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Abstract

Klebsiella pneumoniae biofilm formation is associated with chronic and relapsing infections. Scanning electron microscopy (SEM) is a powerful tool for characterizing biofilm structure and studying their formation. Reliable visualization of biofilm structure requires careful sample preservation, otherwise there may be loss of non-covalent interactions that are susceptible to damage during the dehydration and washing preparation steps. However, no standard procedure has been adopted in literature to fix K. pneumoniae biofilm for scanning electron microscopy studies. This lack of standardization makes it challenging to compare results between studies and determine if optimal fixation methods have been achieved to preserve native structures. To advance this critical area of study, we describe a scanning electron microscopy fixation method optimized for K. pneumoniae biofilm preservation. Our study reveals the importance of optimizing the biofilm preparation steps before scanning electron microscopy to observe differences in biofilm architecture between strains. Using our optimized fixation, we show that although species that overproduce capsular extracellular polysaccharides produced more robust biofilms, K. pneumoniae can form a developed biofilm in the absence of capsular polysaccharides.