Middlebrook 7h10 agar plates

Disease phenotype and severity were evaluated through histopathology and by measuring bacterial growth in the lung and spleen. To determine how well they had been protected, the organs were removed 30 d after infection. For lung histopathology, the right superior lobes were fixed overnight in 10% formalin and embedded in paraffin. Lungs were sectioned at a thickness of 4–5 μm and stained with H&E. For bacterial growth analysis, the lung and spleen were homogenized, and serially diluted samples were plated onto Middlebrook 7H10 agar plates (Becton Dickinson, Franklin Lakes, NJ) supplemented with 10% OADC (Difco Laboratories, Detroit, VA), 2 μg/ml 2-thiophenecarboxylic acid hydrazide (Sigma-Aldrich, St. Louis, MO) and amphotericin B (Sigma-Aldrich). After 4 weeks of incubation at 37 °C, bacterial colonies were counted. Data are presented as log₁₀ colony-forming units (CFU) per organ.

A pure culture of Mycobacteroides abscessus ATCC 19977 (American Type Culture Collection, Manassas, VA, USA) (homotypic synonym Mycobacterium abscessus ATCC 19977) was propagated and maintained at − 80 °C, and the second passage was used for irradiation. The stock solution was cultured in 10 mL total volume of Middlebrook 7H9 broth (Becton Dickenson, Sparks, MD, USA) with 10% (v/v) Middlebrook ADC Enrichment (Becton Dickenson, Sparks, MD, USA) and glycerol (2 mL/L). The tube was shaker-incubated at 37 °C for 46 h to late-log phase. The suspension was washed 3 times in phosphate-buffered solution (PBS, pH 7.4) and diluted to the order of 10⁶ colony-forming units per 1 mL (CFU/mL) in PBS for irradiation. The remaining concentration of viable microorganisms was determined using a CFU assay. One hundred microlitres of the irradiated solution was spread-plated in duplicate onto Middlebrook 7H10 agar plates (Becton Dickenson, Sparks, MD, USA) containing 10% (v/v) Middlebrook OADC Enrichment (Becton Dickenson, Sparks, MD, USA) and glycerol (5 mL/L). Plates were wrapped in Parafilm^® “M” (Bemis, Neenah, WI, USA) and incubated at 37 °C in the dark. Colonies were enumerated after 7 days.

For each experiment, Mycobacterium smegmatis (Trevisan) of the Lehmann and Neumann strain (INCQS 00021, ATCC 607, Coleção de Microrganismos de Referência em Vigilância Sanitária-CMRVS, FIOCRUZ-INCQS, Rio de Janeiro, RJ) was initially grown on Middlebrook 7H10 agar plates (BD Biosciences) supplemented with 0.2% (w/v) glucose, 0.2% (v/v) glycerol, and 15 mM NaCl for three days. For the initial liquid culture, cells were grown in Middlebrook 7H9 broth (BD Biosciences) supplemented with 0.2% (w/v) glucose, 0.2% (v/v) glycerol, 15 mM NaCl, and 0.05% (v/v) tween 80 at 37°C in an orbital shaker at 150 r.p.m. for three days [24 (link)]. For defined carbon sources, cells at the stationary phase of growth, obtained from the initial liquid culture in Middlebrook 7H9 broth, were diluted to O.D.₆₀₀ 0.05 in fresh Middlebrook 7H9 broth, as before, or in minimal media containing asparagine (0.5 g/l), KH₂PO₄ (1.0 g/l), Na₂HPO₄ (2.5 g/l), NH₄Fe(SO₄)₂  • 12H₂O (50 mg/l), MgSO₄  • 7H₂O (0.5 g/l), CaCl₂ (0.5 g/l), ZnSO₄ (0.1 mg/l), 0.2% tyloxapol, vitamin B12 (10 mg/ml), and when appropriate, 0.1% glycerol and/or 0.01% cholesterol, as described previously [8 (link)]. We then worked with five groups: (1) 7H9+Gly; (2) MM+Gly; (3) MM+Gly+Chol; (4) MM+Chol; and (5) MM and growth was monitored by measuring optical density at 600 nm (A₆₀₀) [24 (link)].

M. tb H37Ra (strain ATCC 25177) was obtained from the ATCC (American Type Culture Collection, VA, USA). M. mass strain CIP 108297 (M. mass CIP) was obtained from CIP (Collection of Institut Pasteur, Paris, France). A rough isolate of M. mass (M. mass R) was kindly provided by Dr B. J. Kim (Seoul National University, Seoul, South Korea). M. tb H37Ra and M. mass were cultured in Middlebrook 7H9 medium (BD Biosciences, Franklin Lakes, NJ, USA) supplemented with 10% OADC (BD Biosciences), 0.2% glycerol and 0.05% Tween 80 (Sigma-Aldrich). For all experiments, mycobacteria were cultured in late log phase and a portion of heavy aggregates was excluded by centrifugation. Then, collected mycobacteria were homogenized and stored at −70°C. Representative bacterial vials were thawed and the number of colony-forming units (CFU) viable on Middlebrook 7H10 agar plates (BD Biosciences) were counted. Then, mycobacteria were suspended in RPMI medium containing 10% human sera, and used for infection.

NTM isolates were grown in 50 mL of Middlebrook 7H9 broth (BD, Sparks, MD) containing 0.5% (vol/vol) glycerol and 10% (vol/vol) oleic acid-albumin with aeration (120 rpm) for 4 days at 37°C for M. smegmatis and M. abscessus or 7 days at 37°C for M. avium and M. chimaera. In some cases, 500-mL nephelometry flasks were used to measure and record turbidity as absorbance (540 nm) daily and/or cultures were diluted and plated on Middlebrook 7H10 agar plates (BD, Sparks, MD) containing 0.5% (vol/vol) glycerol and 10% (vol/vol) oleic acid-albumin to determine colony forming units (CFU).