Viability of freeze-dried Tice-substrain BCG by bioluminescent measurement of adenosine triphosphate.

The ATP content of viable cells in a single lot of freeze-dried Tice-substrain Mycobacterium bovis-BCG vaccine was determined after washing of organisms with isotonic buffer, using four extraction methods: boiling in 0.2 M potassium phosphate buffer at pH 7.4 for 12 min; boiling in 0.1 M Tris-EDTA buffer at pH 7.75 for 2 min; n-butanol/1.9% sodium glutamate-0.01 M sodium arsenate buffer, pH 7.0; and n-butanol/0.01 M Tris-EDTA buffer, pH 7.0. Liberated ATP was assayed with a Lumac biocounter by integrated measurement of light produced by firefly luciferin/luciferase. The dose response of internal standards paralleled that of ATP standards in water, and the response of endogenous ATP in BCG was not significantly different from a composite linear internal standard curve above 10 pg ATP (corresponding to about 10(5) viable organisms per ml), the sensitivity of the assay. When the ATP content of BCG was calculated from the composite curve, the n-butanol/Tris-EDTA method was found to be the most precise (CV less than 10%). Butanol extraction procedures were about twice as efficient as boiling methods and yielded an ATP value of about 3.4 fg/CFU, similar to ATP/CFU factors previously reported for other BCG substrains. However, when results were corrected for quench and ATP recovery, which varied with extraction method, the conversion factor increased nearly threefold.