Biofilm Research with the Phenom SEM

Studying Biofilm Formation with the Phenom SEM

"A combination of Phenom ProX and ParticleMetric software enables us to determine cell sizes of rod-shaped bacteria in biofilms."

Many bacteria grow on surfaces and form aggregates called biofilms. Since biofilms are found everywhere in environments and show strong resistance to antibiotics, mechanism of biofilm formation and its regulation have been attracting many researchers. We are using a soil bacterium, Pseudomonas fluorescens, as a model organism to understand how the biofilm formation by this bacterium is regulated.

Dr. Shiro Yoshioka, Institute for Molecular Science, National Institutes of Natural Sciences, Japan

Dr. Shiro Yoshioka, Institute for Molecular Science, National Institutes of Natural Sciences, Japan

Transposon mutagenesis is often used to identify genes required for biofilm formation. If the transposon is inserted into a gene involved in biofilm formation, the mutant will show increased or reduced biofilm formation depending on its role. Using this technique, we identified a gene responsible for synthesis of a bacterial second messenger, c-di-GMP, whose loss caused reduction in biofilm formation compared to the wild type (WT) strain. During this research, we also found that disruption of one of the genes in de novo purine nucleotide biosynthesis resulted in reduced biofilm formation compared to WT. Since the importance of the genes in this pathway has been repeatedly reported for various bacteria, we investigated these mutants to clarify relationship between this biosynthesis pathway and biofilm formation in P. fluorescens.

The Phenom ProX for Biofilm Research

While the biofilm formations by the mutants were reduced to less than half of WT, the cell counting experiments suggested that numbers of the mutant cells in the biofilms were comparable to that of WT. To explain the result, we hypothesized that the cell sizes of the mutants in the biofilms became smaller than that of WT. In other words, the mutations caused the reduction in cell sizes for the biofilm cells. This assumption is not so strange because the mutants cannot synthesize both AMP and GMP, which may lead to slower growth of the mutants.

The scanning electron microscopy (SEM) is one of the best methods to examine the cell sizes of bacteria in biofilms. To obtain information on the cell sizes of bacteria in the biofilms, we employed a desktop SEM, the Phenom ProX, which allowed us to obtain a SEM image within a minute after the sample setting. This fast speed enabled us to prepare several of micrographs for one strain for further detailed analyses using the ParticleMetric software. The figure below shows one of the examples of clear visuals for the biofilm cells of P. fluorescens using the Phenom ProX.

Phenom SEM image of P_fluorescens from Shiro-Yoshioka

Phenom SEM image of biofilm cells of P. fluorescens formed on a surface of polyvinyl chloride

The Power of Phenom ParticleMetric Software

The comparison of the SEM image of the WT with those of the mutants clearly indicated that the size reduction occurred in the mutants. However, it was difficult to emphasize the difference in cell sizes between WT and mutants without numerical comparisons.

The ParticleMetric software for the Phenom ProX is able to generate various parameters for each particle in SEM images. Because P. fluorescens is rod-shaped, the circumscribed circle diameter could be a good approximation for the length of the cells. Using this software, we collected the circumscribed circle diameters of more than 1,500 individual cells from several of SEM images from one strain, created the histograms, and calculated the medians of the circumscribed circle diameters (shown below). The Phenom ParticleMetric software performed this procedure automatically. The only point of attention for us was the fact that the automatic particle detection sometimes treated the aggregated cells as a single cell. ParticleMetric allows us to intuitively remove such cells for reliable calculations from the data collections.

The results of the calculations revealed that the lengths of the mutant cells were reduced by ~25-30% compared to that of WT. Thus, the evidences obtained by Phenom ProX and ParticleMetric software successfully proved our hypothesis that the mutant cells in the biofilms became smaller than that of WT.

Phenom SEM analysis with ParticleMetric of P_fluorescens

A histogram generated by the ParticleMetric software for the biofilm cells of P. fluorescens after collecting the circumscribed circle diameters for more than 1,500 individual cells

The median of the circumscribed circle diameter was calculated to be 1.79 μm. Besides the histogram, the properties of one selected cell are displayed.