Figure 1 : Dual-chamber microreactor fabrication and assembly.
A) Components of chip and assembled device. B) PDMS chips fabrication
process. C) Silicon gaskets fabrication process. D) Interface
establishment - ALI was established when the bottom chamber was filled
with liquid and the top chamber was filled with air, while the LLI was
created when both chambers were filled with liquid.
Device sterilization
Before inoculation, the PDMS chips, acrylic Plexiglas covers, and
silicon gaskets were sonicated with Milli-Q water for 480s, rinsed with
an ethanol solution (80% v/v), and exposed to UV light for
sterilization for 30 min. The PETE membranes and tubing were sterilized
using the autoclave (121°C/ 20min). All the components were assembled
aseptically in the biosafety cabinet.
Device inoculation and biofilm formation
P. aeruginosa from frozen stocks was grown on agar plates for
16-18h at 37°C. To prepare liquid pre-culture, one colony was
transferred into 1mL of CAMHB media (3g/L beef extract, 17.5g/L casein
hydrolysate, 1.5g/L starch, pH 7.0), incubated for 16-18h at 37°C, and
shaken at 200 revolutions per minute (RPM). The overnight pre-culture
was diluted 1:30 v/v in fresh media, incubated for 2h at 37°C, and
shaken at 200 RPM. The inoculum size was optimized by testing two
different standardized densities, which are OD600 = 0.04
and 0.4, respectively, to obtain a fully covered membrane within 48 h of
growth. Before inoculation, the inoculum size controls were also
obtained by viable colony forming units (CFU) counts.
For the device inoculation, 100μl of CAMHB media was injected into the
basal chamber through the basal chamber inlet before 100μl of bacterial
inoculum was injected into the top chamber through the top channel
inlet. After inoculation, the excess media and inoculum were discarded
by the tubing removal, and the chip was sealed with a sterile and
breathable film (AeraSealTM) and incubated at 37°C for 2h under static
conditions to allow bacteria attachment. Two different interface models
were tested – ALI and LLI. For the ALI establishment, the culture media
was removed from the apical chamber after 2h of bacterial adhesion. For
LLI conditions, the apical media was removed after 2h of attachment and
replaced by fresh culture media in the apical chamber. Biofilms were
grown at 37°C for 48h, and the media on the basal chamber was refreshed
after 24h of culture.
Device repeatability test
The repeatability of this microreactor was evaluated by quantifying
biofilm growth under ALI and LLI models, using CFU counts. Three
biological replicates were performed for biofilm formation on ALI and
LLI. Two devices containing six flow cells were inoculated for each test
model, and another device infused with media was used as controls.
Controls for inoculum size were performed after each inoculation by
viable colony counts to ensure that differences in the inoculum size
were minimal. After 48h, each membrane was aseptically removed from the
device, gently dip-washed in 1mL of sterile PBS, and subsequently
transferred to a tube containing 1 mL of sterile PBS. To disrupt the
biofilm, the membranes were sonicated for 280s at 47kHz and 1.8W/cm2,
previously shown to cause no deleterious effect on the colony-forming
ability of P. aeruginosa [18]. Following the disruption, ten-fold
serial dilutions of the detached bacterial suspensions were performed in
sterile PBS, plated on LB agar plates, and incubated at 37 for 16-18h.
Viable colony counts were calculated using equation 1:
\(\frac{\text{CFU}}{\text{mL}=\frac{N\times 10}{10^{-D}}}\) Eq. (1)
where N represents the colony number and D represents the number of 1:10
dilutions. Six randomly selected membranes were analyzed by scanning
electron microscopy (SEM) to confirm that bacteria had been removed from
the surface.
Scanning Electron Microscopy (SEM)
The morphology of the biofilms grown on the devices was visualized using
scanning electron microscopy (SEM, JEOL, JMC-6000 NeoScopeTM Benchtop
SEM, USA) at 15kV. The devices were disassembled, and membranes gently
dip-washed in sterilized water before fixation in a formaldehyde
solution in PBS (4% v/v) for 90min. After fixation, membranes were
gently rinsed with water and air-dried at room temperature. Before
imaging, the membranes were sputter-coated with gold for 2min using a
Smart Coater (JEOL, USA). At least three membranes from each
experimental condition were analyzed.
Confocal laser scanning microscopy (CLSM)
The biofilm formed on different interfaces was investigated using
FilmTracer™ LIVE/DEAD® Biofilm viability kit according to the
manufacturer’s specifications. The samples were gently washed with
sterilized water followed by staining with LIVE/DEAD solution for 25mins
at 37°C in the dark. Excess dye was removed using sterilized water. The
biofilms were then fixed for 40 min by infusing PFA solution (4% v/v in
PBS). The sample was rinsed with sterile water and mounted on a glass
slide. Fluorescence was observed using a confocal laser scanning
microscope (Olympus FluoView, inverted FV 3000RS IX83, 100×
magnification oil immersion objective). The image was acquired using 488
nm and 660nm incident light. The z-stack images were taken with a step
of 1μm, and the resolution was 1,024×1,024 pixels. All experiments were
performed in duplicate. 3D reconstruction view and 3D z-stack projection
images were obtained, and fluorescence intensity measured using ImageJ.
The ratio of live cells to dead cells was compared using the fluorescent
intensity ratio of green to red in 3-D projected z-stack CLSM images to
quantify the difference between biofilm formed on different interfaces.
For analysis, the images were obtained from two replicates which three
images pick on each replicate. Thus, six images in total for each
biofilm model were collected for statistical analysis.
Extracellular polymeric substances (EPS) staining
Alcian blue staining was used as a colourimetric-based approach to
detect the polysaccharides in the extracellular polymeric substances
[19]. The membrane was aseptically removed from the device, gently
washed with sterilized water, and then fixed using 4% (v/v)
formaldehyde solution in PBS for 40min. The membrane was rinsed with
water again and then immersed in Alcian blue (1% w/v in acetic acid,
3% v/v pH 2.5) for 15mins. Excess dye was removed by multiple rinses
with water. Images were obtained
using NanoZoomer-SQ Digital slide scanner C13140-01 (Hamamatsu). Eight
images were collected at 40x magnification from each sample, and the
mean red, green and blue (RGB) ratio of Alcian blue and standard
deviation were obtained using ImageJ.
Antibiotic Treatment Test
Ciprofloxacin (CIP) is a broad-spectrum antibiotic that is effective
against PAO1[20] and hence has been used as the model antibiotic in
this study. To assess the effects of the different interface on P.
aeruginosa susceptibility to CIP, the 48h-old biofilms were exposed to
a range of concentrations (50-1600μg/mL) for 6h. For the antibiotic
treatments, a stock solution of CIP was prepared in water, and working
solutions were freshly prepared by dilution of the stock in culture
media. The antibiotic solution was injected into the basal chamber, and
the biofilm interacts with antibiotics from the substrate side for 6h at
37°C. The susceptibility was assessed by CFU counts according to the
procedure described in section 2.6.
Statistical Analysis
The repeatability of culturing biofilms at different interfaces was
examined by calculating the standard deviations across all experiments.
All data are expressed as Log (CFU/mL). Unpaired t-tests were performed
to determine the significant differences between the biofilm formed at
ALI and LLI. The antibiotic susceptibility of biofilm formed on ALI and
LLI was investigated using one-way ANOVA. Graphs show means and error
bars indicate the standard error of the mean. All the statistical
analysis was performed using Graphpad Prism 7.0.
Results and Discussion
An adaptable dual-chamber microreactor has been developed incorporating
different interfaces and surfaces to investigate the effects on biofilm
development in terms of morphological properties, colony counts, and
responses to antibiotics. Compared to conventional mono-interface
reactors, the dual-chamber device offers significant advantages in
mimicking the relevant physiologic and environmental settings in which
bacteria thrive, offering the potential for more targeted experiments to
deliver precise outcomes.
Device fabrication and assembly
The fabrication process of PDMS chips using 3D printing illustrated in
Fig. 1-C, shows the versatility of the microfluidic system with
affordable equipment and straightforward protocols [21]. PDMS is the
ideal material for the fabrication of microfluidic devices having great
biocompatibility, chemical stability, gas permeability, transparency,
low cost and ease of molding [22]. The intermediary gaskets (Fig.
1-D) fabricated with silicone (a non-toxic, inert, and soft material)
were introduced to cushion the mechanical force created by the screw and
nuts and to prevent leakage from the flow cell. The assembled
dual-chamber device comprises six layers (Fig. 1-A), with two acrylic
Plexiglas cover layers, two PDMS layers containing the flow chamber, and
middle layers of gaskets. The acrylic Plexiglas covers help distribute
the bonding force evenly on the PDMS chips to seal the system. Several
bonding techniques have been used in microfluidic devices such as
surface plasma and corona discharge that permanently bond PDMS layers
with glass or other PDMS layers [23]. However, these techniques
require specialized equipment. Moreover, several end-point sample
analysis methods require the device to be disassembled, which could
potentially disrupt the biofilm matrix, and consequently, the use of an
irreversibly bonded device is limited. For analysis that requires many
replicates, using permanently bonded devices could also be both costly
and time inefficient. Therefore, the reversible mechanical bonding
developed in our device will offer more flexibility and convenience for
endpoint sample manipulation. The ability to fit different membranes in
the same device enables the testing of different surfaces, and the
device can be reused after sterilisation to reduce the cost and time of
production. The establishment of the ALI and LLI methods is illustrated
in Fig.1-D using red and blue dyes. The ALI method was established when
the bottom chamber was filled with liquid and the top chamber filled
with air, while the LLI was created when both chambers were filled with
liquid.
Optimization of inoculum concentration and biofilm attachment
time
Inoculum size optimization was performed to establish the formation of a
mature biofilm throughout the membrane in 48h. The investigated inoculum
density was standardized to OD600=0.04 and
OD600=0.4 with inoculation times of 2h and 24h. Fig. 2
presents the microphotographs of the biofilms grown from different
inoculum densities and with different attachment periods. Biofilms from
low-density inoculum (OD600=0.04) with 2h attachment
time were scattered throughout the membrane, while biofilms from the
same density with attachment time of 24h, completely covered the
membranes. Biofilms grown from high-density inoculum
(OD600=0.4) formed a homogeneous layer on the membranes
after 2h of attachment. This condition was chosen as the optimal
parameter to carry out the experiments throughout this study.