquired phages without tedious phage identification procedure. These features make this method highly applicable to laboratory and industrial processes.