Characterization of Scardovia wiggsiae Biofilm by Original Scanning Electron Microscopy Protocol

Abstract

Early childhood caries (ECC) is a severe manifestation of carious pathology with rapid and disruptive progression. The ECC microbiota includes a wide variety of bacterial species, among which is an anaerobic newly named species, Scardovia wiggsiae, a previously unidentified Bifidobacterium. Our aim was to provide the first ultrastructural characterization of S. wiggsiae and its biofilm by scanning electron microscopy (SEM) using a protocol that faithfully preserved the biofilm architecture and allowed an investigation at very high magnifications (order of nanometers) and with the appropriate resolution. To accomplish this task, we analyzed Streptococcus mutans’ biofilm by conventional SEM and VP-SEM protocols, in addition, we developed an original procedure, named OsO4-RR-TA-IL, which avoids dehydration, drying and sputter coating. This innovative protocol allowed high-resolution and high-magnification imaging (from 10000× to 35000×) in high-vacuum and high-voltage conditions. After comparing three methods, we chose OsO4-RR-TA-IL to investigate S. wiggsiae. It appeared as a fusiform elongated bacterium, without surface specialization, arranged in clusters and submerged in a rich biofilm matrix, which showed a well-developed micro-canalicular system. Our results provide the basis for the development of innovative strategies to quantify the effects of different treatments, in order to establish the best option to counteract ECC in pediatric patients.

Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons

“…Staining with other heavy-metal reagents such as Ruthenium Red and RuO 4 may provide more specific contrasting of ECM components. As stated in [ 50 ], they also confirmed that sample drying using HDMS was a valuable and rapid alternative to conventional chemical fixation and drying. We tested the OsO 4 -RR-TA-IL protocol on a Candida albicans biofilm from an ex vivo sample (unpublished results) and also analyzed the images obtained with a four-quadrant method [ 100 ] by Hitachi 3D Mountains Map software (Digital surf, France).…”

“…Conventional SEM and FESEM are the best methods for biofilm visualization if high magnification and high-resolution images are needed to accurately describe biofilm morphology [ 50 , 51 , 52 ]. In comparative analyses, like the evaluation of the anti-biofilm effects of a drug/treatment, it appears as an extremely useful tool [ 11 ], since the results of SEM imaging are highly correlated with those from other analytical methods [ 53 , 54 , 55 , 56 , 57 , 58 , 59 ].…”

“…An IL covering keeps the biofilm hydrated under high vacuum SEM conditions (IL resists evaporation), so the Eps is preserved, thus avoiding dehydration steps and critical point drying procedures. The protocols used in [ 50 ] are summarized in Table 2 .…”

“…In such cases, scanning electron microscopy may be the best choice for its high image quality, magnification, and resolution, but only if protocols are carefully customized to allow imaging of a hydrated sample under high vacuum and high voltage conditions. In the paper [ 50 ], an innovative protocol (OsO 4 -RR-TA-IL) was tested. The use of the OsO 4 -RR solution ( Table 2 ) in a post-fixation step was discussed above [ 78 , 79 , 102 , 103 , 104 ].…”

“…S. mutans spherical bacterial cells were scattered on biofilm matrix compact surface. Image was captured from the same sample observed in Figure 1 a from [ 50 ]; ( b ) SEM, 5000×. Increasing magnification S. mutans spherical bacterial cells appeared clustered in small groups on the surface of a rough and dense extracellular matrix (Eps).…”