They noted a glycerin-bile-potato mixture grew bacilli that seemed less virulent, and changed the course of their research to see if repeated subculturing would produce a strain that was attenuated enough to be considered for use as a vaccine. The BCG strain was isolated after subculturing 239 times during 13 years from virulent strain on glycerine potato medium.
The researchers observed that a mixture of glycerin, bile, and potatoes produced bacilli that were less harmful. They then focused their research on whether repeated subculturing could create a weakened version of the bacteria suitable for vaccine development. After 239 subcultures over 13 years, they isolated the BCG strain from a virulent strain grown on glycerine potato medium.