The Laryngoscope V C 2009 The American Laryngological, Rhinological and Otological Society, Inc. Targeted Imaging Modality Selection for Bacterial Biofilms in Chronic Rhinosinusitis Andrew Foreman, BMBS (Hons); Deepti Singhal, MS; Alkis J. Psaltis, PhD, MBBS; Peter-John Wormald, MD Objectives/Hypothesis: Biofilms are increas- ingly recognized as having an etiological role in chronic rhinosinusitis (CRS). Research into biofilms in CRS currently relies on microscopic imaging techniques, none of which are universally accepted. This study compares LIVE/DEAD BacLight (Invitro- gen Corp., Carlsbad, CA) staining and fluorescence in situ hybridization (FISH), both utilizing the confocal scanning laser microscope (CSLM) for biofilm determination and characterization in CRS patients. Study Design: Prospective study. Methods: Twenty CRS patients undergoing en- doscopic sinus surgery were recruited for the study. Sinus mucosal tissue harvested at the time of surgery underwent both the BacLight/CSLM and FISH/CSLM protocols for biofilm determination and characterization. Results: Combining the results of both protocols, 18/20 (90%) patients had bacterial biofilms demon- strable on at least one modality. The high biofilm detection rate combining the two techniques suggests the prevalence of biofilms in CRS may be greater than previously reported. The protocols had equiva- lent results in 15/20 patients. Using the differences observed in the remaining five patients, we can high- light the most appropriate use for each technique. Conclusions: BacLight/CSLM and FISH/CSLM are complementary techniques for biofilm determina- tion and characterization. Both protocols are suited to different research areas and the selection of tech- nique used should be based on the specific objectives of the research protocol. In this way we can utilize the advantages of each technique to facilitate effec- tive research. Key Words: Chronic rhinosinusitis, biofilms, BacLight, fluorescence in situ hybridization, Staphylococcus aureus. Laryngoscope, 120:427–431, 2010 INTRODUCTION Late in the 17th century, bacteria were found to exist in two contrastingly different forms, the planktonic and the biofilm form. Not only are these two forms different phenotypically, but it has been shown that they tran- scribe more than 70 different genes as well. Since Koch outlined his famous postulates in the mid-18th century, we have become very familiar with how planktonic bacte- ria cause a wide spectrum of acute infectious diseases. Recently, we have come to learn of the role that the ses- sile biofilm form plays in infections as well. The role of biofilms in chronic inflammatory diseases has become increasingly recognized across a range of medical and surgical specialties, including device-associated infec- tions, 1 cystic fibrosis, 2 chronic prostatitis, 3 and otitis media with effusion. 4 Given its similarities with other biofilm-mediated diseases, the biofilm paradigm has recently been applied to chronic rhinosinusitis (CRS). In recent years a number of microscopic imaging modalities have been employed to attempt to identify biofilms on the sinus mucosal surface of CRS patients. 5–8 Our department has demonstrated that a nucleic acid probing technique using the LIVE/DEAD BacLight Viability Kit (Invitrogen Corp., Carlsbad, CA) imaged under the confocal scanning laser microscope (CSLM) is superior to scanning electron microscopy and transmission electron microscopy 9 for the visualization of biofilms on sinus mucosa. Conclusive evidence now exists for the presence of biofilms on the sinonasal mucosal surface of CRS patients. 5–8,10 In addition, it has been shown that their presence may also be associated with more severe sinus disease clinically and radiologically. 11,12 From this basis, research is now evolving from biofilm identification to- ward biofilm characterization and investigations of the pathogenic role of biofilms in CRS. As the requirements of research in this field change, so too do the require- ments of the imaging modality, with a new focus on From the Department of Surgery–Otorhinolaryngology, Head and Neck Surgery, University of Adelaide and Flinders University, Adelaide, Australia. Editor’s Note: This Manuscript was accepted for publication July 31, 2009. This work was supported by the Garnett Passe and Rodney Wil- liams Memorial Foundation. Send correspondence to Peter-John Wormald, MD, Department of Otorhinolaryngology, Head and Neck Surgery, Queen Elizabeth Hospital, 28, Woodville Road, Woodville, SA 5011, Australia. E-mail: peterj.wormald@adelaide.edu.au DOI: 10.1002/lary.20705 Laryngoscope 120: February 2010 Foreman et al.: Imaging of Bacterial Biofilms in CRS 427