Fluorescence in-situ (FISH) method is often used for detection of pathogenic microflora in drinking water. An important reason for the popularity of FISH is that it is more rapid and, unlike culture- based methods, allows identifying also active but not culturable cells (ABNC). The ability to detect ABNC cells is important as it has been shown that ABNC forms of bacteria are no longer capable of growing on conventional bacteriological media but conserve the pathogenic factors. Analyses of biofilm in drinking water are receiving increasing attention, because biofilm represents majority of biomass in the distribution network. It is quite convenient to detect bacteria in situ on the surface using FISH. The challenge of bacteria detection by FISH on the surface is, however, to reduce the number of processing steps during the hybridization and thus to reduce the risk of cells loss. Another major problem with detection of bacteria in drinking water/biofilm is low signal to noise ratio. It is also preferable that the analysis protocol is as short and simple as possible. PNA probes require less stringent hybridization conditions compared to DNA probes. Moreover, using a PNA probe the time of hybridization can be reduced. As the PNA hybridization occurs at higher temperature, compared to DNA probes, unspecific binding is reduced. The aim of this study was to investigate the effect of washing intensity on the signal and unspecific binding. For this a PNA probe specific against E. coli (developed within SAFER project) was used for hybridization with E. coli, S. paucimobilis and P. fluorescens. S. paucimobilis is a beta-proteobacterium, a class of bacteria that has been connected with membrane filter clogging. P. fluorescens is abundant in drinking water and biofilm samples. The probe binds specifically to E. coli. The traditional PNA hybridization protocol involves a washing step of 30 minutes. Here we show that the specific signal faded considerably with the application of washing step. Already a five minutes of washing reduced almost 50% of the signal intensity. There was no or there was little unspecific binding observed using the cultures of S. paucimobilis and P. fluorescens however the intensity of fluorescence increased as the washing step was applied. As the conclusion, (i) the washing step not only reduces the positive signal, but also may increase the false positive signal, and (ii) its absence would reduce the sample handling time by 30 minutes, thus (iii) using PNA probes the washing is not needed. The new protocol, designed as a result of these experiments can also be applied to the field samples (coupons from river water, the different stages of purification).