Imaging Biofilms in Tissue Specimens - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 32

For example, the authors used a general eubacterial probe, EUB 338 tagged with a
Cy3 fluorophore, to image mouse tissue infected with Staphylococcus aureus . For
certain samples, probing revealed red fluorescent aggregates that resembled
microcolonies of cocci. However, the same aggregates were observed in control
samples (not infected) and cocci were not observed in sections stained using a tissue
Gram stain. This led to the suspicion that the probe was nonspecifically binding to
mast cells present in the tissue. This example serves to underscore the importance of
appropriate controls and complementary alternative staining methods when using
species-specific probes. A study published by Wallner and collaborators ( 1993 )
investigated the effect of several parameters on fluorescent rRNA-targeted oligo-
nucleotide probe binding and specificity. These authors found that excessively high
probe concentrations lead to an increase in nonspecific binding that was likely due
to attachment of probes to cell components rather than mispairing of the probe
sequence to nontarget sequences.

5 Embedding and Sectioning of Tissues

Embedding is the process of casting a tissue section in a selected medium. In this
process, the tissue is infiltrated with a liquid medium, which solidifies, allowing
sectioning into thin slices while preserving tissue structure and morphology. The
selection of an embedding medium depends on the type of tissue, sectioning
technique, and the staining techniques to be used. The medium needs to adhere to
the tissue and be elastic enough to withstand the sectioning procedure, while
impeding the permanent deformation of the tissue to preserve morphology and
structure. A number of embedding media have been developed. Low-temperature
agarose gels and gelatin have been used for embedding tissue to be sectioned with
vibratomes. These media are useful for tissues that can't stand high temperatures.
Plastic resin media, either epoxy or acrylic, have been extensively used for embed-
ding undecalcified tissue, such as bone and teeth and for imaging of tissue-implant
combinations such as stented vessels (Rippstein et al. 2006 ). They also perform
well for sections thinner than the normal 4-6 m usually collected from renal and

bone marrow biopsies (Bruce-Gregorios 2006 ). Epoxy resins allow fast embedding
of the tissue and give good contrast for electron microscopy (Luft 1961 ); however,
there are some disadvantages. The epoxide groups in the resin may reduce the
antigenicity of the tissue, thus impacting immunohistochemistry. In addition, epoxy
resins are toxic, can cause allergic reactions, and the vinylcyclohexane dioxide
component is known to be carcinogenic (Bruce-Gregorios 2006 ). Acrylic resins are
made of esters of acrylic or methacrylic acid and have been preferred for embed-
ding hard tissues due to ease of use and good quality of the subsequent staining
(Bruce-Gregorios 2006 ). The most common medium used for tissue embedding is
paraffin. This can be conducted using automated systems, resulting in a fast and
uniform way of preparing specimens. All water content has to be removed from
tissue prior to paraffin embedding because paraffin is not miscible with water.

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