Imaging Biofilms in Tissue Specimens - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 34

used a commercially available fluorescent Gram staining kit for tissue to examine
chronic wound biopsies for the presence of biofilms. Fixed tissue biopsies from
15 patients were cryoembeded, cryosectioned, stained with ViaGram Red + Bac-

terial Gram-Stain and Viability Kit (Life Technologies, Carlsbad, CA), and exam-
ined using epifluorescence microscopy. The images revealed distributions of
microorganisms varying from scattered bacterial cells to dense biofilms.
The use of fluorescent dyes for detecting biofilms in tissues has increased as new
fluorochromes for general and species-specific staining have been engineered.
However, staining bacteria enmeshed in EPS and attached to tissue remains chal-
lenging. As discussed previously, autofluorescence and nonspecific stain binding to
tissue components can hinder the detection of bacteria. A further challenge is
distinguishing bacterial EPS from the host extracellular matrix (HECM). Often,
the detection of EPS around bacterial cells is desired as evidence of biofilm. HECM
is present in all animal tissues and organs and consists of a diverse group of
proteins, glycoproteins, and proteoglycans that form basement membranes and all
interstitial structures of the body (Byron et al. 2013 ; Hynes 2009 ). EPS is composed
of carbohydrates, proteins, DNA, and may also contain host components. Thus, the
overall composition of EPS is similar to HECM and EPS stains also bind HECM.
Unfortunately, no reliable stains that can differentiate EPS from HECM have yet
been described.

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7 Fluorescent Stain Selection

7.1 General Stains

Pilot experiments are highly recommended for determining appropriate staining
protocols for a particular set of specimens. The type of tissue, amount of biofilm,
and fixation method may influence the outcome of staining, so testing a diverse
array of stains, concentrations, and staining times may result in identifying the best
approach. The combination of SYTO ® 9, a green nucleic acid stain, and propidium
iodide, a red nucleic acid stain has been used for staining of biofilms in tissue by
several groups. Both stains are part of commercially available LIVE/DEAD ® kits
from Life Technologies. SYTO ® 9 stains the nucleic acids of bacteria with either
damaged or intact membranes, while propidium iodide rapidly penetrates bacteria
with damaged membranes. Thus, when these two components are used together to
stain bacteria for an appropriate contact time, bacteria with intact cell membranes
fluoresce green, while bacteria with damaged membranes fluoresce red. Kathju
et al. ( 2012 ) successfully used SYTO ® 9 and propidium iodide to verify the
presence of biofilms in tissue from a patient suffering from hidradenitis
suppurativa. The specimen was examined by CSLM and the images agreed with
the culture results, revealing the presence of cocci and rods which were stained
green (live) and red (dead) and arranged in clusters. Anderson et al. ( 2012 ) also