FIG 2.

Thermo-protection of DNA in intact BCG cells. (A) Effect on DNA yield of heating intact enumerated “declumped” BCG cells in thermo-protection buffer for the times shown. A total of 10⁵ BCG cells was heated at 99°C for 0, 15, 30, 45, or 60 min in 2 M KCl and 25 mM HEPES, pH 7.5, (1× thermo-protection buffer) or PBS (control). The experiment was performed in triplicate, and DNA was extracted post heating. (B) DNA yield obtained when heating intact mycobacteria cells from positive MGIT culture in thermo-protection buffer versus heating in MGIT culture fluid. Data are shown for 13 positive MGIT cultures. The DNA yield obtained after heating for 30 min at 99°C in thermo-protection buffer compared to that after heating in MGIT fluid is plotted. Each dot indicates the total DNA recovered (ng) from 1 ml initial MGIT culture. Numbers above the dots indicate the fold improvement in DNA yield when thermo-protection buffer was used rather than MGIT culture fluid. N/A, a sample where no DNA was recovered after heating in MGIT culture fluid so no fold improvement could be calculated.