7h10 plates

Fish were infected either with a low (~40 cfu) or high (~10,000 cfu) dose of M. marinum four weeks after immunization. M. marinum ATCC 927 was cultured at 29°C in standard mycobacterium medium, 7H9 (BD Biosciences), and prepared for infections as described in [8 (link)]. For infections, fish were anesthetized with 0.02% 3-aminobenzoic acid ethyl ester. The desired dose of M. marinum diluted in 0.2 M sterile KCl was injected intraperitoneally (i.p.). Thereafter, the fish were immediately released into a recovery tank. Infection doses were verified by plating the bacteria onto 7H10 plates (BD Biosciences). Following the infections, the well-being of the fish was monitored daily, and fish showing signs of stress or mycobacterial disease during the experiment follow-up period were euthanized with 0.04% 3-aminobenzoic acid ethyl ester.

Liquid cultures of M. smegmatis (mc² 155) were grown in Luria-Bertani (LB) broth (Lennox; Sigma Aldrich) containing 50 μg/ml carbenicillin (Fisher Chemical), 0.15% (v/v) glycerol and 0.1% (v/v) tween-80 in a shaking incubator at 37°C until the O.D_(600nm) reached 0.8–0.9. The number of M. smegmatis colony forming units (CFUs) per ml was determined by plating different dilutions of the bacterial stock on LB agar plates in triplicates and counting the number of CFUs after incubation at 37°C for 72 h.
Liquid cultures of M. bovis BCG (Pasteur 1173P2) were grown in Middlebrook 7H9 broth (Sigma Aldrich), supplemented with 10% (v/v) albumin dextrose catalase (ADC) and 0.2% (v/v) tween-80. ADC was prepared by dissolving 5 g of bovine albumin fraction V (Fisher Chemical), 2 g dextrose (Fisher Chemical), 0.85 g sodium chloride (Fisher Chemical) and 4 mg catalase from bovine liver (Sigma Aldrich) in 100 ml of sterile water and the solution was passed through a 0.22 μm filter. The growth cultures were kept in a shaking incubator at 37°C for 2–3 weeks until the O.D_(600nm) reached 0.8–0.9. The number of CFUs per ml for stock M. bovis BCG was determined by plating different dilutions of the bacterial stock on 7H10 plates (BD Biosciences) in triplicates and counting the number of CFUs after incubation at 37°C for 2–3 weeks.