A survey of native microbial aggregates on alfalfa, clover and mung bean sprout cotyledons for thickness as determined by confocal scanning laser microscopy

In nature, bacteria are often organized in aggregates or biofilms rather than as solitary cells. Biofilms on inert surfaces have been studied in depth using confocal scanning laser microscopy (CSLM) with a variety of fluorescent probes. In contrast, there have been few studies of native microbial aggregates or biofilms on living surfaces, including plants. In this study we used CSLM in combination with the LIVE/DEAD® BacLight™ Viability Kit (Molecular Probes Inc.) to determine the thickness of native microbial aggregates on alfalfa, clover and mung bean sprouts purchased from retail outlets. A survey of aggregate thickness was made by use of a 20× dry lens primarily due to its large free working distance, broad field of view and the uneven topography and shape of cotyledon surfaces. Values for measured thickness (z-axis) were corrected based on the point spread function of fluorescent latex spheres (1.98 μm in diameter). Aggregates consisted primarily of live bacteria. Aggregates on mung bean cotyledons were significantly (P<0.05) thicker (average=6.4±2.1 μm, median=6.0 μm, range of 3.4-10.6 μm) than those on alfalfa (average=3.3±2.1 μm , median=2.8 μm, range=1.7-12.6 μm) or clover (average=3.0±1.1 μm, median=2.8 μm , range=1.7-5.4 μm). Average thickness was not significantly (P>0.05) different for aggregates on clover and alfalfa. Bacteria in fully hydrated aggregates as imaged by CSLM appeared to be less densely packed when compared to sprout surface biofilms imaged previously in our laboratory with conventional scanning electron microscopy techniques, most likely due to the presence of hydrated bacterial exopolymers. Despite a lack of considerable thickness, aggregates and biofilms on plant surfaces may harbor plant and human pathogens making their eradication more problematic and also protect pathogens and native bacteria from adverse environmental conditions.