Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 133, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/57196
Keywords
Immunology and Infection; Issue 133; Thin Section; Biofilm; Paraffin Embedding; Colony Biofilm; Paraformaldehyde Fixation; Fluorescence Microscopy; Lectin Stain; Pel Polysaccharide; Biofilm Matrix
Categories
Funding
- NSF CAREER AWARD [1553023]
- NIH/NIAID award [R01AI103369]
- Direct For Biological Sciences
- Div Of Molecular and Cellular Bioscience [1553023] Funding Source: National Science Foundation
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Sectioning via paraffin embedding is a broadly established technique in eukaryotic systems. Here we provide a method for the fixation, embedding, and sectioning of intact microbial colony biofilms using perfused paraffin wax. To adapt this method for use on colony biofilms, we developed techniques for maintaining each sample on its growth substrate and laminating it with an agar overlayer, and added lysine to the fixative solution. These optimizations improve sample retention and preservation of micromorphological features. Samples prepared in this manner are amenable to thin sectioning and imaging by light, fluorescence, and transmission electron microscopy. We have applied this technique to colony biofilms of Pseudomonas aeruginosa, Pseudomonas synxantha, Bacillus subtilis, and Vibrio cholerae. The high level of detail visible in samples generated by this method, combined with reporter strain engineering or the use of specific dyes, can provide exciting insights into the physiology and development of microbial communities.
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