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<strong><em><span style="font-size:9pt;line-height:115%;">In vitro</span></em><span style="font-size:9pt;line-height:115%;"> activity test is a critical evaluation to screen the potential biocontrol agent. We developed a selective medium for quantitative <em>in vitro</em> activity evaluation of bacterial biocontrol agents against pathogenic <em>Vibrio</em> in aquaculture. Sensitivity test of bacterial biocontrol and <em>Vibrio</em> spp. to nine antibiotics showed that oxytetracycline inhibited the growth of <em>Vibrio</em> spp., but did not inhibit the growth of the bacterial biocontrol. This selective inhibition activity of oxytetracycline suggested this antibiotic might be supplemented to establish a selective medium. The MIC of oxytetracycline against <em>V</em>. <em>harveyi</em>, <em>V</em>. <em>parahaemolyticus,</em> and <em>V</em>. <em>algosus</em> were 120, 250, and 120 </span><span style="font-size:9pt;line-height:115%;font-family:Symbol;">m</span><span style="font-size:9pt;line-height:115%;">g/ml, respectively. These concentration did not inhibit the growth of bacterial biocontrols. Therefore, oxytetracycline was supplemented at 250 µg/ml in Zobell agar medium. This medium was used as a selective medium to enumerate the density of bacterial biocontrol. <em>In vitro</em> activity test of bacterial biocontrol (RLP1) against <em>V</em>.<em> parahaemolyticus</em> showed that strain RLP1 at density of 10</span><span style="font-size:5pt;line-height:115%;">4 </span><span style="font-size:9pt;line-height:115%;">and 10</span><span style="font-size:5pt;line-height:115%;">6</span><span style="font-size:9pt;line-height:115%;"><span> </span>cells/ml was able to kill <em>V</em>. <em>parahaemolyticus</em> during 6 h incubation. At lower density, 10</span><span style="font-size:5pt;line-height:115%;">2 </span><span style="font-size:9pt;line-height:115%;">cells/ml, this bacterial biocontrol agent was able to kill the pathogenic <em>Vibrio</em> during 12 h incubation. This study discovered a selective medium for the bacterial biocontrol and <em>Vibrio</em> spp. and provided the results of its application in the evaluation of <em>in vitro</em> activity of a bacterial biocontrol agent against <em>V.</em> <em>parahaemolyticus</em>. The results also revealed that strain RLP1 is a potential bacterial biocontrol against vibriosis in marine aquaculture.</span></strong>
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