Lactic acid bacteria (LAB) are among the most important group of bacteria, with a critical role in food, pharmaceutical and medical industry. The fast-growing characteristics of LAB strains, their metabolic activity associated with production of many beneficial compounds and most of all, their GRAS status (Generally Recognized As Safe) recommends them as starter cultures for food biotechnology processes. During last decades, LAB strains have also an extensive prophylactic or therapeutic use as probiotics. Due to the fact that there are current limitations in the use of standard MRS media (de Man Rogosa Sharpe), which is selective especially for lactobacilli and enterococci, the aim of the present study was to optimize the growth medium composition for isolating a wide range of LAB strains with biotechnological potential and to improve the biomass accumulation. For this purpose, it has been evaluated the growth of Lactobacillus (L.) rhamnosus ATCC� 9595�, Streptococcus (S.) salivarius subsp. thermophilus ATCC� 19258�, Pediococcus (P.) acidilactici ATCC� 8042�, Lactococcus (L.) lactis 28 and Enterococcus (E.) faecium FFb CMGB L-18 on MRS broth with various carbon sources (glucose, lactose, galactose, maltose, mannose, ribose, arabinose, sucrose, fructose). The results of the study showed that there are interesting differences in the requirements of each analyzed species. The optimization of standard medium composition will be very useful for growth studies as well as metabolic flux studies.