嗜冷黄杆菌培养基优化研究

Optimization of Culture Medium for Flavobacterium psychrophilum

本研究首先采用Biolog微平板法确定本实验室分离自患病虹鳟Oncorhynchus mykiss病灶和脾脏处的嗜冷黄杆菌Flavobacterium psychrophilum CH06、CH07和CH08株能利用的碳源,选定3株菌利用率最高的碳源——L-脯氨酸开展单因素优化实验。以TYES培养基为基础,分别添加0.3 g/L、0.5 g/L和1.0 g/L L-脯氨酸,将1×108 CFU/mL的嗜冷黄杆菌菌液按1%比例接种至此培养基,于18℃、150 r/min下分别培养24 h、48 h和72 h后检测细菌繁殖数量、菌落形态及致病性。结果表明,在TYES培养基中添加L-脯氨酸后,单位时间内嗜冷黄杆菌的生物量大幅度提高,显著高于TYES组;添加0.5 g/L L-脯氨酸组嗜冷黄杆菌产量最高,且不影响其形态,其平均长度和直径几乎没有变化。人工感染实验表明,在优化培养基上生长的嗜冷黄杆菌毒力无明显变化,肌肉注射感染的虹鳟幼鱼出现细菌性冷水病的典型临床病征。本研究结果可为嗜冷黄杆菌的体外培养及规模化生产提供参考。

In this study,Biolog microplate method was used to determine the available carbon sources of CH06,CH07 and CH08 strains of Flavobacterium psychrophilum isolated from lesions and spleen of the diseased rainbow trout(Oncorhynchus mykiss)and the most available carbon source,L-proline,for the three strains,was added into the TYES medium at a dose of 0 g/L,0.3 g/L,0.5 g/L and 1.0 g/L in the four single-factor tests.Then the number of bacterial colonies,colony morphology and pathogenicity of F.psychrophilum were detected in the TYES medium being inoculated with 1×108 CFU/mL of F.psychrophilum at 1%inoculation volume and cultured at 18℃,150 r/min for 24 h,48 h,and 72 h for optimization of the culture medium of F.psychrophilum.The results showed that the addition of L-proline substantially led to increase the biomass of the bacterium per unit of time,significantly higher than that in the TYES group in all time periods.In particular,the maximal yield of F.psychrophilum was observed in the TYES with addition of L-proline addition at a concentration of 0.5 g/L resulted,and L-proline addition did not affect the morphology,average length and diameter of F.psychrophilum.The artificial infection tests did not showed significant change in the virulence of F.psychrophilum grown on optimised medium,and the juvenile rainbow trout with challenged by intramuscular injection had the typical clinical signs of bacterial coldwater disease.The findings have important implications for the in vitro culture and large-scale production of F.psychrophilum.