The medium developed for the original isolation studies on Lactobacillus sanfrancisco has been further refined to provide a basis for development of media for commercial preparation of cell concentrates. Addition of salts, purines, pyrimidines, and optimization of casein hydrolysate and Tween 801 levels resulted in markedly improved growth but did not eliminate the need for freshly prepared yeast extractives (FYE). Commercial yeast, liver, corn steep, and protein hydrolysate preparations tested were inadequate substitutes for the FYE. High maltose levels appear to stabilize viable counts over longer incubation periods. Some differences between strains in growth requirements were observed. Strains L and T were the least fastidious nutritionally, appeared indifferent to oxygen, and had a minimal requirement for Tween 80, but a primary requirement for purines-pyrimidines. Strains B and C were inhibited by oxygen and had a primary requirement for trace element supplementation and Tween 80. Strain L which showed the most rapid and heaviest growth has been selected as the type strain and the one to be utilized in initial commercial tests as a starter culture preparation.