Quantification of Vibrio alginolyticus in Mussel Samples

Mussel samples were transported to the laboratory in refrigerated conditions and processed within 6 h from the collection. Samples were externally cleaned with potable water. In aseptic working conditions, about 10 individuals were opened and the flesh meat and intervalvular fluids were pooled together, and 25 g of bivalve sample were homogenized in a blender and tenfold diluted with alkaline saline peptone water (ASPW) (10⁻¹ dilution). Sediment samples (10 g) were tenfold diluted with ASPW and vortex-mixed for 15 min at 2000× g with the Multi Reax (Heidolph, Germany) (10⁻¹ dilution). Water and phytoplankton-net samples were vortex-mixed for 15 min at 2000× g with the Multi Reax. From seawater and net-phytoplankton raw samples and the 10⁻¹ dilution of mussel and sediment samples further serial dilutions (10⁻¹ to 10⁻⁴) were prepared in ASPW (1 + 9 mL); then, 1 mL of each dilution was surface-spread plated on TCBS agar (Difco Laboratories, Detroit, MI, USA) and incubated at 37 ± 1 °C for 18 ± 3 h. After incubation, yellow colonies growing on TCBS agar plates were counted (only plates with 3 to 150 colonies were considered) for V. alginolyticus enumeration [51 (link),52 (link)]. The results were reported as UFC per gram of mussels/sediment or UFC per milliliter of water/phytoplankton-net sample.