Formation of transconjugants on plating media following in situ conjugation experiments

Abstract

Conjugation on agar plates following mating trials can cause transconjugant numbers to be overestimated. Numbers of transconjugants detected after incubating donors and recipients together were compared with those observed when donors and recipients were incubated separately and then mixed immediately prior to plating on selective agar. Mating comparisons were conducted using broth, a soil slurry, and nonsterile soil. Nalidixic acid was added to selective agar plates to investigate its use as an inhibitor of plate mating. The number of transconjugants from broth matings did not significantly differ from the number of transconjugants produced by plate mating on selective media lacking nalidixic acid. Addition of nalidixic acid to selective media reduced the number of transconjugants from broth matings by 10-fold and the number of transconjugants from plate mating after incubation in broth by 100-fold. The number of transconjugants detected from mating experiments in soil slurries was significantly greater than the corresponding plate matings (p = 0.0073). Furthermore, the addition of nalidixic acid to selective agar eliminated all plate matings. In nonsterile soil matings, transconjugants were detected immediately after the inoculation of donors and recipients into soil only when nalidixic acid was absent from the medium. Key words: transconjugants, in situ conjugation, gene exchange in soil, plate matings.