副溶血弧菌在玻璃表面生物菌膜的生长特性及超声波法解离作用

Growth Characteristics of Vibrio parahaemolyticus Biofilm on Glass Surfaces and Biofilm Detachment under Ultrasound Treatment

摘要:

本文研究了副溶血弧菌（Vibrio parahaemolyticus）在玻璃表面生物菌膜（biofilm，BF）的生长特性，生长过程中环境因素的影响及超声波对其的解离作用。采用结晶紫染色法观察生物菌膜在玻璃表面的生长形态；酶标仪在595 nm波长处测量不同培养条件下生物菌膜的生物量；平板菌落计数法衡量超声波对菌膜的解离效果。结果表明：结晶紫染色法可直观清晰观察副溶血弧菌在玻璃表面形成的生物菌膜，且随着培养时间的延长，副溶血弧菌生物菌膜形成的网状结构越来越致密。根据酶标仪测得的生物菌膜生物量的大小可得到在玻璃表面菌膜生长的最佳条件，当培养基盐度为3%，培养时间为24 h，培养时转速为70 r/min得到的副溶血弧菌生物菌膜已经成熟且菌体个数达到2.56×107 CFU/cm2。用50 kHz超声波，采用间歇式超声波处理方法（每作用30 s间隔30 s）作用总时间4 min在保持菌体活性的同时能达到最佳解离效果。

Abstract:

The growth characteristics of Vibrio parahaemolyticus biofilms on glass surfaces, the effects of environmental factors during biofilm formation, and biofilm detachment under ultrasound treatment were investigated. The morphology of V. parahaemolyticus biofilms on the glass surface was observed by crystal violet staining, biofilm biomass was measured using an enzyme-linked immunosorbent assay (ELISA) plate reader at 595 nm, and the extent of biofilm detachment was measured by counting colony-forming units (CFU) using the plate count method. V. parahaemolyticus biofilms formed on the glass surface, and could be directly observed using crystal violet staining. The reticular structure formed by the V. parahaemolyticus biofilm became denser with increasing cultivation time. The optimum parameters for V. parahaemolyticus biofilm formation with respect to biomass were determined using the plate reader. For a culture medium salinity of 3%, a rotation speed of 70 r/min, and an incubation time of 24 h, the number of bacteria in the mature V. parahaemolyticus biofilm was 2.56 × 107 CFU/cm2. Optimal biofilm detachment was obtained under pulsed ultrasound treatment (30-s sonication at intervals of 30 s) applying a frequency at 50 kHz for four minutes, and the viability of bacteria was maintained under these conditions.