CRYOSECTIONING OF BIOFILMS FOR MICROSCOPIC EXAMINATION

A method for rapid and minimally disruptive embedding and sectioning o f bacterial biofilms has been developed and applied to binary populati on biofilms of Klebsiella pneumoniae and Pseudomonas aeruginosa grown on stainless steel surfaces in continuous flow annular reactors. Biofi lms were cryoembedded using a commercial tissue embedding medium. Froz en embedded biofilms could be removed easily from the substratum by ge ntly flexing the steel coupon. Microscopic examination of the substrat um surface after biofilm removal indicated that less than a monolayer of attached cells remained. Five um thick frozen sections were cut wit h a cryostat and examined by light or fluorescence microscopy. The cry oembedding technique preserved biofilm structural features including a n irregular surface, water channels, local protrusions up to 500 mu m thick, and a well-defined substratum interface. The method requires mi nimal sample processing without dehydration or prolonged fixation, and can be completed in less than 24 h.