The collected samples were placed in sterile bags and transported in refrigerated trucks (12 °C) to the laboratory to be processed for analysis in less than one hour. Samples were removed from the bags and washed in running potable water. Dead shellfish, or those with broken shells, were discarded.
For the microbiological analysis of Vibrio, 25 g of bivalve samples obtained from 12 to 15 individuals (meat and liquor) were weighted, and 225 mL of alkaline saline peptone water (ASPW, OXOID, Hampshire, UK) were added. The matrix was homogenized for 2 min in a Stomacher (IUL, Barcelona, Spain), and directly incubated at 37 °C for 18 h. After incubation, a 3 mm loopful from the top 1 cm of each broth showing growth was streaked on different plates: thiosulfate-citrate-bile salt-sucrose (TCBS) agar (Oxoid, Hampshire, UK), and ChromAgar Vibrio (ChromAgar, Paris, France). The plates were incubated at 37 °C for 24 h. At least five typical sucrose-negative and positive colonies from each plate (TCBS), and mauve and white colonies from ChromAgar Vibrio were isolated and subjected to identification by biochemical tests on API 20E strips (bioMérieux, Marcy-l’Etoile, France) and other complementary biochemical assays. The isolated strains were stored at −80 °C for further analysis.
For the microbiological analysis of Vibrio, 25 g of bivalve samples obtained from 12 to 15 individuals (meat and liquor) were weighted, and 225 mL of alkaline saline peptone water (ASPW, OXOID, Hampshire, UK) were added. The matrix was homogenized for 2 min in a Stomacher (IUL, Barcelona, Spain), and directly incubated at 37 °C for 18 h. After incubation, a 3 mm loopful from the top 1 cm of each broth showing growth was streaked on different plates: thiosulfate-citrate-bile salt-sucrose (TCBS) agar (Oxoid, Hampshire, UK), and ChromAgar Vibrio (ChromAgar, Paris, France). The plates were incubated at 37 °C for 24 h. At least five typical sucrose-negative and positive colonies from each plate (TCBS), and mauve and white colonies from ChromAgar Vibrio were isolated and subjected to identification by biochemical tests on API 20E strips (bioMérieux, Marcy-l’Etoile, France) and other complementary biochemical assays. The isolated strains were stored at −80 °C for further analysis.