Microbubbles reveal chiral fluid flows in
Yilin Wua,b, Basarab G. Hosua,b, and Howard C. Berga,b,1
aRowland Institute at Harvard andbDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
Edited by Tom C. Lubensky, University of Pennsylvania, Philadelphia, PA, and approved December 30, 2010 (received for review November 5, 2010)
Flagellated bacteria can swim within a thin film of fluid that coats
a solid surface, such as agar; this is a means for colony expansion
known as swarming. We found that micrometer-sized bubbles
form explosively when small aliquots of an aqueous suspension of
dropletsof a water-insoluble surfactant(Span 83) are placed on the
droplets are exposed to air. Using these bubbles, we discovered an
extensive stream (or river) of swarm fluid flowing clockwise along
the leading edge of an Escherichiacoli swarm, at speedsof order 10
μm/s,about threetimesfasterthan the swarmexpansion. Theflow
is generated by the action of counterclockwise rotating flagella of
cells stuck to the substratum, which drives fluid clockwise around
isolated cells (when viewed from above), counterclockwise be-
tween cells in dilute arrays, and clockwise in front of cells at the
swarm edge. The river provides an avenue for long-range commu-
that diffuse poorly. These findings broaden our understanding of
swarming dynamics and have implications for the engineering of
bacterial-driven microfluidic devices.
bacterial motility|flagellar rotation|microscopic bubbles
colonize the surface by swimming outward in densely packed
groups within a thin layer of fluid, a process known as swarming
(1–3). Swarming promotes invasiveness and virulence of in-
fectious pathogens (2) and is regulated by pathways that enable
cells to choose between swimming or forming sessile biofilms (4).
A model system that we study is the bacterium Escherichia coli,
which was shown to swarm by Harshey and Matsuyama (5). Our
focus is on swarm mechanics, how cells move and how this motion
is promoted by flagellar rotation (6–8, but see also refs. 9–11).
However, little is known about the swarm fluid, despite its
significance to the expansion and physiology of swarms. The
swarm fluid is important for swarm expansion, not only because
it supports the operation of flagella (12), but also because it
carries nutrients that support growth or molecules that regulate
cellular functions, such as signals involved in quorum-sensing
(13, 14). Quorum-sensing has been found to regulate the syn-
thesis of biosurfactants that facilitate swarming in Bacillus subtilis
(15, 16), Serratia liquefaciens (17), and Pseudomonas aeruginosa
(18). The quorum-sensing molecules involved in these systems
are released from cells and diffuse in the swarm fluid before
entering cells again.
The layer of swarm fluid can be as thin as the width of a cell
(∼1 μm) and even thinner near the swarm edge (8), which makes
it difficult to probe the hydrodynamics of swarm fluid with con-
ventional microfluid markers, such as polystyrene microspheres
(19). Large markers tend to be entrained by cells within the body
of the swarm, but small ones become embedded in the agar. We
solved this problem by developing buoyant fluid markers: micro-
bubbles that form spontaneously when a suspension of droplets
of a water-insoluble surfactant is exposed to an air-liquid in-
terface. With these unique markers, we discovered an extended
stream (or river) flowing clockwise along the leading edge of
hen grown on a moist nutrient-rich medium, many flag-
ellated bacteria elongate, produce wetting agents, and
times the rate of swarm spreading) persist over long distances,
providing the swarm with an avenue for long-range communica-
tion. The findings shed new light on how swarming bacteria might
regulate material transport, broaden our understanding of fla-
gellar hydrodynamics (20), and have implications for the engi-
neering of bacterial-driven microfluidic devices (21, 22).
Microbubble Formation. When a drop of a suspension of Span 83
(a water-insoluble surfactant with nonionic head groups derived
from sorbitol, and hydrophobic tails derived from common fatty
acids, primarily oleic acid) (Materials and Methods) is placed on
an agar surface a few centimeters in front of an E. coli swarm, the
water is absorbed by the agar and the Span 83 droplets are ex-
posed to air. The droplets explode into large films, which soon
contract and transform into arrays of micrometer-sized objects
by a cascade of sudden events, as shown in Fig. 1 (see also Movie
S1) or into a single micrometer-sized object that can be much
larger than the original droplet, as shown in Fig. S1 (see also
Movie S2). These objects appear to be air bubbles, air-filled
capsules with surfactant walls. The initial explosion of surfactant
droplets usually is complete within 1/15 s (two video frames), but
the contraction and bubble formation takes variable amounts of
time. The films that form appear to be similar to the macroscopic
ones studied by Matar and Troian (23), but the subsequent
contraction and bubble formation appears to be unique and
merits further study. The explosions are reminiscent of those
described recently for large interfacial air bubbles by Bird et al.
(24) and might share a similar mechanism. In any event, the
microbubbles formed in this way move continuously among cells
within the swarm, indicating that they can easily follow fluid flow.
In contrast, untransformed surfactant droplets of a similar size
show only intermittent movement within swarms or get stuck in
Long-Range Clockwise Flow Along E. coli Swarm Edges. Astheswarm
approaches, the bubbles move quite smoothly along the swarm
edge; although, the bodies of cells near the swarm edge show
limited or even no movement. Surprisingly, the direction of net
movement of the bubbles is always clockwise (CW) when the
swarm is viewed from above (found with >30 swarms). An ex-
ample is shown in Fig. 2 A and B (see also Movie S3). This move-
ment can persist over long distances. We observed one micro-
bubble moving CW along the swarm edge at an average speed of
∼8 μm/s for a distance of over 0.5 mm. These observations sug-
gest the existence of long-range unidirectional flow (i.e., a river,
and H.C.B. contributed new reagents/analytic tools; Y.W. and B.G.H. analyzed data; and
Y.W., B.G.H., and H.C.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| March 8, 2011
| vol. 108
| no. 10
along the edge of E. coli swarms). Such a river might establish
right-handed chirality in swarms if a colony branches, because
cells tend to move onto virgin agar where there is sufficient liquid
for them to spin their flagella. Indeed, we observe such right-
handedness in young swarms of E. coli, as shown in Fig. 2C.
Submicron bubbles (<1.0 μm in diameter) continuously varied
their speed and distance from the swarm edge (defined as the
distance from the bubble to the nearest cell body) and provided
a tool for probing the flow-speed profile of the river. From the
trajectory for each bubble, we measured the velocity and the
distance to the swarm edge in every video frame (30 times per
second). The velocity was divided into tangential and radial
components (along the swarm edge and in the direction of swarm
expansion, respectively). The average tangential and radial ve-
locities at a specific distance from the swarm edge were taken as
the average tangential and radial flow speeds of the river at that
distance from the swarm edge.
We collected such data for each of three E. coli swarms (strain
HCB1668). The average tangential and radial flow-speed profiles
for one of these swarms are plotted in Fig. 3 A and B (see also
Fig. S2). The tangential flow profile peaks at a distance of 2 to
2.5 μm, about half of the average length of the flagellar filaments
(7), at a flow speed of ∼8 μm/s. The other two swarms showed
similar tangential flow profiles. The tangential flow speed drops
to nearly zero at the distance of ∼7 μm, suggesting that the av-
erage width of the river is ∼7 μm. Because cells at the swarm
edge tend to project their flagella outwards onto the virgin agar
when they get stuck (7, 9), the river flow is most likely generated
by the rotating flagella of these stuck cells, which form a special
kind of bacterial carpet (25). The radial flow speed within the
distance of one flagellar length is close to the expansion rate of
the swarm (3.3 μm/s). This finding supports the notion that
swarm expansion is aided by flagella pumping fluid outwards
from the edge of the colony (6, 7, 9).
Local Chiral Flow Patterns. We found other asymmetric flow pat-
terns on the scale of single cells. When the edge of a dense E. coli
swarm (with microbubbles already formed) is diluted by a small
move in exclusively counterclockwise (CCW) swirls in between
the immobile cells (>50 samples). Fig. S3 shows such an example,
per second; see also Movie S4). The rotational frequency of the
likely depending upon the amount of surrounding fluid.
To better understand the CW river and the CCW swirls just
described, we wanted to probe the flow generated by single cells
near the swarm edge. However, the swarm edge is densely packed
with cells, and it is impossible to discern the flow generated by
individual cells. Instead, we collected cells at the swarm edge in
motility buffer and dropped this cell suspension on a coverslip
coated with a thin layer of agar (Materials and Methods), creating
a sparse distribution of isolated cells and a denser array of cells
near the drying edge of the drop. Like cells near the swarm edge,
the cells near the drying edge of the drop have limited movement,
most likely because of the lack of surrounding liquid, but they also
create long-range CW flows along the edge of the cell array and
CCW swirls in between cells (Movie S5). Therefore, this system
(referred to as the “agar slip model”) allows one to observe iso-
lated cells under conditions that mimic those observed near a real
By tracking the motion of microbubbles around isolated cells
in the agar slip model, we found that bubbles circle around cell
bodies exclusively CW (>50 samples, one of which is shown in
Fig. S4; see also Movie S6), which suggests CW flow circulations
around cells residing in a thin liquid layer on a solid surface.
CCW swirls in between several immobile cells can be explained
as the net effect of CW circulations around each individual cell.
Similarly, the long-range CW flow along a swarm edge can be
explained as the net effect of CW flows around cells that are
lined up along the swarm edge.
seen by bright-phase microscopy at the center of A first explodes into a less
bright or even invisible film that spreads over a much larger area than the
size of the droplet (B and C). The film then contracts and bubbles form at the
periphery of the film (D and E). The film contraction proceeds and a second
round of bubble formation takes place (E and F), resulting in many bright air
bubbles all over the area previously occupied by the film. The bubbles shown
here have a minimum diameter of ∼1.0 μm and a maximum diameter of ∼2.0
μm. They remain stable for up to 5 h on agar under our experimental con-
ditions (Materials and Methods). (Scale bar in F, 10 μm.) See also Movie S1.
Bubble formation cascade. The highly refractive surfactant droplet
of Span 83 was placed at a distance of 3 to 5 cm ahead of an E. coli swarm
(HCB1668). Microbubble formation was complete within ∼10 min but the
swarm took 3 to 5 h to reach the bubbles. By this time the water in the Span
83 suspension had been absorbed by the agar and thus had little effect on
the swarm. (A and B) A small section of the advancing swarm edge, with the
swarm moving from left to right. One microbubble (2.2 μm in diameter,
indicated by the white arrows) moves downward along the swarm edge, or
CW when the swarm is viewed from above. The bubble traveled a distance
of 87.4 μm over 6.23 s at an average speed of 14.0 μm/s. For comparison, the
swarm expansion rate was 3.2 μm/s. (Scale bar in B , 10 μm.) See Movie S3. (C)
The right-handed colony pattern of a young swarm of E. coli AW405. Most
branches of the swarm curve clockwise. (Scale bar, 0.5 cm.)
Asymmetric flow and colony patterns. A 0.2-μL drop of a suspension
speed versus the distance from the swarm edge, computed from 28 bubble
trajectories. The dashed line is a Giddings peak function fit using the soft-
ware Origin 6.1 (OriginLab Corp.). Two other swarms showed similar tan-
gential flow profiles (Fig. S2). (B) Average radial flow speed versus the dis-
tance form the swarm edge. Error bars are SDs for the ensemble of bubble
Flow profile of the river for one swarm. (A) Average tangential flow
| www.pnas.org/cgi/doi/10.1073/pnas.1016693108 Wu et al.
All of the flow patterns observed with E. coli were seen as well
with strains of B. subtilis (DS3610) (26) and Serratia marcescens
(ATCC274) (27) grown on Eiken agar (Materials and Methods).
These observations suggest that such flows generally occur with
peritrichously-flagellated bacteria near solid surfaces.
Asymmetric Flows Correlate with CCW-Biased Flagellar Rotation. To
find out the origin of CW circulation around individual cells, we
imaged microbubbles and cell bodies in phase-contrast and fla-
gellar filaments in fluorescence (Materials and Methods). In the
agar-slip model, as a swarm cell approaches shallow fluid near
the edge of the drop and becomes stuck, the flagella unbundle,
and the filaments project outwards and roll CW, as shown in Fig.
4 (see also Movies S7 and S8). Although the flagella fail to
propel cells under these circumstances, they continue to rotate
and frequently change their directions of rotation. The sense of
this rotation could be inferred from the direction of the roll and
the shape of the filaments. The flagella appear to have a CCW
rotational bias similar to that found with swimming cells, because
they prefer to roll CW (conspicuous in 10 of the 15 examples), as
expected for normal (left-handed) helices spinning CCW close to
a solid surface (28). This observation suggests two ways in which
CW flow around the cells might be generated: when a CCW-
rotating filament approaches the surface, it is swept CW, which
moves fluid CW; once a filament is oriented in this way, it will
pump fluid CW, because a left-handed helical filament spinning
CCW moves fluid away from its base in a direction parallel to the
helical axis. A mechanism of this kind has been proposed for the
generation of flows around stuck cells of B. subtilis (29). Cells in
a swarm swim without curving strongly to the right or the left (6),
suggesting that they move between two fixed surfaces, apparently
proposition was proved by the relative immobility of small par-
ticles of MgO smoke deposited on the upper surface of the swarm
(8). However, cells near the edge of a swarm or in the agar slip
model are stuck to the agar and not swimming freely, so it is likely
observed with cells lacking flagellar filaments (HCB1688 grown
without a fliC inducer) or with cells having paralyzed flagella
(HCB84), so rotating filaments are absolutely required.
We found a unique method for making microbubbles that serve
as tracers for fluid flow in bacterial swarms, and with these
tracers discovered an array of interesting chiral flow patterns:
a river flowing CW in front of the swarm, CW swirls around
isolated cells, and CCW swirls in between cells in sparse arrays.
These flow patterns arise from CCW-biased flagellar rotation,
when left-handed helical filaments projecting out from cells and
spinning CCW roll CW over the underlying surface and pump
As noted earlier, the layer of fluid in a swarm provides not
only an environment that enables cells to spin their flagella, but
also a vehicle for the transport of nutrients or molecules secreted
by cells (e.g., those involved in quorum sensing). Long-distance
transport is generally more effective when it occurs by bulk flow
than by diffusion alone. With bulk flow, displacement is pro-
portional to time; with diffusion, displacement is proportional to
the square root of the time. Therefore, given enough time, bulk
flow always wins (30). A small molecule carried by a stream
flowing at 8 μm/s at the swarm edge will outdistance one diffusing
such transport will not work for a stream flowing over agar, be-
cause the small molecule will diffuse out of the stream into the
underlying agar. Thus, we find it very interesting that some of the
signaling molecules involved in quorum sensing are packaged in
membrane vesicles (31), and that membrane vesicles are com-
monly seen in biofilms (32). Cells of Pseudomonas aeruginosa
produce hydrophobic quinolone signals (PQS) involved in the
regulation of the synthesis of biosurfactants that facilitate
swarming (33). PQS have been shown to be packaged in secretory
membrane vesicles, which then traffic the signals between cells to
coordinate group behavior (31). Membrane vesicles of this size
(diameter of order 100 nm) are produced by many bacterial spe-
cies, including E. coli (32). Because vesicle diffusion coefficients
are quite small, flow of swarm fluid will greatly enhance their
transport, providing an avenue for long-range communication in
the swarming colony. If a swarm is facing environmental stress,
long-range communication could enable unaffected regions of
the colony to turn on specific responses in ample time. Cells in
swarms are known to have higher resistance to a variety of anti-
microbial agents than cells in liquid cultures (34–36). We wonder
whether colonization by swarming of pathogenic bacteria might
be blocked by modification of surface properties required for
the generation of fluid flows, instead of through perturbation of
Materials and Methods
Bacterial Strains. The E. coli strains used for this study were AW405 (wild-
type), HCB84 (motA448) (37), and HCB1668 (FliC S353C), an AW405 de-
rivative whose flagellar filaments can be labeled with thiol-reactive fluo-
rescent dyes (7). B. subtilus (DS3610) (26) and S. marcescens (ATCC274) (27)
were also used. Single-colony isolates were grown overnight in LB medium
(1% Bacto tryptone, 0.5% yeast extract, and 0.5% NaCl, pH 7.5) at 30 °C to
stationary phase. For E. coli HCB1668, kanamycin (50 μg/mL), chloramphen-
icol (34 μg/mL), and arabinose (0.5%) were added to the growth me-
dium. These cultures were diluted 10−5to provide cells for inoculation of
Swarm Plates. For E. coli, the swarm agar was 0.45% (AW405) or 0.6%
(HCB1668) Eiken agar in 1% Bacto peptone, 0.3% beef extract, and 0.5%
NaCl. For B. subtilus and S. marcescens, Eiken agar was respectively 0.8% and
0.6% in the medium just described. When grown on 0.8% Eiken agar, B.
subtilus swarms exhibited a cell density at the swarm edge comparable to
that of E. coli grown on 0.45% Eiken agar. The swarm agar was autoclaved
and stored at room temperature. Before use, the agar was melted in a mi-
crowave oven, cooled to ∼60 °C, and pipetted in 25-mL aliquots into 150 ×
gellar filaments. In A to E the flagellar filaments (bright hairy structures)
move CW around the cell body (the rod-shaped object at the center of each
panel), which is stuck to the agar slip. This movement is most evident when
following the bundle of filaments indicated by the white arrow. Meanwhile,
a microbubble (the gray spot) circulates CW around the cell. The phase-
contrast image of the cell body was cropped from the image taken for A, and
its contrast was enhanced and smoothed in ImageJ (http://rsbweb.nih.gov/ij/);
the enhanced image was superimposed on the fluorescence images shown
in A to E. Phase-contrast images of the microbubble were plotted directly
onto the corresponding fluorescence images and highlighted as gray spots.
(F) The processed phase-contrast image of the cell and microbubble (high-
lighted as a white spot) at the time of 0.4 s, with the bubble trajectory from A
to E plotted as a black curve. (Scale bar in F, 5 μm.) See Movies S7 and S8.
Correlation between CW bubble circulation and CW rolling of fla-
Wu et al.PNAS
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15-mm polystyrene Petri plates. For E. coli HCB1668, antibiotics and arabi-
nose were added to the liquefied swarm agar before pipetting at the con-
centrations used in liquid cultures. The plates were swirled gently to ensure
complete wetting, and then cooled for 30 min without a lid inside a large
Plexiglas box. Drops of diluted cell culture (2 μL, described above) were in-
oculated at a distance of 2 to 3 cm from the edges of the plates, and the
plates were dried for another 30 min without a lid, covered, and incubated
overnight at 30 °C and 100% relative humidity until the swarms grew to
Preparation of Surfactant Suspensions. A drop of the water-insoluble sur-
factant Span 83 (Sorbitan sesquioleate, S3386; Sigma-Aldrich) was mixed
with de-ionized water in a glass bottle at a wt/wt ratio of 0.03% to 0.04%. Air
was injected at the bottom of the mixture through a narrow plastic tube,
resulting in an uprising of bubbles that accelerated the breakdown of Span
83 droplets. This process was done for >1 h, until the surfactant suspension
became milky. When viewed with a phase-contrast microscope, the sus-
pension appeared full of refractile Span 83 droplets with diameters ranging
from a fraction of a micrometer to a few micrometers.
Preparation of Agar Coverslip Samples (Agar Slip Model). Swarm cells were
collected by gently rinsing the leading edge of the swarm with motility
medium (0.01 M potassium phosphate pH 7.0, 0.067 M NaCl, 10−4M EDTA),
as described previously (7), except that Tween 20 was not used. When we
needed to visualize flagellar dynamics, cells of E. coli HCB1668 were fluo-
rescently labeled at this point with Alexa Fluor 532 C5maleimide (Invitrogen-
Molecular Probes), as described previously (7), except that Tween 20 was not
used. We placed 1 μL of Span 83 suspension on top of a 20 × 20-mm patch of
thin agar (0.6%) that had been freshly poured on a 24 × 60-mm No. 1
coverslip (#48393–106; VWR), precleaned with saturated solution of KOH
in ethanol. We put the sample in a 100% relative-humidity chamber and
waited 0.5 to 1 h until the drop was absorbed by the agar and the micro-
bubbles formed. Then 2 μL of cell suspension was added where the Span 83
had been added, and the sample was immediately covered with a 1 × 3-inch
microscope slide (#48300–047; VWR) placed on 1-mm thick (10 × 24-mm)
shims cut from a similar slide. Then this preparation was transferred from
the humidity chamber to the microscope stage for imaging. Cells could be
imaged for at least 10 min without noticeable evaporation.
Phase Contrast and Epifluorescence Imaging. The motion of microbubbles and
filaments around cell bodies stuck to the agar substrate were observed si-
multaneously in phase contrast and epifluorescence, respectively, with a 40×
phase-contrast objective mounted on a Nikon Diaphot 200 inverted micro-
scope. Phase-contrast illumination was provided by a yellow LED, and fluo-
rescence excitation was provided by an argon-ion laser, as described below,
switched on and off in synchrony with alternate lines of a 2:1-interlaced
video recording. Recordings were made with a CCTV camera (model KPC-
650BH; KT&C) and a digital tape recorder (model GV-D1000; Sony). The
video sequences were transferred to a PC as “avi” files and uncompressed
using the free software VirtualDub (http://www.virtualdub.org/). Separate
phase-contrast and fluorescence sequences were assembled by deinterlacing
with custom software written in Matlab (The MathWorks, Inc.), replacing the
missing lines with pixels that were the arithmetic means of the pixels above
and the pixels below. Two synchronized multipage tagged-image file format
images were generated for further analysis.
The phase-contrast light source was a yellow LED (# OVLGY0C9B9; TT
Electronics/Optek Technology) powered by custom-built electronics and
placed about 75 mm above the phase ring of the microscope condenser. The
epifluorescence light source was an argon-ion laser (Stabilite 2017; Spectra-
Physics) tuned to 514 nm. The laser exposure time was controlled with an
electro-optical deflector (EOD, model # 310A; Conoptics), which deflected
the laser beam through the aperture of a pinhole (“on”) or away from this
aperture (“off”). The EOD was driven by two power supplies (HP 6515A) set
to 750 V via a custom-built three-state switch (designed by Winfield Hill,
model # RIS-688, Rowland Institute at Harvard, Cambridge, MA): +1,500 V
for “on”, −1,500 V for “off”, and 0 V in between experiments to protect the
The microscope was modified for laser illumination by rotating the
fluorescence cube in a custom-designed mount 90° around the vertical axis to
allow excitation from the right side. We used a dichroic mirror (527DCLP;
Chroma Technology Corp.) without excitation or emission filters; however,
the excitation light was blocked in the emission channel with a thin-film,
single-notch filter (NF01-514U-25; Semrock). Custom-built electronics con-
trolled the timing and the exposure times (i.e., the length of the “on” states
of the two light sources), in synchrony with the video vertical sync pulse. The
exposure time for both phase and laser illumination varied between 40 and
Data Analysis. Microbubbles were tracked in the phase-contrast video
sequences manually using the MTrackJ plugin (Erik Meijering, http://www.
imagescience.org/meijering/software/mtrackj/) developed for ImageJ (http://
rsbweb.nih.gov/ij/). The track data were then analyzed to yield the bubble
speed and the distance from bubble to swarm edge. We inferred the size of
microbubbles by doing Gaussian fits to the light-intensity profile of a line
crossing the bubble center plotted in ImageJ. The widths of Gaussian fits (2σ)
were taken as the diameter of bubbles. As a validation to this method, it
yields values for cell widths consistent with other experimental data, about
ACKNOWLEDGMENTS. We thank Peko Hosoi, Daniel Kearns, and Richard
Losick for comments on the manuscript, and Linda Turner, Rongjing Zhang,
and Junhua Yuan for discussions and help with the experiments. This work
was supported by Grants AI065540 and AI016478 from the National
Institutes of Health.
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