Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

Abstract

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.

The Pseudomonas aeruginosa Complement of Lactate Dehydrogenases Enables Use of d - and l -Lactate and Metabolic Cross-Feeding

“…Thin sections of P. aeruginosa colonies were prepared as described previously ( 44 ). Briefly, to produce bilayer plates, a 4.5-mm-thick bottom layer of medium (1% agar,1% tryptone) was poured and allowed to solidify before a 1.5-mm-thick top layer was poured.…”

Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence

“…After three days, bright field images and fluorescence images were visualized with a Zeiss Axio Zoom.V16 fluorescence stereo zoom microscope. Thin sections of colony biofilms (n ≥ 3 biological replicates) were prepared as described previously 14 . Differential interference contrast (DIC) and fluorescent confocal images were captured using an LSM800 confocal microscope (Zeiss).…”

“…PA14 single recombinants were selected on M9 minimal medium agar plates (47.8 mM Na2HPO4•7H2O, 22 mM KH2PO4, 8.6 mM NaCl, 18.6 mM NH4Cl, 1 mM MgSO4, 0.1 mM CaCl2, 20 mM sodium citrate, 1.5% agar) containing 100 µg/ml gentamicin. The plasmid backbone was resolved out of PA14 using FLP-FRT recombination by introduction of the pFLP2 plasmid 32 Thin sections of PA14 colony biofilms were prepared as described previously 20 . Briefly, after three days of growth as described above, biofilms were overlaid with 1% agar and sandwiched biofilms were lifted from the bottom layer and fixed overnight in 4% paraformaldehyde in PBS at 25°C for 24 h in the dark.…”

“…Because biofilm development leads to the formation of steep chemical gradients that can affect gene expression patterns along the z-axis 22 , we chose to visualize P rebP1 activity across colony depth. To do this, we employed a thin sectioning protocol to prepare vertical sections taken from the center region of three-day-old colony biofilms 23 . Confocal microscopy of the dual reporter (P rebP1 -mScarlet P PA1/04/03 -gfp) biofilm sections revealed that cells expressing rebP1 are present in thin striations such that both bright and dark cells are situated at the same depth (red fluorescence in Figure 3d).…”

The Pseudomonas aeruginosa complement of lactate dehydrogenases enables use of D- and L-lactate and metabolic crossfeeding

“…Thin sectioning and preparation for microscopic analyses. Thin-sections of P. aeruginosa colonies were prepared as described in (39). Briefly, to produce bilayer plates, a 4.5-mm thick bottom layer of medium (1% agar,1% tryptone) was poured and allowed to solidify before 355 pouring a 1.5-mm thick top layer.…”