Utilization of ram horn hydrolysate as a supplement for recovery of heat- and freeze-injured bacteria

Tryptic soy agar (TSA) has been widely and successfully used as a resuscitation medium for injured bacteria. The purpose of this study was to examine the use of ram horn hydrolysate (RHH) as a supplement for recovery of injured bacteria, to compare it to TSA and to evaluate its usefulness as a possible alternative supplement in microbial media. The TSA was enriched with RHH at various concentrations. 2% RHH was found to be optimal for growth of healthy bacteria. The enriched medium (TSA + 20 ml RHH in 1–l) was termed enriched tryptic soy agar (ETSA). TSA and ETSA were tested for the recovery of heat- and freeze-stressed bacteria. Four species of bacteria (Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and Pseudomonas aeruginosa) were heat-treated at 55 °C in physiological saline by a capillary tube method. For the freeze-injury studies, 5 ml of the tryptic soy broth cultures were placed in a freezer at −25 °C for 7 days. The numbers of uninjured bacteria on TSA were evaluated as control. The number of viable bacteria on TSA and ETSA was determined by the pour plate method. Recovery rates of bacteria on ETSA were higher than that of TSA. The RHH appears to be a valuable supplement for microbial media.