Mating assay: Plating below a cell density threshold is required for unbiased estimation of transfer frequency or transfer rate

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Abstract
Mating assays are common laboratory experiments for measuring the rate, frequency, or efficiency at which a plasmid transfers from a population of donor cells to a population of recipient cells. Selective plating remains a widely used quantification method to enumerate transconjugants at the end of such assays. However, transfer frequencies or rates may be inaccurately estimated because plasmid transfer can occur on transconjugant-selective plates rather than only during the intended mating period. We investigated the influence of cell density on this phenomenon. We conducted mating experiments with IncPα plasmid RP4 at a range of cell densities and mating conditions and compared the results to a model of cell-to-cell distance distribution. Our findings suggest that irrespective of the mating mode (solid vs liquid), the enumeration of transconjugants is significantly biased if the plated cell density exceeds 20 Colony Forming Unit (CFU) /mm2 (or 1.2x105 CFU per standard 9 cm Petri dish). Liquid mating assays were more sensitive to this bias because the transfer frequency of RP4 is several orders of magnitude lower in suspension compared to surface mating. Therefore, if selective plating is used, we recommend to plate below this density threshold and that negative controls are performed where donors and recipients are briefly mixed before plating at the same dilutions as for the actual mating assay.

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