Bioluminescent Imaging of Bacterial Biofilm Infections In Vivo - Bacterial Pathogenesis: Methods and Protocols - page 215

3. Centrifuge and then sequentially wash cells with 50 mL, 25 mL and 5 mL
ice-cold 10% glycerol solution (centrifuging between washes).
4. Resuspend the electrocompetent cells in 500 μL of ice-cold 10% glycerol.
5. Add one 1 μL of 1 μg/μL plasmid pXen-5 (pAUL-A Tn 4001 luxABCDE Km R )
to 200 μL of electrocompetent S. aureus and mix ( see Note 2 ).
6. Transfer the bacteria/plasmid mixture into a 1-cm ice-cold cuvette (Bio-Rad)
and electroporate at 25 μF, 100 , and 2.5 kV (GenePulser II; Bio-Rad).
7. Immediately add 0.8 mL of BHI, gently invert cuvette and incubate statically at
37 °C for 1h.
8. Plate 200 μL volumes of the transformation mix on BHI agar plates containing
0.3 μg/mL of erythromycin and incubate plates at 37 °C for 24 h.
9. Patch colonies ( 1cm 2 ) onto BHI agar plates containing 0.3 μg/mL of

erythromycin and incubated at 37 °C overnight.
10. Uniformly streak a quantity of each patch (10 μL loopful of cell growth consisting
of

∼

10 8 -10 9 cells) over the entire area of a BHI agar plate containing 200 μg/mL
of kanamycin and incubated plates at 37 °C for 24 h.
11. Screen plates for bioluminescent transposants using a highly sensitive low-light
optical imaging system.
12. Select a single highly bioluminescent colony and characterize to ensure that
it is stable (e.g., bioluminescent photon counts correspond to CFUs) and
behaves similarly to the parental strain, both in vitro and in vivo (e.g., not
attenuated).

∼

3.4. In vitro Catheter-Associated Biofilm
1. Grow the bioluminescent biofilm-forming bacterial strain overnight in TSB

supplemented with 0.25% glucose (TSBG) in an orbital shaker at 37 °C.
2. Dilute the culture 1:10 in fresh TSBG and further incubate for 1.5 h at
37 °C.
3. Adjust OD at 600 nm to reach a standardized cell suspension of 10 5 CFUs/mL in
TSBG ( see Note 3 ).
4. Cut teflon intravenous catheter 14-gauge into 1-cm segments (soft tissue model),
polyethylene catheter PE 50 into 6-mm segments (UTI) and sterilize each piece
with 70% ethanol. Air-dry catheters overnight.
5. Develop bacterial biofilms on the cut, sterile catheters by placing individual
segments into Eppendorf tubes containing 1.0 mL of bacterial cell suspension in
TSBG with 10 5 CFUs/mL in the exponential phase of growth.
6. Vortex well to ensure the removal of air bubbles from inside catheter, allowing it
to remain immersed during incubation ( see Note 4 for incubation times for each
pathogen).
7. After incubating for appropriate time intervals at 37 °C recover colonized catheters
aseptically and rinse once gently in TSBG to remove unbound bacteria. Flush out
lumen of catheter with sterile TSBG. Image catheters for bioluminescence prior
to implant to ensure proper colonization.