Difco middlebrook 7h9 broth

H37Ra and M. smegmatis were grown to mid-to-late-logarithmic phase in Difco™ Middlebrook 7H9 Broth (Becton, Dickinson and Company; Cat. #271310) supplemented with ADC enrichment media (Remel™, ThermoFisher Scientific, Cat. #R450592), 0.2% glycerol and 0.05% Tween 80 at 37°C. For assays, bacterial cultures were adjusted to a total OD₆₀₀ of 1.2 reconstituted in 1 mL of phosphate buffered saline (PBS) prior to addition to wells.

Seven mycobacteria species [M. abscessus (Abs), M. bollettii (Bol), M. massiliense (Mas), M. avium (Avi), M. intracellulare (Int), M. chimaera (Chim) and M. bovis (BCG))] were used for all experiments and culture conditions [21 (link)]. The considered species belong to three different Mycobacterium complexes: (1) M. tuberculosis complex (MTB), which includes BCG; (2) M. avium complex (MAC), which includes Avi, Int, and Chim; (3) M. abscessus complex (MAB), which includes Abs, Mas, and Bol. All species were cultured aerobically (30 mL, 37 °C, 200 rpm shaking) in Difco Middlebrook 7H9 Broth (Becton Dickinson, Franklin Lakes, NJ, USA) containing Tween 80, glycerol and 10% Difco Middlebrook ADC enrichment (BD, Franklin Lakes, NJ, USA) placed into storage at −80 °C until use in this study. Bacterial growth conditions were the same as reported in [21 (link)]. Briefly, the bacterial growth was evaluated by measuring the optical density of the culture at 600 nm (OD₆₀₀) (Helios Omega UV/Vis (Thermo Fisher, Waltham, MA, USA). After an OD₆₀₀ of 2.0–2.5 was reached, cultures were transferred to 50 mL conical flasks, placed on ice to stop the metabolism, and centrifuged (8000 rpm, 4 °C, 10 min). In total, 5 mL of culture supernatant was transferred to a 20 mL air-tight glass headspace vial after centrifugation. Six biological replicates were prepared for each sample.

M. bovis BCG-medac/Vejicur (Bacillus Calmette-Guérin) and with pSMT3-dsRed+hygromycin (Prof Zakaria Hmama, Vancouver) transformed BCG-RFP were grown to log-phase in Difco Middlebrook 7H9 broth (Becton Dickinson, BD), supplemented with 10% Middlebrook OADC Enrichment (Becton Dickinson, BD) and 0.5% Glycerol, with the addition of 50 µg/ml Hygromycin B (Streptomyces sp., 400052, Merck Millipore) for BCG-RFP. BCG was heat-fixed by incubating for 30 min at 80 °C on a shaker.

All the necessary experimental procedures were undertaken inside BSC class II facility in accordance with the guidelines of “Institutional Bio-safety Committee” of KaviKrishna Laboratory. BCG strain (ATCC^® 35737™) was obtained from the American Type Culture Collection (ATCC) and grown in Difco™ Middlebrook 7H9 broth (Becton Dickinson). The media contained 0.5% glycerol, 0.06% Tween 80, and 10% oleic acid albumin dextrose catalase (OADC) (BD, USA). Streptomycin-auxotrophic mutant Mtb strain18b (gifted by Prof. Stewart T. Cole, EcolePolytechinqueFederale de Lausanne, Lausanne, Switzerland) was cultured in 7H9 medium (Difco, BD Biosciences, Franklin Lakes, NJ, USA) supplemented with Middlebrook albumin-dextrose-catalase, 0.5% glycerol, and 0.06% Tween 80 (Sigma, St. Louis, MO, USA) until an Optical Density (OD) of around 1 was obtained. We added streptomycin sulfate (50μg/ml) into the Mtb-m18b culture medium for bacterial growth. Green fluorescent protein (GFP) tagging procedure was performed as described earlier. The bacteria were prepared as single-cell suspension in RPMI media as previously described (16 (link)), being used to infect CSCs.

The Mycobacteria bovis Bacillus Calmette–Guerin (BCG) Danish strain (ATCC 35733) and M. tuberculosis H37Rv were grown at 37°C in Difco Middlebrook 7H9 broth (Becton Dickinson) or on Middlebrook 7H10 agar supplemented with 10% oleic acid-albumin-dextrose-catalase-enriched Middlebrook (OADC, BD), 0.2% glycerol and 0.05% Tween-80 for 3–4 weeks. Mycobacteria were cultured in the ABSL-II level lab of the Shanghai Pulmonary Hospital of Tongji University.

The inhibitory potential of the compounds was evaluated against M. tuberculosis H37Rv reference strain (ATCC 27294) by the resazurin reduction microplate assay (REMA) as previously described²² (link). Stock solutions (0.5 mg mL⁻¹ for 8-MG (1) and 2 mg mL⁻¹ for all other test compounds) were made in neat DMSO (Sigma-Aldrich) and aliquots were stored at −20 °C. The assays were performed in Difco™ Middlebrook 7H9 broth (Becton Dickinson – BD) supplemented with 10% (v/v) BBL™ Middlebrook ADC enrichment (albumin, dextrose and catalase – BD) and 2.5% (v/v) DMSO. The maximum concentration tested varied among compounds due to differences in solubility (2.5 − 40 µg mL⁻¹). The minimal inhibitory concentration (MIC) was determined by performing 10-point 2-fold serial dilutions for each compound. Three independent experiments were performed, and MIC was considered as the lowest compound concentration that prevented the resazurin (Sigma-Aldrich) colour conversion from blue (inhibition) to pink (growth). The MIC values stated for the compounds were the most frequent values among the three experiments, or the highest value observed.

The Mtb H37Ra used in this study was obtained from the American Type Culture Collection (ATCC25177) and maintained in Difco Middlebrook 7H9 broth (Becton Dickinson), supplemented with 0.5% glycerol, 0.05% Tween 80, and 10% oleic acid albumin dextrose catalase (OADC, BD, United States) at 37°C. For bacterial culture and infection, all experiments were carried out in a biosafety level 2 laboratory following the appropriate biosafety standard operating procedures.