A previously described and widely used conjugation protocol was used as the starting point for development of our improved method [2] (link). 200 μl samples of C. difficile overnight cultures were heated, as above, and incubated at 37 °C for 2 min. 1 ml of overnight E. coli conjugant donor (CA434) culture was harvested by centrifugation at 4000g for 2 min and transferred into the anaerobic workstation. E. coli cell pellets were then gently resuspended in 200 μl of heat treated or untreated C. difficile culture. This mixed cell suspension was then pipetted onto well-dried, non-selective agar plates (10 × 10 μl spots) and allowed to dry. BHI agar was used routinely but BHIS (BHI agar supplemented with 0.1% (w/v) cysteine and 0.5% (w/v) yeast extract), TY [5] (link) and Brazier’s (Brazier’s CCEY media, 1% (v/v) defibrinated horse blood, 4% (v/v) egg yolk emulsion) agar were also tested. All solid media contained 1.5% agar. Conjugations were then incubated for 8–24 h following which growth was harvested using 900 μl of TY broth, serially diluted and spread on plates containing either cycloserine (for total C. difficile CFU counts), or cycloserine and thiamphenicol (to select for transconjugants). Approximate conjugation efficiency was then calculated as transconjugant CFU/total C. difficile CFU. These experiments were performed using biological duplicates with technical triplicates. Statistical significance of these results was determined using either individual student t-tests or in combination with one way analysis of variance (ANOVA), performed using GraphPad Prism 6. P values of<0.05 were considered statistically significant.
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