Imaging Biofilms in Tissue Specimens - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 30

treatments. Biofilms consist of tridimensional microbial communities where spatial
relationships between bacterial populations and host tissue may be important for
biofilm function and interactions with host cells. Thus, it is desirable to image tissue
specimens with minimal manipulation to preserve biofilm architecture. Biofilm
bacteria are embedded in extracellular polymeric substances (EPS). An ideal
technique for imaging biofilms in tissues would allow the visualization of encasing
EPS as well as microbial cells.
Imaging of bacteria and fungi in tissue has been performed for many years using
methods and dyes developed for conventional pathology. In fact, histopathology of
infectious diseases has played an important role on the identification of etiologic
agents (Gupta et al. 2009 ). Classic techniques like Brown and Brenn Gram staining
for tissue have been applied for more than 80 years (Engbaek et al. 1979 ), but
despite their proven value they require that the tissue be exposed to a series of
chemical rinses. In addition, while bacterial cells can be differentiated from host
cells and structures by means of morphology, imaging of EPS poses a challenge.
In this chapter, certain aspects that influence biofilm imaging in tissues will be
discussed including sample collection, fixation, embedding and sectioning,
staining, and microscopy. Although electron microscopy can be used for high-
resolution analysis of tissue samples, this chapter focuses primarily on fluorescence
microscopy. Specific examples from published studies of microorganisms and
biofilms in tissue are used when possible.
2 Specimen Collection
There are a variety of methods for collecting tissue samples for biofilm analysis and
method choice is at the discretion of the clinician. If surgical debridement of tissue
is necessary, the debrided material is a convenient source of specimens for analysis.
Punch biopsies of various diameters can also be collected. One advantage with both
of these methods is that they provide underlying tissue from the wound. This can be
helpful for showing biofilm attachment to the tissue and the extent of tissue
invasion. However, both of these methods are invasive and the necessity of the
procedures and potential risks to the subject must be considered. Wound specimens
can also be collected by curettage, which is a less invasive procedure and routinely
conducted during standard wound care. Topical anesthetics, such as a lidocaine gel,
can be used to prevent or reduce pain during specimen collection. Typically,
specimens are collected at the advancing edge of the wound, although the microbial
populations can vary at different locations within the wound. Specimens should be
placed in an appropriate fixative as soon as possible after collection.