Individuals of R. decussatus and R. philippinarum were collected in the Óbidos Lagoon (Caldas da Rainha, Portugal, 39°24′44.1″ N 9°12′54.0″ W, in November of 2019). And other individuals of R. decussatus were collected in Ria Formosa (Algarve, Portugal, 37°01′05.4″ N 8°00′11.6″ W, in January of 2020) (Figure 1 ). The clams were transported, under controlled temperature (10 °C), to the laboratory and immediately processed upon arrival.
Enumeration of Vibrionaceae and coliforms was performed by serial dilutions, of the molluscs edible part and inter-valve liquid, in saline solution (0.85% w/v) and plating in thiosulfate citrate bile salts sucrose agar (TCBS; VWR, Radnor, PA, USA) and Chromocult® Coliform agar (Merck, Darmstadt, Germany), respectively.
Plates were incubated for 24 h at 28 °C for Vibrio spp. and at 37 °C for coliforms. Colony-Forming Units (CFUs) with distinct morphology on TCBS agar were inoculated onto Tryptic Soy Agar (TSA; Biolife, Milano, Italy) supplemented with NaCl (1.5% w/v), and incubated for 24 h at 28 °C. CFU that had distinct morphology on Chromocult® were inoculated onto Nutrient Agar (NA; VWR, Radnor, PA, USA), and incubated for 24 h at 37 °C. The isolates were conserved at −80 °C in cryovials (VWR, Radnor, PA, USA).
Enumeration of Vibrionaceae and coliforms was performed by serial dilutions, of the molluscs edible part and inter-valve liquid, in saline solution (0.85% w/v) and plating in thiosulfate citrate bile salts sucrose agar (TCBS; VWR, Radnor, PA, USA) and Chromocult® Coliform agar (Merck, Darmstadt, Germany), respectively.
Plates were incubated for 24 h at 28 °C for Vibrio spp. and at 37 °C for coliforms. Colony-Forming Units (CFUs) with distinct morphology on TCBS agar were inoculated onto Tryptic Soy Agar (TSA; Biolife, Milano, Italy) supplemented with NaCl (1.5% w/v), and incubated for 24 h at 28 °C. CFU that had distinct morphology on Chromocult® were inoculated onto Nutrient Agar (NA; VWR, Radnor, PA, USA), and incubated for 24 h at 37 °C. The isolates were conserved at −80 °C in cryovials (VWR, Radnor, PA, USA).