Oadc enrichment broth

All experimental work using M.tb was performed in an approved containment level 3 (CL3) laboratory. Frozen 1 ml aliquots of strain stocks were thawed and grown in 100 ml Middlebrook 7H9 media with 10% OADC to mid-log phase then divided into 1 ml aliquots and stored at −80 °C until required. Mycobacterial stocks were titrated by serially diluting in supplemented 7H9 media and culturing in BACTEC MGIT tubes supplemented with PANTA antibiotics and OADC enrichment broth (Becton Dickinson, UK), as previously described^{42 (link)}. In addition, the number of colony forming units (cfu) for each dilution was determined by solid culture on 7H10 agar plates. A standard curve of time-to-positivity (TTP) against cfu was derived and linear regression analysis generated an equation to convert experimental TTP to cfu.

This assay was performed as previously described^{19 (link)}. Duplicate tubes containing 300 μl of whole blood were incubated with 300 μl of RPMI (containing 10% pooled human serum, 2mM L-glutamine and 25 mM HEPES; Sigma, UK) inoculated with ~150 cfu BCG or M.tb on a 360° rotator at 37 °C for 96 hours (volume of the mycobacterial stock was calculated to give a TTP of 6.5 days, previously determined to give optimal differential responses). Cells were then lysed with sterile water, the mycobacteria resuspended in 7H9 media and transferred to a BACTEC MGIT tube supplemented with PANTA antibiotics and OADC enrichment broth (Becton Dickinson, UK). Tubes were placed in the BACTEC 960 machine and incubated at 37 °C until the detection of positivity by fluorescence (TTP). In addition, on day 0, duplicate viability control tubes were set up by directly inoculating supplemented BACTEC MGIT tubes with the same volume of mycobacteria as the samples. The mean TTP for duplicates was converted to a cfu count (as described above) and net growth ratio was calculated as Log₁₀(sample cfu/control cfu). A smaller net growth value indicates less bacillary replication and therefore represents greater mycobacterial control. Samples failing to meet pre-defined reproducibility criteria of ΔTTP < 6 hours between duplicates, as determined by initial experiments, were excluded.

The in-tube direct PBMC MGIA was performed as previously described (Fletcher et al., 2013 (link)). Briefly, 2 ml screw-cap tubes containing 1 × 10⁶ PBMC and ~500 CFU BCG Pasteur (unless otherwise specified) in a total volume of 600 μl RPMI (containing 10% pooled human serum, 2 mM l-glutamine and 25 mM HEPES) were incubated on a 360° rotator (VWR International) at 37 °C for 96 h. Where specified, cells were lysed at the end of the culture period by centrifuging tubes at 12000 rpm for 10 min, removing supernatant and adding 500 μl sterile water (or other lysis agent as specified) to the pellet before pulse vortexing and transferring to BACTEC MGIT tubes supplemented with PANTA antibiotics (polymyxin B, amphotericin B, nalidixic acid, trimethoprim and azlocillin) and OADC enrichment broth (Becton Dickinson, UK). For experiments where cells were not lysed, cultures at the end of the 96 h period were transferred directly to supplemented BACTEC MGIT tubes.