Api 50 chb e medium

Colony morphology of the strains MS586^T and MS82 was determined after growth on NBY agar plates. Gram staining was performed as described previously (Murray, Doetsch, & Robinow, 1994); cell morphology and flagellation types were observed with a transmission electron microscope (TEM) using routine negative glutaraldehyde staining; and the production of fluorescent pigments was tested on King B medium (King, Ward, & Raney, 1954). Optical density (OD600) metrics recorded for NBY liquid cultures were used to evaluate optimal growth and pH, at temperatures from 4°C to 40°C, with an interval of 4°C for 24 hr, and at pH 4.0–10.0.
Physiological and biochemical tests were conducted as described previously (Peix, Berge, Rivas, Abril, & Velázquez, 2005). Cellular fatty acids were identified using the Sherlock 6.1 system (Microbial IDentification Inc.) and the library RTSBA6 (Sasser, 1990). Biochemical features and enzyme activities were determined using API 20 NE and API 50 CH strips with API 50 CHB/E medium (bioMerieux), as well as Biology GENIII Microplates (Biolog) as directed in the manufacturer's instructions; results were recorded after incubation for 48 hr at 28°C.

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.

To evaluate carbohydrate metabolism in Renuspore^®, API 50 CH, a standardized system, comprising 49 biochemical tests, was used. Several pure identical single colonies of Renuspore^® were picked and suspended in an ampule of API NaCl 0.85% medium (Biomérieux, Ref 70700) until turbidity was equivalent to 2 McFarland (Biomérieux, Ref 70900). The prepared suspension was transferred to an ampule of API 50 CHB/E medium (Biomérieux, Ref 50430). Two hundred microliters of the inoculated API 50 CHB/E medium were added into the strip wells and incubated for 48 h. A positive test corresponded to acidification revealed by the phenol red indicator contained in the medium changing to a yellow color. Control strain was not indicated in the assay since carbohydrate fermentation was performed to analyze the potential probiotic properties of Renuspore^®.