Thiosulfate citrate bile salts sucrose agar tcbs

Live feed cultures were retained on a 65-µm mesh, aseptically concentrated and collected using sterile Falcon tubes (approximately a volume of five mL of concentrated live prey), centrifuged at 4700× g for 10 min, and the supernatants were eliminated with a micropipette. The pellets obtained were aseptically weighed and ground by hand using a sterile glass digester with 1 mL of sterile physiological saline (0.85%) (PS) added to obtain a homogenate. Appropriate 10-fold dilutions of the homogenates in PS were prepared and 0.1-mL aliquots were inoculated in triplicate onto agar plates. Culturable counts of heterotrophic and florfenicol-resistant bacteria were determined using a spread plate method with Plate Count Agar (PCA, Becton Dickinson, Sparks, MD, USA), with 2% NaCl added, whereas Vibrio spp. counts were determined using Thiosulfate-Citrate-Bile Salts Sucrose agar (TCBS, Becton Dickinson, Sparks, MD, USA) prepared using 50% microfiltered (0.22 µm) aged seawater [50 (link)]. Plates with added florfenicol (30 µg/mL, Sigma-Aldrich^® (St. Louis, MD, USA) were used to determine the florfenicol-resistant bacteria [50 (link),51 (link)]. All plates were incubated at 22 °C for 5 days and the bacterial numbers per gram of sample were calculated as described in Miranda and Rojas [51 (link)].

The bacterial strains VPAP36 and VPAP40 were isolated from settled moribund larval samples of the Chilean scallop (Argopecten purpuratus) during two different mass mortality events that occurred in two geographically distant commercial hatcheries located in the north of Chile, in Inglesa Bay (27°05′30″ S; 70°51′51″ W) and Tongoy Bay (30°15′22″ S; 71°29′46″ W), respectively (Figure 1). Triplicate larval samples were aseptically collected from the bottom of the culture ponds during water exchange using a sterile container, transported to the Aquatic Pathobiology Lab of the Universidad Católica del Norte, and immediately processed. The larval samples were centrifuged at 2000× g using an Eppendorf Model 5415D centrifuge and homogenized in 5 mL of sterile seawater (0.2 μm). The homogenized sample obtained was inoculated in triplicate on Soy Trypticase agar supplemented with 2% NaCl (TSA2, Becton-Dickinson, Sparks, MD, USA), and on Thiosulfate-Citrate-Bile-Salts Sucrose agar (TCBS, Becton-Dickinson) prepared with 50% aged micro-filtered (0.45 μm) seawater. The plates were incubated at 20 °C for 48 h. The predominant colonies that developed were purified using TSA2 and stored at −85 °C in CryoBank^TM vials (Mast Diagnostica, Reinfeld, Germany). The strains were grown on TSA2 agar at 20 °C for 24 h prior to use.