SETTING: A tuberculosis (TB) research laboratory in the Netherlands. OBJECTIVE: The concentration of Mycobacterium tuberculosis cells from sputum is almost universally performed by centrifugation after chemical liquefaction. These methods are thus dependent on the effective sedimentation of mycobacterial cells, and the buoyant density of these cells relative to sputum is therefore of critical importance. DESIGN: We cultured M. tuberculosis in different systems and measured their buoyant density. We also calculated the centrifuge times and speeds needed to effectively pellet the mycobacteria. RESULTS: In contrast to earlier reports, we were unable to identify cells with a buoyant density <1 g/cm(3). The measured buoyant density of the cells ranged from 1.13 to 1.02 g/cm(3), and we suspect that the less dense cells are more likely to reflect clinically derived mycobacterial cells. CONCLUSION: Based on our results, this means that for effective sedimentation in a typical universal centrifuge, centrifugation for 22 min at 3200 x g would be required. A limitation of this study is that cultured M. tuberculosis was studied. The data from this study should be confirmed in clinical samples. However, based on our results, centrifugation at lower speed for less time is unlikely to result in effective recovery.
SETTING: A tuberculosis (TB) research laboratory in the Netherlands. OBJECTIVE: The concentration of Mycobacterium tuberculosis cells from sputum is almost universally performed by centrifugation after chemical liquefaction. These methods are thus dependent on the effective sedimentation of mycobacterial cells, and the buoyant density of these cells relative to sputum is therefore of critical importance. DESIGN: We cultured M. tuberculosis in different systems and measured their buoyant density. We also calculated the centrifuge times and speeds needed to effectively pellet the mycobacteria. RESULTS: In contrast to earlier reports, we were unable to identify cells with a buoyant density <1 g/cm(3). The measured buoyant density of the cells ranged from 1.13 to 1.02 g/cm(3), and we suspect that the less dense cells are more likely to reflect clinically derived mycobacterial cells. CONCLUSION: Based on our results, this means that for effective sedimentation in a typical universal centrifuge, centrifugation for 22 min at 3200 x g would be required. A limitation of this study is that cultured M. tuberculosis was studied. The data from this study should be confirmed in clinical samples. However, based on our results, centrifugation at lower speed for less time is unlikely to result in effective recovery.
Authors: Peter Daley; Joy Sarojini Michael; Kalaiselvan S; Asha Latha; Dilip Mathai; K R John; Madhukar Pai Journal: PLoS One Date: 2009-05-20 Impact factor: 3.240