Imaging Biofilms in Tissue Specimens - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 36

Fig. 1 Epifluorescence
micrograph of a thin section
from a human wound
specimen biopsy stained
with Sytox ® green and the
fluorescent lectin, wheat
germ agglutinin conjugated
with Texas Red ® . The host
extracellular matrix (and
likely also bacterial
extracellular polymeric
substances) appears red due
to lectin binding and is
colonized by biofilms of
cocci ( green spheres )
complication in IF. Again, adequate controls and complementary imaging methods
can be used to avoid erroneous results. For the detection of particular species within
biofilms, IF and FISH are attractive complementary techniques for microscopic
analysis.
7.3 Fluorescent In Situ Hybridization
The use of molecular techniques to fluorescently tag specific microorganisms is an
attractive approach. Particularly because imaging the spatial distribution of targeted
microorganisms in relation to tissue structures and other microorganisms is possi-
ble. FISH is a molecular technique that uses fluorescent-labeled rRNA oligonucle-
otide probes which combined with microscopy or flow cytometry allows the
detection of microorganisms. This technique facilitates the rapid and specific
identification of microbial cells in their natural environments and can be used in
phylogenetic, ecological, diagnostic, and environmental studies (Amann
et al. 2001 ; Bottari et al. 2006 ; Moter and G¨bel 2000 ). As discussed previously,
controlling specimen and fixation-induced autofluorescence as well as nonspecific
binding are important aspects of FISH. Moter and G¨bel ( 2000 ) wrote a compre-
hensive review of FISH. Although emphasizing the many applications of FISH for
detection of microorganisms in systems ranging from water to environment to
medicine, the authors described a series of pitfalls of the method. The drawbacks
discussed included the occurrence of false positive results due to autofluorescence
of bacteria or tissue and the nonspecific binding of the probes. False negative results
were also highlighted, particularly the problems with penetration of the probe into
bacterial cells (particularly Gram negative bacteria) and the detection of cells with
low rRNA contents. Bacteria of low RNA content is of particular concern for
analysis of biofilms in tissue samples where the bacteria may have a low metabolic