Depletion attractions drive bacterial capture on both non-fouling and adhesive surfaces, enhancing cell orientation

Abstract

Depletion attractions, occurring between surfaces immersed in a polymer solution, drive bacteria adhesion to a variety of surfaces. The latter include the surfaces of non-fouling coatings such as hydrated polyethylene glycol (PEG) layers but also, as demonstrated in this work, surfaces that are bacteria-adhesive, such as that of glass. Employing a flagella free E. coli strain, we demonstrate that cell adhesion on glass is enhanced by dissolved polyethylene oxide (PEO), exhibiting a faster rate and greater numbers of captured cells compared with the slower capture of the same cells on glass from a buffer solution. After removal of depletant, any cell retention appears to be governed by the substrate, with cells immediately released from non-fouling PEG surfaces but retained on glass. A distinguishing feature of cells captured by depletion on PEG surfaces is their orientation parallel to the surface and very strong alignment with flow. This suggests that, in the moments of capture, cells are able to rotate as they adhere. By contrast on glass, captured cells are substantially more upright and less aligned by flow. On glass the free polymer exerts forces that slightly tip cells towards the surface. Free polymer also holds cells still on adhesive and non-fouling surfaces alike but, upon removal of free PEO, cells retained on glass tend to be held by one end and exhibit a Brownian type rotational rocking.

Figure 1.

Polymers in solution exert osmotic attractive depletion forces on particles when they do not adsorb. Osmotic attractions may also produce depletion aggregation when the polymer adsorbs to the particles, as long as there is substantial free polymer in solution to produce an adequate osmotic pressure to drive particles together.

Figure 2.

(A). Five different runs tracking cell capture kinetics for E. coli cells on a non-adhesive PEG brush surface. Cells initially flow past the surface for 20 minutes, the last 2 minutes of which are included, demonstrating a lack of adhesion. Then, upon addition of 1 wt% PEO depletant to the bulk solution, cell capture initiates and levels off. (B). Comparison of PEO depletant-driven capture on a PEG surface (blue points), PEO depletant-enhanced capture on glass (purple points), and surface chemistry-driven capture on bare glass from buffer (red points). The field of view, in which cells are counted, is 178 μm × 267 μm.

Figure 3.

(A) Kinetics of cell release from a non-adhesive PEG brush surface, after replacement of PEO by buffer, superposing 5 runs. (B) Cell retention on glass, comparing retention after removal of PEO depletant (purple) to retention after initial capture from PBS without depletant. An additional control, cell retention on PEG brush surfaces after removal of PEO depletant (in Part A) is also included. Example video frames for the three experiments are placed below each set of bar graphs. Within each panel, the darkest border shows cell counts before rinsing or removal of depletant, the middle shade shows retention after rinsing at 5 s⁻¹, and the light-bordered panel shows retained cells after a subsequent increase in wall shear rate to 110 s⁻¹. (C) Reflectometry establishing PEO adsorption timescale on glass.

Figure 4.

(A) Definition of standing/ leaning/tipped cells and typical micrographs showing examples of each, along with cell alignment angles. Each panel in B-D includes a schematic, an example micrograph, a pie chart summarizing standing/leaning/tipped data from 15–20 different surface regions for 3 separate runs on separate days, and histograms for flow alignment of same cells. Four conditions are compared: (A) PEO-depletion driven capture on a PEG brush surface (B) adhesive cell capture on glass from buffer (C) cell capture on glass enhanced by PEO depletant and (D) PEO enhanced cell capture on glass after removal of PEO depletant. For all data, there is flow at a wall shear rate of 5 s⁻¹. Color coding of frames matches Figures 2 and 3.

Figure 5.

Examples of diffusive cell rotation motion and wiggling. (A) A lack of motion for cells adhered to a glass surface in the presence of PEO and (B) motion of the same cells after replacement of PEO by buffer. The time stamps show when the image was recorded, relative to the time of the first image in each of the two cases. The green dots indicates the part of cells appearing fixed during about a minute in which the cells orientation varied through Brownian rotation and wiggling after PEO was rinsed in part B. The same points for the same cells in part A show the positions of the immobile points at the time each cell was fully immobile on the surface in the presence of PEO.