3.4. Confocal laser scanning microscopy imaging
The images of biofilm structures were shown in Fig. 4, and the thickness also was quantified ( Table 3). Coincident with the results of crystal violet staining, in times of 2 and 8 h only occurred bac-teria adhesion and microcolonies formation, independent of tem-perature. Similar results also have been demonstrated by Enos- Berlage, Guvener, Keenan, and McCarter (2005) and Shime- Hattori et al. (2006) in V. parahaemolyticus. As the incubation time increases, the biofilm architecture transformed from micro-colonies to bacterial clusters and aggregate into honeycomb-like structure, and the thickness increased accordingly. Besides, the contact surfaces were completely covered by dense and homoge-neous biofilm when cultured to 48 h at 15 _C or 24 h at 25 _C, and strains exhibited the highest biofilm thickness at these conditions. The CLSM images validated the results using crystal violet staining that the developmental process of biofilm was disparated at these temperatures, the biofilm architecture was denser at 25 _C when compared to biofilm architecture at 15 _C and 37 _C. The results also demonstrated that temperature significantly influence biofilm-forming process and the morphological structures of biofilm, and 25 _C is more appropriate for biofilm formation