环境因子对水霉菌生物膜形成的影响

Effects of environmental factors on biofilm formation of Saprolegnia

【目的】体外构建水霉菌(Saprolegnia)生物膜(Biofilm,BF),研究环境因子对其生物膜形成的影响。【方法】采用改良的微孔板法研究静置培养条件下寄生水霉(Saprolegnia parasitica)ATCC200013在96孔酶标板上的成膜情况,CCK-8法(Cell Counting Kit-8)定量检测生物膜中水霉菌的活力。【结果】水霉菌的生物膜的OD450值在培养24 h达到峰值,48 h后趋于稳定。随着初始孢子浓度升高,水霉菌生物膜OD450值升高,差异显著(P<0.05)。20-25°C生物膜形成量最多,OD450值显著高于其他温度组(P<0.05)。在起始pH值为4-11的沙氏葡萄糖液体培养基中,水霉菌均能形成生物膜。在培养基中加入0.12 mmol/L以上CaCl2,能促进生物膜形成;添加0.03-2.00 mmol/L MgCl2,水霉菌生物膜形成量与未添加MgCl2对照组无显著性差异;Cu2+对水霉菌生物膜的形成有显著影响,0.5 mmol/L以上添加处理几乎不形成生物膜;NaCl能明显抑制水霉菌生物膜的形成,当NaCl质量分数低于0.12%时,对生物膜形成的影响较小(P>0.05)。水霉菌在鲫皮和肌肉提取液包被后生物膜形成量与对照组相比无显著性差异,而鲫表皮黏液、鳃黏液包被后生物膜的形成量明显减少。【结论】研究首次采用微孔板法体外构建水霉菌生物膜,发现其生物膜的形成与多种环境因素有着密切的关系。这为了解水霉菌生物膜的形成规律提供了一定参考,为水霉菌生物膜的进一步研究奠定了基础。

[Objective] Biofilm of Saprolegnia was developed in vitro and the effects of the environmental factors were studied. [Methods] The characteristics of biofilm formation of Saprolegnia parasitica ATCC200013 were investigated by modified microtiter-plate test, and the viability of Saprolegnia in the biofilms was detected by CCK-8. [Results] The OD450 value of Saprolegnia biofilm reached the peak after 24 h and stabilized after 48 h. OD450 value of biofilm significantly elevated with densities of initial Saprolegnia spores（P〈0.05）. The largest biofilm quantity was at 20 °C and 25 °C, which were significantly higher than the others（P〈0.05）.Saprolegnia can develop biofilm at initial pH between 4 and 11. When more than 0.12 mmol/L CaCl2 was added, biofilm formation of Saprolegnia was promoted. However, it played no significant role when 0.03 to 2.00 mmol/L MgCl2 was added. Cu2＋ significantly affected the biofilm formation, while no evident biofilm was found when more than 0.5 mmol/L was added. Additionally, the biofilm formation was significantly inhibited by NaCl, but it played no significant role when less than 0.12%was added（P〈0.05）. Compared with the control group, the biofilms developed on the surface of the microtiter plate coated by the extract of skin or muscle showed no remarkable difference, while the biofilms on the microtiter plate coated by skin or gill mucus decreased. [Conclusion] A new method to form and measure Saprolegnia biofilm is successfully established. It describes for the first time that Saprolegnia biofilm formation can be affected by different environmental factors, which is beneficial to understand the growth of Saprolegnia biofilm and study further.