Middlebrook 7h10 medium

M. bovis BCG Connaught (ATCC 35745) and M. brumae (ATCC 51384^TM) were grown on Middlebrook 7H10 medium (Difco Laboratories, Surrey, UK) supplemented with 10% oleic-albumin-dextrose-catalase enrichment, for 4 or 1 week, respectively, at 37 °C. For in vitro stimulation of splenocytes and for coating enzyme-linked immunosorbent assay (ELISA) plates, suspensions of M. brumae or BCG were heat-killed at 121 °C for 30 minutes^{39 (link)}. Mycobacteria emulsified in OO (Sigma) were prepared as previously described^{12 (link)}. For tumor induction, MB49 BC murine cells were cultured as previously described^{11 (link),26 (link)}.

Using the pAL5000-TOPO vector, an EGFP-expressing Mycobacterium-Escherichia coli shuttle vector was generated as previous study (51 (link)). Briefly, the EGFP gene was amplified from the pIRES2-EGFP vector (Clontech, Mountain View, CA, USA; Cat No., 6029–1), and the hsp65 promoter gene was amplified from genomic DNA of M. bovis BCG. The EGFP gene with the hsp65 promoter was amplified from the pMyong2-EGFP^h vector and ligated into pAL5000-TOPO using HindIII and BamHI restriction sites. The hsp65promoter and EGFP gene were amplified by overlapping PCR. To generate three different types of recombinant strains expressing EGFP (Asan 50594, M. smegmatis and BCG), the pAL5000-TOPO-EGFP plasmid was electroporated into competent bacteria (Asan 50594, M. smegmatis and BCG) using the Gene Pulser II electroporation apparatus (Bio-Rad, Hercules, CA, USA). Transformants were selected on Middlebrook 7H10 medium (Difco Laboratories, Detroit, MI, USA) (52 (link)) supplemented with OADC and containing 100 μg/ml kanamycin. Typically, colonies of transformants were selected from the plates, transferred into 7H9 broth medium (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% glycerol, 0.05% Tween-80, 10% ADC and 100 μg/ml kanamycin and cultured for 3~5 days. The growth rate of the recombinant mycobacteria strains was determined by measuring the medium OD at 600 nm.

M. bovis BCG Connaught (ATCC 35745) and M. brumae (ATCC 51384^T) were grown on Middlebrook 7H10 medium (Difco Laboratories, Detroit, Michigan) supplemented with 10% Oleic-Acid-Dextrose-Catalase (OADC) enrichment for 3 or 1 week, respectively, at 37 ℃. BCG and M. brumae cells were resuspended in sterile phosphate buffered saline (PBS) and adjusted to a 1.0 McFarland turbidity standard as previously described [26 (link)] in order to get the required concentration for each treatment. To confirm the dose of each experiment, representative bacterial suspensions of each treatment were serially diluted in PBS, and CFU were counted after plating on Middlebrook 7H10 medium as previously described [27 (link)]. Irradiation of M. brumae for intravenous infection and intravesical treatment was performed at Aragogamma S.L. as previously described [5 (link)].

As previously described13 (link), the 7H9 liquid culture medium for M. smegmatis strains consisted of Middlebrook 7H9 medium (Becton Dickinson, Franklin Lakes, New Jersey, U.S.) supplemented with 10% ADS (5% (w/v) bovine serum albumin fraction V, 2% (w/v) dextrose and 8.1% (w/v) NaCl), 0.5% (v/v) glycerol, and 0.05% (v/v) Tween80. 7H10 media containing Middlebrook 7H10 medium (Becton Dickinson, Franklin Lakes, New Jersey, U.S.), 10% ADS, and 0.5% (v/v) glycerol was used for solid culture to examine growth status. Hygromycin (Hyg) was purchased from GenView, erythromycin (EM) from Merck, hydrogen peroxide (H₂O₂) and kanamycin (Kan) from Sigma. Restriction enzymes such as Van91I, AlwNI, MfeI, and PacI were purchased from Fermentas. T4 DNA ligase and Q5 DNA polymerase were purchased from New England Biolabs.

The culture of MSP and MSW was conducted in 5 mL of Middlebrook7H9 medium supplemented with 10% ADC and 500 mg sterile 2-mm glass beads at 37°C under continuous shaking. The cultures isolated during the log phase were centrifuged at 500 g in order to remove clumps, and the cells were washed twice with sterile saline. The cell suspension was adjusted to an OD₆₀₀ of 0.8, corresponding to a concentration of 5 × 10⁷ CFU/mL. Female Balb/c mice (6–8-week-old) were infected intravenously with 0.1 mL of the bacterial suspension. The size of inoculum was confirmed by CFU counts.
The ability of survival and dissemination of each strain in vivo was assessed in six mice per group that were sacrificed by cervical dislocation on 0, 3, 6, 9, 12, 15, 20, and 35 days after infection. Lung, spleen, and kidney tissue specimens were weighed and homogenized in saline. The specimens were cultured in Middlebrook 7H10 medium (Becton Dickinson, United States) supplemented with 10% OADC following appropriate dilutions. CFUs were enumerated following 3–4 days of incubation at 37°C. All of the animal procedures were approved by the animal care committee of the Fudan University.