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This study aimed to determine the survival and growth of Escherichia coli O157:H7 and Salmonella enterica subsp. enterica in a medium supporting the growth of a Lactic Acid Bacteria (LAB) food antimicrobial culture.Foodborne pathogens and LAB were cultured individually in tryptic soy broth (TSB), tryptic soy broth supplemented with one g l−1 Tween 80® (TSB-T80), and de Man, Rogosa and Sharpe (MRS) broth. Growth of E. coli O157:H7 and Salmonella was similar in TSB and TSB-T80 but was significantly less in MRS. Conversely, LAB growth was similar in MRS and TSB-T80 but was significantly less in TSB.Supplementation of TSB with Tween 80® allows growth of LAB to levels similar to that observed with MRS but does not inhibit the growth of E. coli O157:H7 and Salmonella. We present the formulation of a medium useful in studies useful for evaluating competitive inhibition of foodborne pathogens by LAB in vitro.This study reports the utility of TSB-T80 for the completion of in vitro competitive inhibition assays incorporating a Lactic Acid Bacteria food safety culture.