We used the BACTEC system to evaluate the effects of several decontamination methods and the addition of antibiotics on the viability of Mycobacterium ulcerans. The effects of polyoxyethylene stearate or egg yolk as supplements were also evaluated to determine their impact on the growth of M. ulcerans. Strains of different geographic origins were subjected to Petroff, reversed Petroff, oxalic acid, and mild HCI treatments. After treatment, the viability of each strain was assessed in the BACTEC system. All of the decontamination methods tested adversely affected bacterial viability. Treatment with mild HCl gave the best results, allowing better growth rates with some strains and causing a delay in growth with others, depending on the geographic origin of the strain. A mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin did not significantly inhibit growth. Supplementing BACTEC medium with egg yolk markedly improved the recovery of M. ulcerans following the use of each of the decontamination methods. Our findings demonstrate a detrimental impact on the viability of M. ulcerans by all of the decontamination methods currently in common use. This explains, at least in part, the difficulty often experienced in cultivating this organism from clinical specimens. Egg yolk should be added to enhance the rate of successful primary cultivation of M. ulcerans in the BACTEC system.