Furthermore, FISH is not a stand-alone technique in the diagnostic setting, as culture is still used for antibiotic susceptibility testing. While traditionally the probes for FISH were based on single MLN0128 ic50 stranded DNA, another set of probes increasingly used in diagnostics are based on a polyamide ‘peptide’ backbone (Egholm et al., 1993; Bjarnsholt et al., 2008). PNA FISH probes abide by Watson/Crick

pairing but possess unique hybridization characteristics because of their uncharged chemical backbone, including rapid and stronger binding to complementary targets compared with traditional DNA probes. PNA probes can also be used with unfixed biological samples; however, only a limited number of probes are currently available, restricting the use of PNA FISH for the present. CLSM and FISH emphasize that demonstrating biofilm spatial organization is extremely important to: (1) identify whether the bacteria present are aggregated, (2) indicate a polymicrobial nature of a biofilm, (3) indicate the extent of biofilm on a surface that CFU may vastly underestimate, and (4) to show biofilm EPS that may comprise a greater

part of the biofilm than cells alone. On nonbiological, flat surfaces, biofilm spatial organization can best be measured by various parameters using image analysis software. The most common program is comstat that yields a number of spatial parameters including thickness, biovolume, selleck and roughness (Heydorn et al., 2000). Quantification of biofilm spatial organization is harder Lepirudin however in clinical specimens that usually have a complicated and convoluted surface geometry, and currently is largely descriptive

or qualitative in these samples – that is, data showing cells or clusters per unit area without a good method to quantify spatial dimensions. As comstat thresholding does not work well on tissue backgrounds, quantifying the biofilm involves a manual rendering of biofilm images in other software to resolve bacteria and laborious cell counting, particularly if NA probes are used because they stain host cell nuclei as well as bacterial DNA (Nistico et al., 2011). Resolving biofilm spatial organization is also made more difficult because of the spatial scales involved. For example to be able to resolve individual bacteria in an image, the field of view needs to be on the order of 100 μm2, while the specimen might be on the order of cm2 (1 million fields) for tissue or even 100s of cm2 (over 100 million fields) for large orthopedic implants making microscopic data from a small proportion of the sample often the only practical method to demonstrate biofilm in situ. Finally, because biofilms may also be extremely localized, it is difficult to quantify by averaging several images on the surface, because heterogeneity leads to extensive sample variability.