Imaging Biofilms in Tissue Specimens - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 33

Dehydration is typically accomplished with a graded ethanol series, followed by
infiltration with xylene or a xylene substitute such as SafeClear .

Cryoembedding and cryosectioning of tissue has been used routinely in the
medical field, especially when a fast diagnosis of pathology is required, such as
during surgery. These techniques do not provide the best preservation of tissue
morphology but are often suitable for specific applications. Cryoembedding of
tissues has the advantages of being a fast and simple technique for preparing
samples for sectioning and requires virtually no reagents other than the embedding
medium. Like other embedding media, the water content of the samples needs to be
replaced prior to embedding. In cryoembedding, this prevents ice crystal formation
within the tissue during freezing.
For biofilm analysis in tissue specimens, cryosectioning and cryoembedding
have proven to be suitable methods (Han et al. 2011 ). After fixation, the tissue
specimen was dehydrated in a 30 % sucrose solution until it sank to the bottom of
the flask, which was an indication that the solution had penetrated the entire
specimen. The sample was then placed in a small metal or plastic disposable tray
with a freezing medium (Tissue-Tek ® O.C.T

™

Compound) and snap-frozen on a
block of dry ice. Sections were then cut with a cryostat, a refrigerated microtome
with temperature of about

™

30 C. After cutting, the tissue sections were
picked up on plus microscope slides and then stained or frozen for later analysis.

20 to

6 Staining

Both conventional and fluorescent stains have been used for staining bacteria in
tissue. Hematoxylin and eosin (H&E) is a traditional histological stain that has been
used to demonstrate the presence of microorganisms in tissue. Hansen et al. ( 2006 )
and Marx and Tursun ( 2012 ) applied H&E to biopsies of patients with osteoradio-
necrosis and detected the presence of large aggregates of bacteria next to areas of
necrotic bone. Gram staining of tissue has also been used for many years, and
several techniques, such as the Brown and Brenn Gram stain and Brown and Hopps
Gram stain, have been tested and compared (Engbaek et al. 1979 ). Sizemore
et al. ( 1990 ) used fluorescently labeled wheat germ agglutinin to label Gram-
positive bacteria. This lectin binds specifically to N -acetylglucosamine in the
peptidoglycan layer of Gram-positive microbes and does not stain Gram-negative
bacteria due to their outer membrane. Although conventional Gram staining can
provide good differentiation of Gram-positive bacteria, the identification of Gram-
negative bacteria can be impaired by coloring of the host tissue. As an alternative,
several attempts were made to develop fluorescent dyes to differentially Gram stain
microorganisms. Mason et al. ( 1998 ) developed a combination of hexidium iodide
(HI) and SYTO 13 to Gram label bacteria for both flow cytometry and fluorescence
microscopy. When used in combination, Gram negatives were stained green by
SYTO 13, while Gram positives were stained by both SYTO 13 and HI, resulting in
red-orange fluorescence due to the quenching properties of HI. Han et al. ( 2011 )