Slicing overcomes the bacterial cell wall barrier to fluorescence in situ hybridization

Simultaneous visualization of all bacterial species in a polymicrobial biofilm by fluorescence in situ hybridization (FISH) remains a challenge because bacterial taxa respond differently to the hybridization procedure. This heterogeneity is due, in part, to the cell wall barrier of Gram-positive taxa. Enzymatic procedures required to permeabilize the cell walls of Gram-positive microbes can result in the disruption or loss of Gram-negative bacteria. Here, we demonstrate a procedure that enables the hybridization of difficult-to-hybridize bacteria while preserving the microarchitecture of all bacteria within a biofilm. The key feature of the procedure is to physically cut through the cell wall, thus allowing probe entry. We first embed the sample in a covalently crosslinked, glycol methacrylate resin to preserve the structure of the biofilm. Embedment is followed by sectioning and hybridization of material with the methacrylate still in place. We tested the procedure on common oral species, including difficult Gram-positive taxa, and found marked improvement in hybridization; both average signal intensity and homogeneity of hybridization were improved as compared to standard whole cell mount procedures. Our results confirm that the cell wall is the major barrier preventing efficient hybridization in whole mount samples. By physically overcoming the cell wall barrier, our protocol provides a universal procedure to visualize all bacteria in a polymicrobial community.

It has long been recognized that the major barrier to efficient in situ hybridization of bacteria is the cell wall, with Gram-positive bacteria generally being the most problematic. Because enzymatic methods that facilitate hybridization of Gram-positive bacteria can result in the loss of Gram-negative bacteria, visualization of both kinds of bacteria simultaneously is often not feasible. In this study, we use embedding and sectioning to establish a universal approach for the simultaneous visualization of all bacteria within a microbial community while preserving its microarchitecture. We show that the mechanism underlying the approach is the physical slicing of the bacterial cell, thus obviating the barrier posed by the cell wall. These findings will benefit researchers within the microbiology community interested in complex microbial communities.