Imaging Biofilms in Tissue Specimens - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 35

used SYTO ® 9 and propidium iodide to study the effect of different strains of
S. aureus on vaginal mucosa. Although the dyes also stained the mucosal cells,
there was morphological evidence of coccal micro-colonies between the epithelial
cells. More recently, Tsai et al. ( 2013 ) examined biopsies from patients suffering
from nasopharyngeal cancer who developed osteoradionecrosis for the presence of
biofilm. Clusters of bacteria were detected using SYTO ® 9 and propidium iodide
and the authors observed that far more samples were positive for biofilm according
to imaging than through culturing techniques.
The authors have observed that the combination of Sytox ® green and wheat
germ agglutinin conjugated with Texas Red ® (WGR-TR) allowed the detection of
microorganisms in tissue, regardless of type, fixative, embedding medium, and
sectioning technique (Fig. 1 ). These dyes are components of a commercially
available kit, ViaGram

Red + Bacterial Gram-Stain and Viability Kit (Life
Technologies, Carlsbad, CA). Sytox ® green is a high affinity nucleic acid stain,
and in fixed tissue samples, stains all Gram-negative and Gram-positive microor-
ganisms and the nuclei of mammalian cells, but not HECM. The bacteria are readily
differentiated from the mammalian cells and other structures by size and morphol-
ogy. The fluorescently labeled lectin, WGA-TR, stained HECM and probably also
EPS. When both green and red channel images taken separately on a
epi-fluorescence or CSLM are combined using imaging software, the resulting
image allows the clear visualization of bacterial cells as well as their spatial
localization on the tissue.

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7.2 Immunofluorescence

Immunofluorescence (IF) utilizes species-specific antibodies labeled with
fluorophores to tag microorganisms. This can involve directly labeled primary
antibodies or detection of unlabeled primary antibodies with fluorescently labeled
secondary antibodies. In order to successfully bind, antibodies need to have spec-
ificity and be applied in the correct concentration to antigens with accessible
epitopes (Harlow and Lane 1999 ). Furthermore, to be able to bind to targets within
a biofilm, the antibody needs to penetrate the EPS that surrounds the microbial
cells. Nonetheless, antibodies against group A Streptococci have been successfully
used to image these bacteria in mouse wounds (Connolly et al. 2011 ) and within the
crypts of the palatine tonsils from children (Roberts et al. 2012 ). The authors have
used immunofluorescence and CSLM to image S. aureus biofilm cryosections of
mouse tissue samples (Figs. 2 and 3 ). Transmitted light microscopy was used as an
alternate imaging method. As with other imaging techniques, it is important to
include alternate imaging methods as well as both positive and negative controls to
confirm results. For IF, nonspecific binding of both primary and secondary anti-
bodies is possible. Nonspecific binding can be reduced using a variety of blocking
agents including serum, albumin, and skim milk powder. The binding of antibodies
to nontarget bacteria with similar epitopes to target bacteria is also a possible