Imaging Biofilms in Tissue Specimens - Antibiofilm Agents: From Diagnosis to Treatment and Prevention - page 39

virtual slice or plane, many micrometers deep, within a sample, without the
disturbing light emissions from slices out of the focal plane (Nwaneshiudu
et al. 2012 ). The specimen is moved in the z direction and the stack of images
collected is then combined using computer software. The resulting three-
dimensional reconstructed image provides a high degree of resolution and detail.
Computer image analysis can be combined with CSLM for the determination of
various parameters including number of cells, area of coverage, individual dimen-
sion measurements, and spatial orientation (Lawrence and Thomas 1999 ).
For several years CSLM has been used for analysis of suspected bacteria-
harboring tissue in addition to culturing and molecular techniques to confirm the
presence of biofilms. Neut and collaborators ( 2003 ) evaluated joint explants from
revision surgeries in which infection was suspected. The analysis involved tissue
and biomaterial culturing as well as CSLM of the prosthesis. The samples were
stained with LIVE/DEAD ® BacLight Bacterial Viability Kits and showed the

presence of green (live) cell clusters. A CSLM-based study was also conducted by
Hall-Stoodley et al. ( 2006 ) to test the hypothesis that chronic otitis media is a
biofilm-related infection. Fifty-two biopsies from 26 children suffering from otitis
media with either effusion or recurrent disease were analyzed in addition to
16 samples from control subjects. The mucosa samples were stained using generic
dyes, species-specific probes, and antibodies and underwent thorough examination.
All control samples were negative for biofilm presence, while 46 samples from the
test group were positive for biofilms according to either a general or specific
staining method. Bacteria in the tissue were clearly identifiable in the samples
and ranged from micro-colonies to large clusters. A case report published in 2008
also reported the use of CLSM to investigate the cause of a persistent infection
following a total joint arthroplasty (Stoodley et al. 2008 ). The patient had recurring
pain and infection episodes for years despite several interventions. CSLM analysis
of fluid, tissue, and cement collected at his final surgical revision revealed the
presence of biofilm. These examples show that CSLM can be a powerful tool for the
diagnosis of biofilm-related diseases. However, as stated by Stoodley et al. ( 2008 ),
it involves expensive equipment that is not generally available in hospitals for
examinations. It also requires an experienced operator and the analysis of samples
is a time-consuming process. These drawbacks limit the routine use of CSLM for
medical diagnostics.

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9 Defining and Quantifying Biofilm

Biofilms are generally defined as communities of microorganisms attached to a
surface and enmeshed in an extracellular polymer matrix. For practical purposes
related to the microscopic examination of tissue specimens, the presence of micro-
bial aggregates closely associated with tissue is usually accepted as adequate
evidence of biofilm. Although demonstrating the presence of EPS would provide
further evidence of biofilm, as discussed previously, distinguishing EPS from the