Real-Time Approach to Flow Cell Imaging of Candida albicans Biofilm Development

For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the analysis of scientific images. We discuss the origins, challenges and solutions of these two programs, and how their history can serve to advise and inform other software projects.

With the advent of digital cameras and motorization of mechanical components, computer control of microscopes has become increasingly important. Software for microscope image acquisition should not only be easy to use, but also enable and encourage novel approaches. The open-source software package µManager aims to fulfill those goals. This unit provides step-by-step protocols describing how to get started working with µManager, as well as some starting points for advanced use of the software. © 2010 by John Wiley & Sons, Inc.

The structural organization of four microbial communities was analysed by a novel computer program, COMSTAT, which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria, Pseudomonas: putida, P. aureofaciens, P. fluorescens and P. aeruginosa, tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure - mean thickness, roughness, substratum coverage and surface to volume ratio - showed that the four Pseudomonas: strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies, which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum, and formed flat, uniform biofilms. P. aureofaciens resembled P. aeruginosa, but had a stronger tendency to form micro-colonies. Finally, the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0.03 mM, 0.1 mM or 0.5 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover, biofilm roughness increased with lower citrate concentrations, whereas surface to volume ratio increased with higher citrate concentrations.