Brilliant green phenol red lactose sucrose agar

The samples (chicken feces, environmental samples, and pest animal contents) were diluted to 9 times their volume with buffered peptone water (BPW) (Merck^®, Germany) for incubation at 37°C for 24 h. One milliliter of the BPW broth was transferred into 9 mL of the enrichment broth (Rappaport Vassiliadis soya [RVS] broth) (Merck^®) for additional incubation at 37°C for 24 h. One loop of the RVS broth was streaked on Brilliant-green Phenol-red Lactose Sucrose agar (Merck^®) to isolate Salmonella. The suspicious Salmonella colonies were picked after incubation at 37°C for 24 h. Subsequent biochemical identification was performed as previously described by Tran et al. [21 (link)].

Qualitative analysis of Salmonella spp. was performed according to EN ISO 6579-1:2020-08 standard protocol [37 (link)]. The basic homogenate, which contained the agar slice and 100 mL of buffered peptone water broth (Merck KGaA, Darmstadt, Germany), homogenized with a stomacher, provided the basis for pre-enrichment. Deviating from the ISO standard protocol after incubation of the basic homogenate at 37 °C for 24 h for pre-enrichment, enrichment broths, Rappaport Vassiliadis Bouillon (RV) (Merck KGaA, Darmstadt, Germany) and Muller–Kauffmann Tetrathionate-Novobiocin Broth (MKTTn) (Merck KGaA, Darmstadt, Germany) were incubated for 24 h at 37 °C for MKTTn and at 42 °C for RV, before streaking out on the two selective agars, Brilliant-Green Phenol-Red Lactose Sucrose Agar (Merck KGaA, Darmstadt, Germany) and Miller and Mallinson ChromoSelect Agar (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated for 24 h at 37 °C. Biochemical testing for dulcitol, o-nitrophenyl-β-D-galactopyranoside (ONPG), malonate, and salicin was carried out for further investigation of the isolates. Typical and suspect colonies were tested using agglutination tests (sifin diagnostics gmbh, Berlin, Germany), and Salmonella spp. confirmation was carried out using polymerase chain reaction tests, performed at the German Federal Institute for Risk Assessment (BfR) in Berlin, Germany.