Vitamin Free Yeast Base is recommended for classification of yeasts based on vitamin requirement. It contains all essential nutrients and necessary inorganic salts for the cultivation of yeasts. Use a highly diluted inoculum and incubate the tubes for 7 days at 25-28°C, since with the inoculum, vitamins may also be transported. Yeast themselves are also able to carry traces of vitamins, and therefore a second inoculation in Vitamin Free Yeast Base must be performed following the same procedure as for the first inoculation. Then incubate at 25-28°C for 7 days.