The importance of microscopic characterization of membrane biofilms in an unconfined environment

Confocal laser scanning microscopy (CLSM) is often used to evaluate biofilm development or biofouling mitigation in membrane systems. However, several methods of CLSM sample preparation exist. In this paper, we evaluate the effects of three preparation techniques - dry, confined (wet), and unconfined (immersed) mounting - on CLSM-derived biofilm architecture and dimensions. Although placing a wet or dry biofilm between a slide and a coverslip before viewing is relatively common, our results show that this confinement significantly alters the biofilm observed. Therefore, biofilms should be viewed in an unconfined and hydrated state that allows for full extension of the biofilm structure in a media-filled viewing well of fixed depth (~. 250. μm). Pseudomonas aeruginosa biofilms were grown on thin-film composite reverse osmosis membranes and glass coupons. Dry and confined mounting of 24 and 48. h biofilms resulted in biofilms with low 3-D complexity and thickness (14 and 18. μm, respectively). Measured biofilm thickness was significantly higher on samples prepared using unconfined mounting (55. μm). Additionally, the reduction in biofilm thickness and biovolume observed after treatment with biocidal compounds was significantly less on the dry and confined biofilms than the unconfined samples. Our results strongly suggest that biofilms on membranes be prepared for microscopy using unconfined mounting to accurately assess biofilm structure and dimensions. Unconfined mounting will allow for accurate CLSM assessment of membrane biofilm structure, dimensions, and biofouling mitigation measures in membrane systems.