1 This article was published in Biofouling, 32(2), 179-190, 2016 http://dx.doi.org/10.1080/08927014.2015.1131821 Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH) Diana Vilas Boas a , Carina Almeida a,b , Sanna Sillankorva a , Ana Nicolau a , Joana Azeredo a and Nuno F. Azevedo b a LiBRo –Laboratório de investigação em Biofilmes Rosário oliveira, Centre of Biological Engineering, university of Minho, Braga, Portugal; b Laboratory for Process, Environment, Biotechnology and Energy Engineering (LEP ABE), Department of Chemical Engineering, f aculty of Engineering, university of Porto, Porto, Portugal ABSTRACT Bacteriophage–host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA- FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations. Introduction Plaque counts (or plaque forming units; PFU) on agar plates, originally developed by d’Herelle (1917), have been used as the gold standard to enumerate phage particles. While this method has been extensively implemented in laboratories worldwide, it also presents important drawbacks, such as: (1) poor reproducibility (between experiments and between laboratories); (2) long incubation periods (usually 18 to 24 h); (3) inability to perform multiplex experiments (eg inability to detect/enumerate different phages within the same sample); and (4) inability to assess the spatial distribution of phages or to visualize cell–phage interactions (Doolittle et al. 1996; Allen et al. 2011; Yoon et al. 2011; Pankaj 2013). Some authors have attempted to develop culture-independent techniques that might avoid at least some of the limitations of PFU. Doolittle et al. (1996), for instance, used different dyes to stain cells and phages in biofilm structures – differentiated communities of sessile microorganisms surrounded by a complex matrix (Doolittle et al. 1996; Costerton 1999; Cerqueira et al. 2013).