Diffusion of copper-oxide nanowire bundles carrying antibiotics through the
biofilm pores produced by Pseudomonas aeruginosa
METHODS AND APPROACH
Pseudomonas aeruginosa Biofilm Synthesis
We will follow Harrison’s (2006) procedure shown in Figure 1 below to grow Pseudomonas
aeruginosa biofilms with the Calgary Biofilm Device (CBD) and examine the biofilm’s structure
using a confocal laser scanning microscope (CLSM).
Figure 1. The experimental design to synthesize Pseudomonas aeruginosa biofilms, using LuriaBurtani Broth (LB) and the Calgary Biofilm Device (CBD) system; and examination of the
biofilm’s structure using confocal laser scanning microscopy (CLSM) equipment (Harrison,
2006).
Biofilm synthesis procedure (Harrison, 2006):
Day 0
Grow a pure culture of Pseudomonas aeruginosa from the stock sample.
Streak an LB agar plate from the stock sample and incubate at 35℃ for 24 hr
(first sub-culture) (Fig. 1A).
Day 1
Streak an LB agar plate from a single colony on the first sub-culture plate and
incubate at 35℃ for 24 hr (second sub-culture) (Fig. 1A).
Day 2
Prepare a 30-fold dilution (1.0 McFarland LB standard inoculum) from the
second sub-culture plate (Fig. 1B). Inoculate and incubate microtiter plates.
Insert CBD peg lid into the 96-well microtiter plate containing 150 µL of
inoculum in each well (Fig. 1C). Place microtiter plates on a gyrorotary shaker
(~150 rpm) in a humidified incubator and incubate at 35℃ for 24 hr (Fig. 1C).
Day 3
Rinse biofilms to remove loosely adherent cells.
Place a CBD peg lid into a 96-well microtiter plate containing 200 µL of 0.9%
NaCl in each well for 2 min (Fig. 1D). Remove CBD pegs using flamed pliers
for biofilm structure examination (Fig. 1E). CLSM preparation. Immerse pegs
in a 96-well microtiter plate containing acridine orange stain in each well and
then mount each peg on a glass coverslip with 2 drops of 0.9% NaCl (Fig. 1I).
Examine coverslip with CLSM (Fig. 1J). Visualize 3D image (Fig. 1K).
Copper-oxide Nanowire Synthesis
We will use (researcher’s name) procedure to create copper-oxide nanowires.
Antibiotic and Nanowire Fusion
We will use electrostatics.
Experimental Design
Sample size
Figure 2. A microtiter plate illustrating the regions of the treatment parameters control (A), antibiotic
only (B), nanowire only (C), and antibiotic fused nanowire (D) (1, 2, 3, and 4), and the antibiotic fused
nanowire dilution 101, 102, 103, 104, 105, and 106 aliquots (I, II, III, IV, V, and VI) (Ceri, 1999).
Controls and test samples
Each experimental group will be exposed to Pseudomonas aeruginosa biofilms in addition to the
parameters listed in Table 1 below:
Table 1. Experimental group parameters tested with Pseudomonas aeruginosa biofilms.
EXPERIMENTAL
GROUP
EXPERIMENTAL TREATMENT
PARAMETERS
EXPECTED
RESULT
A
Control


No antibiotic
No copper-oxide nanowire
Growth
B
Antibiotic only


Antibiotic
No copper-oxide nanowire
Growth
C
Nanowire only


No antibiotic
Copper-oxide nanowire
D
Nanowire + Antibiotic

Antibiotic fused nanowire
No growth
(copper toxicity)
No growth
(antibiotic
penetration)
Experimental treatment parameter preparations:
Control
Add 200 µL of 0.9% NaCl to 24 wells of a 96-well microtiter plate
of a randomly selected region (1, 2, 3, or 4) (Fig. 2)
Antibiotic only
Add 200 µL of antibiotic to 24 wells of a 96-well microtiter plate of
a randomly selected region (1, 2, 3, or 4) (Fig. 2)
Nanowire only
Add copper-oxide nanowires to 24 wells of a 96-well microtiter
plate containing 200 µL of 0.9% NaCl of a randomly selected region
(1, 2, 3, or 4) (Fig. 2)
Nanowire + Antibiotic
Prepare 6 dilutions of antibiotic fused copper-oxide nanowires by
factors of 10 using 200 µL of 0.9% NaCl.
Add 200 µL of diluted antibiotic fused copper-oxide nanowires to 4
wells of a 96-well microtiter plate of randomly selected regions (1,
2, 3, or 4) and (I, II, III, IV, V, VI) (Fig. 2)
Results
We will use Harrison’s (2006) viable cell counting procedure to determine Pseudomonas
aeruginosa growth following each treatment parameter discussed previously.
Viable cell count procedure (Harrison, 2006):
1. After conducting treatment parameters, rinse biofilms by placing the CBD peg lid
into a 96-well microtiter plate containing 200 µL of 0.9% NaCl in each well for 2 min
(Fig. 1D).
2. Remove CBD pegs from lid using flamed pliers and place pegs in microtiter plate
containing 200 µL of 0.9% NaCl in each well (Fig. 1E).
3. Sonicate. Use Aquasonic 250HT ultrasonic cleaner (60 Hz for 5 min.) to remove
bacterial cells from the peg surface.
4. Serially dilute bacterial cells in 0.9% NaCl, plate on LB agar medium, and incubate at
35℃ for 24 hr.
5. Count colony forming units (CFUs) on each plate and record.
Experimental procedure
DAY
EXPERIMENTAL PROCEDURE
0
-
Streak an agar plate with Pseudomonas aeruginosa from a stock culture and
incubate at 35℃ for 24 hr
1
-
Streak an agar plate with Pseudomonas aeruginosa from a single colony (Day
0 agar plate) and incubate at 35℃ for 24 hr
Self-assemble copper-oxide nanowires
2
-
3
-
4
-
5
-
Grow Pseudomonas aeruginosa biofilm on CBD peg lid for all treatment
parameters (A, B, C, and D) (Table 1)
Use an aliquot to look for the presence of copper-oxide on nanowires using
TEM and XRD
Set half of the copper-oxide nanoparticles sample aside for treatment
parameter C (Table 1)
Fuse antibiotic to copper-oxide nanowire using electrostatics
Use an aliquot to look for antibiotic coupling using fluorescence microscopy
Use an aliquot to look for Pseudomonas aeruginosa biofilm using CLSM
Prepare 6 dilutions of antibiotic coupled nanowires by factors of 10 for
treatment parameter D (I, II, III, IV, V, and VI) (Fig. 2)
Prepare experimental treatment parameters A, B, C, and D (Table 1) and add
to a microtiter plate randomly to regions 1, 2, 3, and 4 (Fig. 2)
Randomly divide parameter D into parts I, II, III, IV, V, and VI (Fig. 2)
Insert CBD peg lid with Pseudomonas aeruginosa biofilm growth onto
microtiter plate and incubate at 35℃ for 24 hr
Remove treatment sample pegs from CBD lid keeping each treatment group
separate
Sonicate, dilute, and plate each treatment sample separately
Incubate plates for 24 hr at 35℃
Perform plate counts on each treatment sample and record the number of
colony forming units (CFUs)
- Grow Pseudomonas aeruginosa biofilm on CBD peg lid for all treatment
parameters (A, B, C, and D) (Table 1)
- Self-assemble copper-oxide nanowires
Repeat Days 2-5 (Fig. 3).
Note: To keep a pure culture of Pseudomonas aeruginosa over the 2 year duration we will
re-streak Day 1 culture plates from a single colony every 2-3 days.
Figure 3. Diagram of experimental procedure from Days 2-5 (Herrmann, 2010).
Analysis
Interpretation
Conclusion