Effect of Quantity and Configuration of Attached Bacteria on Bacterial Propulsion of Micro-Beads

Supplementary Information
Effect of Quantity and Configuration of Attached
Bacteria on Bacterial Propulsion of Micro-Beads

Bahareh Behkam and Metin Sitti
NanoRobotics Laboratory, Department of Mechanical Engineering,
Carnegie Mellon University, Pittsburgh, PA 15213, USA

1. Fabrication of Unpatterned Polystyrene Micro-Beads
The 10 µm pure polystyrene (PS) micro-beads (72986, Sigma-Aldrich, St. Louis,
MO) used in this research work were electrostatically stabilized with an alkyl sulfonate. It
was observed that the surfactant interfered with the particle-protein binding leading to
weak adhesion of bacteria at very small densities (approximately 1 bacterium/100 µm2).
Unpatterned beads were prepared by first removing their surfactant coating. 10%
polystyrene beads were diluted in deionized (DI) water 5 times. The suspension was then
centrifuged at 800g and resuspened in 1:1 DI water/Isopropyl alcohol (IPA). The wash
procedure was repeated five times to ensure removal of substantial amount of the
adsorbed surfactant, thus, causing the bacteria to uniformly coat the surface of the
unpatterned beads at an adhesion density of approximately 1 bacterium/7 µm 2 (Fig.
S1(a)).


2. Fabrication of Patterned Polystyrene Micro-Beads
A plasma-based patterning technique was developed to prepare the patterned
micro-beads. Depending on plasma power, process time, and the type of gas used,
exposure of polystyrene to gas plasma leads to etching of up to few nanometers from the
surface.S1,S2 Moreover, plasma treatment provides a rich variety of chemical
functionalities at the surface, leading to attraction of proteins to the surface by
electrostatic or dipolar interactions. 10% polystyrene bead suspension was diluted in DI
water 10 times and the diluted suspension was washed 3 times. For plasma etching, a
monolayer of PS beads, approximately 4 mm in diameter was prepared on a glass

coverslip, using a previously developed particle self-assembly method.S3 Briefly, a 3 mm
thick layer of polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning, Midland, MI)
was prepared according to the manufacturer's instruction. A circle, approximately 3 mm
in diameter was cut out of the PDMS layer. The perimeter of the circle was cut slantwise
to allow wetting of the walls and to provide a concave air-water interface. The PDMS
ring was then placed on a glass slide and an aliquot portion of the 1% PS bead suspension
(the volume was calculated to provide a dense monolayer of the beads after evaporation
of water) was deposited within the circle. The assembly was placed in a 10 cm Petri dish
and covered to reduce the evaporation rate, thus allowing for the formation of the
monolayer. After 24 hours, the glass slide was taken out and the PDMS ring was
removed. To pattern the beads, the monolayer bead sample was placed in an air plasma
cleaner (PDC-32G, Harrick Plasma, Ithaca, NY) at 18 W RF coil power for 2.5 minutes.
This process etched the portion of the micro-bead that was not masked by the underlying


glass slide or the adjacent beads, revealing a functionalized hydrophilic surface.
Afterwards, the sample was immediately placed in motility medium (0.01 M potassium
phosphate, 0.067 M sodium chloride, 10-4 M EDTA, 0.01 M glucose, and 0.002% Tween20, pH 7.0)

S4

and sonicated for 2.5 minutes to release the beads from the glass slide.

Bacteria attach to the plasma treated regions of the patterned beads at a density of
approximately 1 bacterium/12 µm2; while very few or none adhere to the untreated area
(Fig. S1(b)).

REFERENCES
S1. E. Bonaccurso, B. Cappella, K. Graf, J. Phys. Chem. 110, 17918 (2006).

S2. X. M. Zhao, Y. N. Xia, O. Schueller, D. Qin, G. M. Whitesides, Sensor. Actuat. APhys. 65, 209 (1998).

S3. N. Denkov, O. Velev, P. Kralchevsky, I. Ivanov, H. Yoshimura, K. Nagayama,
Langmuir 8, 3183 (1992).

S4. N. Darnton, L. Turner, K. Breuer, H. Berg, Biophyis. J. 86, 1863 (2004).


Fig. S1. Optical microscopy images and schematics of 10 µm polystyrene beads with S.
marcescens bacteria attached in different configurations: (a) Bacteria uniformly adhere to the
unpatterned micro-bead; (b) Bacteria only adhere to the plasma treated region of the patterned
micro-bead.