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 glycerin-bile-potato mixture yielded bacilli that were less virulent and subsequently investigated whether repeated subculturing could create a weakened strain suitable for vaccine development. The BCG strain was obtained after 239 subcultures over 13 years from a virulent strain on glycerine potato medium.