Attachment of Pseudomonas fluorescens to glass and influence of electrolytes on bacterium-substratum separation distance

Abstract

The influence of Na+, Ca2+, La3+, and Fe3+ on the adhesion of Pseudomonasfluorescens H2 and H2S was investigated with interference reflection microscopy (IRM). IRM is a light microscopy technique which allows (i) visualization of the adhesive sites of living bacteria as they attach to a glass cover slip surface and (ii) evaluation of the bacterium-glass surface separation distance within a range of 0 to ca. 100 nm. The addition of each cation caused changes in IRM images consistent with a decrease in the separation distance, and minimum effective concentrations were as follows: Na+, 1 mM; Ca2+, 1 mM; La3+, 50 I,M; and Fe3+, 50 ,uM.With strain H2, the effects of Na+, Ca2+, and La3+ were fully reversible in that the separation distance increased again when the electrolyte was replaced with distilled water. However, with strain H2S, a spontaneous mutant of H2 with increased attachment ability, only the effect of Na+ was fully reversible, and the effects of Ca2' and La3+ were only partially reversible or irreversible. The effect of Fe3+ was irreversible with both strains, but this may be related not only to the electrolytic nature of Fe3+ but also to the decrease in solution pH to 3.5 caused by its addition. It is proposed that the electrolytes caused a decrease in separation distance by neutralizing negative charges on bacterial surface polymers and that the different effects obtained with the two strains are related to their different adhesion abilities.When bacteria in an aqueous suspension are exposed to a solid surface, many of those bacteria may adhere to the surface and become permanently attached. Investigators of bacterial attachment have attempted to determine the adhesion mechanism(s) of different bacteria or to test the effects of different environmental or physiological conditions on attachment (14). However, there has been much variation in results from these studies with respect to the ability of different bacterial species, strains, or phenotypes to attach and the susceptibility of their adhesion to modifying factors. Because of this variation, it has been extremely difficult to determine the mechanism by which a given factor can modify the adhesion process.Factors which influence attachment include those which influence the physicochemistry of adhesion, such as electrolyte species or concentration (10,15,17), pH (17, 23), temperature (4, 11), and surface (3,20) Meaningful direct observation of attaching cells has been impossible because of the limiting resolution of light microscopy and the inability to view the bacterial surface-substratum interface during adhesion.However, with interference reflection microscopy (IRM), the adhesive sites on the bacterial surface can be visualized while attachment to a solid surface is established. With this technique, it is possible to observe living cells as they become loosely associated (i.e., within 100 nm) with a glass cover slip surface and to evaluate the change in distance between the cell and surface as firm adhesion is established. With IRM, the darknes...

Reversible and Irreversible Adhesion of Motile Escherichia coli Cells Analyzed by Total Internal Reflection Aqueous Fluorescence Microscopy

“…The data presented here quantitatively demonstrate a distance change associated with motile cells proceeding from a surfaceassociated state where they can swim relatively far from, but parallel to, the surface, to a more firmly attached state, closer to the surface, where they become immobilized. Other researchers have observed cells become attached to the surface (see, for example, reference 22) and have also observed, qualitatively, that immobilized cells are closer to the surface at high ionic strength than at low ionic strength (10,11). In the present work, these results have been augmented with more quantitative measurement of distances, as well as the observation of motile cells.…”

Interference reflection microscopic study of sites of association between gliding bacteria and glass substrata

“…3). It is interesting that these movements are possible even at the small cell-substratum separation distances similar to those previously found to be characteristic of firm adhesion by other bacteria and by eucaryotic cells (10,12,16,27 (19). The sense of rotation of U67 appeared to be sinistral, with a helix of 790 pitch.…”

“…Bar = 2 p.m. (2, 9, 12, 16). Nevertheless, variations in film thickness, i.e., separation distance, ranging from 0 to 100 nm appear as interpretable relative-intensity differences (16) (9,10,12,16,27). When applied to the study of motility of fibroblasts (1) and amoebae (27), IRM demonstrated multiple small sites of adhesion (dark areas) which remained stationary with reference to the substratum as these eucaryotic cells moved.…”

“…IRM was developed to visualize surface contact sites on adherent eucaryotic cells (9) and has been applied to evaluate bacterium-substratum separation distance (10,11 (33).…”

Review on biofilm formation and its control options

“…Characteristics of aqueous medium, such as pH, nutrient levels, ionic strength, and temperature, may also play a role in the rate of microbial attachment to a surface (Donlan, 2002). For example, an increase in the number of attached bacterial cells was observed as a result of an increase in nutrient concentration in medium (Cowan et al, 1991) and an increase in the concentration of several cations (Fletcher, 1988). Additionally, hydrodynamic properties of the aqueous medium such as velocity characteristics of the liquid influence the rate and extent of attachment (Characklis, 1990).…”