A Modified Plating Technique for the Recovery and Enumeration of Stressed Salmonella typhimurium Hf .

A plating technique that would allow for recovery and enumeration of stressed Salmonella typhimurium was developed. The strain of S. typhimurium used in this study was isolated from an outbreak of salmonellosis caused by the consumption of contaminated pasteurized milk. Cultures propagated for 18 h at 37°C in Tryptic Soy Broth (TSB) were enumerated on Brilliant Green Agar (BGA), Salmonella-Shigella agar (SS), Xylose Lysine Desoxycholate Agar (XLD), and Hektoen Enteric Agar (HE). Recovery of 10 to 10 fewer cells was seen on the selective agars as compared to recovery on Tryptic Soy Agar (TSA). When the culture was spread plated onto TSA, allowed to stand at room temperature for 4 h, overlaid with XLD, HE, or SS, and incubated at 37°C for 24 h, recovery levels were comparable to recovery on TSA. Enhanced recovery of S. typhimurium cells that had been injured by freezing in TSB or in blended whole egg, or by heating at 54°C was also seen. In some cases recovery was at a higher level when the overlay procedure was used than on control TSA plates. Recovery of S. typhimurium from cheese that had been artificially contaminated with S. typhimurium , Staphylococcus aureus , and Pseudomonas fragi exceed control MPN values when the overlay procedure was used, although the recovery rate was within the MPN 95% confidence limits. The selective and differential properties of XLD, HE, and SS were maintained. Salmonella colonies appeared black, while the starter culture bacteria, S. aureus and P. fragi appeared as pinpoint white colonies. The potential exists for this procedure to be used for the direct enumeration of sublethally stressed salmonellae if their numbers exceed 100 per gram.