Mycobacterium tuberculosis culture, a critical technique for routine diagnosis of tuberculosis, takes more than two weeks. Here, step-by-step improvements in the protocol including a new medium, microaerophlic atmosphere or ascorbic-acid supplement and autofluorescence detection dramatically shortened this delay. In the best case, primary culture and rifampicin susceptibility testing were achieved in 72 hours when specimens were inoculated directly on the medium supplemented by antibiotic at the beginning of the culture.

While there is evidence for the persistence of Mycobacterium bovis in soil, there are no reports for the other Mycobacterium tuberculosis complex (MTC) mycobacteria. Here, soil was inoculated with 10 8 c.f.u. g "1 M. tuberculosis, M. bovis and M. canettii and subcultured monthly for 12 months. The pathogenicity of mycobacterial colonies, identified by using matrix-assisted laser desorption/ionization time of flight mass spectrometry, was assessed in a mouse model. Moreover, mice were fed with food that contained 16.7 % M. tuberculosis-contaminated soil. The three tested MTC species survived in soil for 12 months with a final inoculum of 2¾10 3 c.f.u. g "1 for M. tuberculosis, 150 c.f.u. g "1 for M. bovis and 2¾10 4 c.f.u. g "1 for M. canettii. In an experiment that included negative controls, all (5/5) mice inoculated with such M. tuberculosis and M. canettii developed 0.03-0.3 granulomas mm "2 in their lungs and spleen and grew mycobacteria; five mice that were inoculated with M. bovis from soil did not develop granulomas but grew mycobacteria. Furthermore, 0.2-0.4 granulomas mm "2 were observed in the lungs and spleen of 3/5 mice fed with M. tuberculosis-contaminated soil in the presence of two negative control mice. M. tuberculosis grew in the stomach, intestine, spleen and lung in 5/5 challenged mice, whereas the negative controls remained M. tuberculosis-free (P50.008, Fisher exact test). This study provides clear evidence that MTC mycobacteria survive in soil, and that M. tuberculosis remains virulent while in the soil, outside its hosts, for extended periods of time.