An improved method of ammonia determination, applicable to amidases and other ammonia-producing enzyme systems of mycobacteria.

The colorimetric estimation of amidase activity, using both qualitative and quantitative determinations of ammonia, is widely used for the differentiation of mycobacteria. At present the generally used phenol-hypochlorite method requires heating of the test solution to 90 degrees C for 30 min or to boiling for 5 min. At room temperature at least 2 h are necessary to obtain a full and stable color. Heating is also disadvantageous because it increases the vaporization of toxic phenol vapors and it may lead to the formation of insoluble manganese dioxide, which interacts with the photometric determination. We found that the addition of ketones (preferably acetone) to the catalyst solution (MnSO4) accelerates the reaction in such a manner that heating is not necessary and the full color development can be obtained within 6 min. The proposed method is superior to the conventional ones because (1) the fully developed color can be obtained after 6 minutes without heating; (2) boiling, which increases the volatilization of phenol and creates dangers for the laboratory staff and the equipment, can now be reduced; and (3) the formation of manganese dioxide in the test solution is avoided.