Enumeration of viable tubercle bacilli (BCG) by the roll-tube method; use of a serumagar medium.

A serum-agar medium for the enumeration of viable tubercle bacilli (BCG) by the roll-tube method is described. The only ingredients required are distilled water, serum, phosphates, agar, and, depending on the type of agar used, iron. The serum is used as diluent for the bacillary suspension under test, so that it and the inoculum are added together to the agar solution in the roll-tubes. The phosphates are essential for growth, aid the formation of a firm agar gel, and serve to buffer the medium at about pH 6.3. The phosphate buffer solution is used as solvent for the agar. Eight out of nine commercial agar preparations tested yielded media which would not support optimal growth unless extra iron (ferric chloride) was added; a concentration of 33 mug of additional iron per ml in the final medium proved satisfactory.Using the index of dispersion as criterion, the variability between replicate counts in a series of 200 observations was found to be of the expected order.