Middlebrook 7h9

The bacterial strains used in this study, Mycobacterium smegmatis mc² 155 and Mycobacterium tuberculosis H37Ra, were obtained from Jaya Tyagi at the All India Institute of Medical Sciences, Delhi, and from Vinay Nandicoori at National Institute of Immunology, Delhi, respectively. M. smegmatis was cultured to an OD₆₀₀ (optical density at 600 nm) of 0.5 in Middlebrook 7H9 (HiMedia) broth, supplemented with 0.44% glycerol (Rankem) and 0.15% Tween 80 (HiMedia), at 37°C and 200 rpm. M. tuberculosis H37Ra, cultured in a biosafety level 2+ facility, was grown in Middlebrook 7H9 medium, supplemented with 0.44% glycerol, 0.15% Tween 80, and 10% albumin-dextrose-NaCl solution (ADN), at 37°C and 180 rpm. The 0.5-OD₆₀₀ cultures were used for testing the efficacy of compounds, membrane permeability assays, and infection.

MIC values were determined using the MABA; rifampicin and isoniazid were employed as references. The mycobacteria strain has been cultured at 37 °C for two weeks in Middlebrook 7H9 (Himedia, Mumbai, India) supplemented with 0.05% (v/v), 2% glycerol and 10% OADC (oleic acid–albumin–dextrose–catalase of Liofilchem s.r.l, Roseto degli Abruzzi, Italy). The 96-well plates received 100 μL of Middlebrook 7H9 broth and serial dilution of compounds was made directly on the plate with drug concentrations of 0.244–250 μg/mL and 5000 to 4.882 μg/mL for extracts. Plates were covered and sealed with parafilm and incubated at 37 °C for 14 days. Then, 40 μL of freshly prepared 1:1 mixture of alamar blue reagent and 7% Tween 80 (Himedia, Mumbai, India) was added to the plate and incubated for 24 h. A blue colour in the well was interpreted as no bacterial growth and pink colour was scored as growth. The MIC was defined as the lowest drug concentration, which prevented colour change from blue to pink. The results of antitubercular activity are depicted in Tables 2 and 3. Minimum inhibitory concentration (MIC) was determined by the broth micro-dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (2011 ).

A complete list of strains used in this study is mentioned in Table 1. Cloning and plasmid propagation were done using E. coli strain DH5α. For protein expression, E. coli BL21 (DE3) was used. Both strains were grown in Luria Bertani (LB) medium at 37°C. Mbovis BCG Pasteur 1173P2, Msmeg mc²155, and the recombinant strains were grown in Middlebrook 7H9 (Himedia) broth supplemented with 10% OADC (oleic albumin dextrose catalase) (Himedia), 0.2% glycerol, and 0.05% Tween80 (20% stock) or on 7H10 agar without Tween80 at 37°C. Kanamycin (50 μg/ml) (Sigma-Aldrich), ampicillin (100 μg/ml) (Sigma-Aldrich), and hygromycin (50 μg/ml) (Invitrogen) were used as and when required.