© 2010 Nature America, Inc. All rights reserved. PROTOCOL 1460 | VOL.5 NO.8 | 2010 | NATURE PROTOCOLS INTRODUCTION Clinicians who deal with device-related and other chronic bacterial infections increasingly face a new category of infectious diseases that differs radically from acute epidemic bacterial infections. These diseases are much less aggressive than acute infections, they often persist for months or years, and they progress through periods of latency that alternate with periods of acute exacerbation. Moreover, although traditional antibiotic therapy provides some relief during such exacerbations, antibiotics fail to resolve the infections 1 . Chronic bacterial infections with Pseudomonas aeruginosa have an important role in pulmonary infection in cystic fibrosis (CF) 2 . The majority of people with CF acquire P. aeruginosa, and in these people, chronic lung infection, repeated exacerbations and progres- sive deterioration in lung function remain major causes of morbidity and mortality. In the chronically infected CF lung, P. aeruginosa adopts a biofilm mode of growth with the formation of structured microbial communities that grow within microcolonies embedded in an extracellular matrix. It has been shown that with the forma- tion of bacterial biofilms, it becomes extremely difficult to eradicate the infection 3 . Biofilm bacteria are much more resistant to antibi- otic treatment, as well as to the host immune response, and we are only beginning to understand the reasons for this biofilm recalci- trance 4–7 . The formation of bacterial biofilms profoundly influences the biological activities of the constituting bacteria in a way that is not easily predicted on the basis of our current knowledge 8,9 . Despite evidence that P. aeruginosa grows within microcolonies in the airways of people with CF, conventional clinical susceptibility test- ing involves the culture of planktonically grown bacteria that have been recovered from the respiratory tract. Consequently, antibiotic therapy is directed by these susceptibility test results to treat symptomatic indi- viduals affected by CF with chronic infections. However, it seems rea- sonable that the antibiotic susceptibilities of planktonic populations, as determined by minimal inhibitory concentration (MIC) methodolo- gies, do not necessarily reflect the actual resistance profile in vivo 10 . One may argue that the strategies of susceptibility testing should account for differences in growth behavior within biofilms, in which the bacteria grow slowly and are densely packed in a microaerophilic environment 11,12 . Previous studies comparing the antibiotic resistance profile of biofilm- versus planktonic-grown P. aeruginosa have revealed that there is obviously not a single agent or combination of agents that dominates the biofilm susceptibility profile of CF isolates 5,13 , and others have shown that people treated with biofilm-effective therapy had improved clinical outcomes 14 . This implies a need for individualized biofilm susceptibility testing in the clinical setting. Nevertheless, even if antimicrobial biofilm resistance is strain specific, it is indispensable to clearly show in clinical trials that biofilm susceptibility testing results in sufficiently different treat- ment regimes and that these treatment regimes benefit people with CF 15 . However, the major problem faced in evaluating the benefit of alternative antibiotic treatment has been the lack of a suitable assay that could provide clinicians with an antibiotic resistance profile of biofilm-grown bacteria. The use of a standardized and reliable high- throughput system to monitor biofilm growth in the presence of various antibiotics would overcome this hindrance. In 1999, a 96-well-plate–based assay to monitor biofilm formation and perform subsequent quantitative microbiology was established in Ceri’s laboratory, and since then it has been widely used to test antimicrobial resistance profiles of biofilm-grown clinical isolates in several bacterial pathogens 16–21 . However, this test system is very material- and time-consuming and is hardly applicable to antimi- crobial resistance testing in routine diagnostics. Other alternative methods to quantify microbial biofilms have been tested for vari- ous organisms. One of these alternative methods for the testing of drug effects on biofilm cells is the colorimetric determination of metabolic activity after drug exposure 22–28 , which requires less post- processing of samples and correlates with cell viability (as opposed, for example, to crystal violet staining of the bacterial biofilm mass) 29 . A 96-well-plate–based optical method for the quantitative and qualitative evaluation of Pseudomonas aeruginosa biofilm formation and its application to susceptibility testing Mathias Müsken 1,4 , Stefano Di Fiore 2 , Ute Römling 3 & Susanne Häussler 1,4 1 Chronic Pseudomonas Infection Research Group, Helmholtz Center for Infection Research, Braunschweig, Germany. 2 Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen, Germany. 3 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden. 4 Pathophysiology of Bacterial Biofilms, Twincore, Center for Experimental and Clinical Infection Research, a joint venture of the Helmholtz Center for Infection Research and the Medical School Hannover, Hannover, Germany. Correspondence should be addressed to S.H. (susanne.haeussler@helmholtz-hzi.de). Published online 29 July 2010; doi:10.1038/nprot.2010.110 A major reason for bacterial persistence during chronic infections is the survival of bacteria within biofilm structures, which protect cells from environmental stresses, host immune responses and antimicrobial therapy. Thus, there is concern that laboratory methods developed to measure the antibiotic susceptibility of planktonic bacteria may not be relevant to chronic biofilm infections, and it has been suggested that alternative methods should test antibiotic susceptibility within a biofilm. In this paper, we describe a fast and reliable protocol for using 96-well microtiter plates for the formation of Pseudomonas aeruginosa biofilms; the method is easily adaptable for antimicrobial susceptibility testing. This method is based on bacterial viability staining in combination with automated confocal laser scanning microscopy. The procedure simplifies qualitative and quantitative evaluation of biofilms and has proven to be effective for standardized determination of antibiotic efficiency on P. aeruginosa biofilms. The protocol can be performed within ~60 h.