Microscopy Workshop Conference Q

Microscopy is one of the best ways to interrogate biofilms, and the workshop will highlight applications of laser confocal microscopy to biofilm research. There will be two independent sessions; one from 8:30 AM to 12 PM, and one from 1:30 PM to 5 PM. We will have four confocal microscopes on-site, kindly provided by leading companies in microscope development: Leica, Nikon, Olympus, and Perkin-Elmer. During the first hour of each session, the company representatives will describe their instruments and provide updates on new developments in confocal hardware and software. Attendees can then proceed to any one of four separate rooms in which the instrumentation is on display and in which demonstrations by Rob Palmer (NIDCR/NIH), Janus Haagensen (Danish Technical Univ.) and Morten Hentzer (Danish Technical Univ.) will show how confocal microscopy is used in biofilm research.

Dr. Palmer will be examining undisturbed supragingival dental plaque (from his own mouth!) collected on retrievable enamel chips. Using fluorescently labeled primary antibodies, he will demonstrate that plaque consists intially of scattered single cells and occasional clusters of cells; in the afternoon, the plaque will consist primarily of mixed-species microcolonies (Palmer et al., 2003; J Bacteriol 185:3400-3409).

Dr. Haagensen will demonstrate the very heterogeneous, organized and dynamic nature of monospecies biofilms. Different biofilm morphologies will be shown using Acinetobacter C6 wild type and variants grown in flow chambers. Using confocal microscopy, it will be possible to follow structural dynamics (streamers, group-coordinated movement of microcolonies etc.) in the development of the wild type and variant biofilms. Dr. Haagensen will also demonstrate how, during changing environmental conditions, it is important that the cells be able to regulate their adherence and to switch between planktonic and sessile states. Using flow chambers and confocal microscopy, he will demonstrate how some Pseudomonas putida biofilms dissolve in response to short-term carbon starvation, whereas transposon mutants deficient in biofilm dissolution are able to maintain their structure in the changing environment.

Dr. Hentzer, together with colleague Herb Schweizer (Colorado State Univ.), will demonstrate the application of in situ reporter techniques in biofilm research. One reporter system for monitoring cellular growth is a transposon cassette carrying a fusion between the growth-rate-regulated Escherichia coli rrnB P1 promoter and the gene encoding an unstable variant of Green Fluorescent Protein (GFP). The P1 promoter is regulated in the same way in E. coli and in Pseudomonas aeruginosa, making it useful for monitoring growth in organisms besides enteric bacteria. Construction of fusions to a gene encoding unstable GFP allows determination of rRNA synthesis rates and, in this way, allows on-line determination of the distribution of growth activity in a complex community at the level of individual cells, cell clusters, and microcolonies. A P. aeruginosa strain carrying the rrnB P1-gfp fusion on the chromosome will be grown as a biofilm and will be inspected for rRNA sysnthesis (GFP synthesis) using confocal laser microscopy. Drs. Hentzer and Schweizer will also demonstrate a genetic system for detection of the P. aeruginosa quorum sensing signal molecules which has been constructed by fusing the quorum-sensing-controlled lasB promoter to a gfp gene. A P. aeruginosa PAO1 strain bearing this reporter construct will be grown as a biofilm in a flow chamber and quorum-sensing-activity will be monitored at the single-cell level by detection of GFP fluorescence using confocal laser microscopy. Also, use of the computer software COMSTAT for semiautomatic quantitative analysis of confocal image data (Heydorn et al., 2000; Microbiology 146:2395-2407) will be demonstrated.

If you would like to bring a sample for examination in the workshop, please contact Robert Palmer (rjpalmer@dir.nidcr.nih.gov). Particularly useful would be prepared material, such as FISH-stained biofilm sections. All instruments have at least two fluorescence channels (i.e., “FITC/TRITC”) and are thus useful for multicolor analysis.

Equipment                                          Information

Leica TCS SL                                      www.confocal-microscopy.com

Nikon C1                                            www.nikonusa.com/usa_group/group.jsp?cat=5&grp=23

Olympus FluoView FV500                     www.olympusamerica.com/seg_section/seg_confocal.asp

Perkin-Elmer UltraVIEW RS                  www.liveCELLimaging.com

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