Api 50ch kits

The strains used for comparative analysis included V. hibernica sp. nova (Vh) strain B1.19, V. litoralis DSM17657 (Vl), A. fischeri MJ11 (Af), V. scophthalmi DSM19140 (Vs), V. parahaemolyticus RIMD 2210633 (Vp) and V. gallaecicus DSM23502 (Vg). The temperature growth range was assessed on TSB and TSA (Merck), Marine Agar (MA) (BD Difco), Brain Heart Infusion (BHI) (Merck) and Nutrient Agar with NaCl (NA) (Peptone (Merck) 5 g/l, Meat Extract (Sigma) 3 g/l and Agar (Sigma) 15g/l at 4°C, 23°C, 30°C, and 37°C. Single colonies were tested for catalase activity with 10% hydrogen peroxide (Sigma) and cultured on Columbia Blood Agar (CBA) (Fannin) at 23°C for 48 h to assay hemolysis. Salt tolerance was conducted in TSB (Merck) supplemented with 0, 3, 6, 10, and 20% additional NaCl (Sigma) and grown at 23°C with agitation (180 rpm) for 24 h. To assess the biochemical profile of the organisms, an API 20NE kit (BioMerieux) was used with API NaCl Medium (BioMerieux). To profile the carbohydrate metabolism range of the bacteria, API 50CH kits (BioMerieux) were used in conjunction with API 50 CHB/E Medium (BioMerieux). Both the API 20NE and API 50CH were used with manufacturers guidelines with the exception of the temperatures used, 4 and 23°C. All assays were completed with three independent biological replicates.

Gram-stained morphology was observed using a phase contrast microscope (Eclipse Ci-L, Nikon, Tokyo, Japan). The outer and inner structures of strain ICN-92133^T were observed using the scanning electron microscope (SEM) and energy-filtered transmission electron microscope (EFTEM) acquired on a Regulus 8100 (Hitachi, Tokyo, Japan) and LIBRA 120 (Carl Zeiss, Germany), respectively. The main characteristics, such as oxidase, catalase, nitrate reduction, and sporulation activities, of strain ICN-92133^T and phylogenetically closest type strains were compared. The optimal growth conditions were determined by culturing at different temperatures (20, 26, 30, 37, and 45 °C), pH (5, 5.5, 6, 6.5, 7, 7.5, 8.5), and osmotic conditions (0, 1, 3, 5, 10, 15 and 20 g of NaCl/L). According to the manufacturer's instructions, enzymatic activities and substrate utilization properties were investigated using API ZYM, API 20A, and API 50CH kits (BioMérieux) at 30 °C.

Lactococcus spp. and S. agalactiae were initially grown anaerobically at 37°C for 24 h on Wilkins–Chalgren agar without the soya peptone addition. Production of acid connected to pH change, based on the ability to utilize carbohydrates, was determined by using API 50 CH kits (bioMérieux, France) after incubation at 37°C for 48 h. API 20 STREP kits (bioMérieux) were used to determine carbohydrate utilization and enzymatic assay after incubation at 37°C for 4 and 24 h. Enzymatic activity was assessed using API ZYM and Rapid 32 ID A kits (both bioMérieux) after incubation at 37°C for 4–4.5 h. All kits were used according to the instructions of the supplier. The ability of tested strains to induce hemolysis was tested on Tryptone Soya Agar and Columbia agar with 5% of sheep blood (both Oxoid) under aerobic and anaerobic conditions at 37°C for 24 h. All strains were tested at least in two or three biological replicates.