Verification of optimal pre-enrichment culture inoculum size for tetrathionate broth and selenite broth to isolate Salmonella enterica by spike test and field trial.

Abstract

The isolation efficiency of Salmonella enterica using selective enrichment media varies depending on culture conditions. Tetrathionate broth (TTB) and selenite broth (SB) differ in selectivity and growth support, yet practical guidance on optimizing their use remains limited. To identify appropriate conditions for improving recovery, we first conducted a spike test in which a defined amount of S. enterica was added to Salmonella negative cattle feces. TTB showed improved isolation rates when the inoculum size was reduced and the incubation period was extended, indicating that each factor independently contributed to recovery. In contrast, SB exhibited enhanced recovery when a smaller inoculum was combined with an incubation period appropriate for that inoculum size. We then validated these observations using naturally contaminated chicken liver, pig feces, and crow droppings. After pre‑enrichment, samples were inoculated into TTB, SB, modified semi‑solid Rappaport agar for stab culture (MSRAsc), and Rappaport-Vassiliadis broth under various culture conditions. Although results varied among sample types, TTB and SB generally showed improved isolation rates when cultured under the optimized conditions. MSRAsc achieved the highest overall recovery, but some strains, likely sensitive to dyes, failed to grow. These results indicate that although optimizing TTB and SB enhances isolation efficiency, no single medium is sufficient to recover a broad range of serovars. Therefore, a combined approach using dye‑free TTB alongside MSRAsc is recommended, and SB should also be used when strains exhibiting poor growth in other selective media are suspected.