Mycobacterium Isolation from Tissue Samples

Tissue samples for microbiological culture were processed in a Containment Level 3 (CL3) facility at the University of Nottingham, Sutton Bonington Campus. Samples were processed according to^{22 (link)}. Briefly tissue pools or lesions were gently ground with sterile sand and 2 ml phosphate buffer saline (PBS; Dulbecco A, Oxoid). Samples were mixed with an equal amount of 5% oxalic acid (Fisher Scientific) and incubated at room temperature for 10 min to reduce non-mycobacterial contamination. The decontaminated pools (200 µl) were inoculated onto Stonebrink Selective agar supplemented with PACT (BD Diagnostics) and Middlebrook 7H11 agar slopes supplemented with PANTA (BD Diagnostics), and incubated at 37 °C for a minimum of 12 weeks. Cultures were examined approximately weekly for the appearance of colonies suggestive of mycobacteria.