Biology Reference
In-Depth Information
3.11. Separation of Bacteria From Catheter
1. After the final imaging time-point, kill mice humanely (in accordance with the
guidelines of the Institutional Animal Care and Use Committee) and surgically
remove the infected catheters for enumeration of bacteria by both bioluminescence
imaging and conventional plate count method.
2. Image the harvested catheters for bioluminescence.
3. Transfer the catheters to tubes containing 1 mL of TSB, place tubes in an ultrasonic
bath (VWR), and sonicate for 5-10 min (38.5-40.5 kHz).
4. Vortex the tubes for 1 min to remove the biofilm bacteria from the support surface
and image catheters to confirm the loss of bioluminescence and thus the complete
removal of the biofilm.
5. Repeat sonicating-vortexing procedure if necessary. Note, repeating the procedure
for >10 min may affect the viability of certain pathogens.
6. Dilute the suspension of bacteria removed form the catheter, plate on trypticase
soy agar, and incubate at 37 °C for colony counting.
Correlation between CFUs and bioluminescent signal (Photons/Sec) is deter-
mined by plotting Photons/Sec/Catheter versus CFUs/Catheter.
3.12. Imaging
1. Prior to and during imaging, mice are anesthetized with 2-2.5% isoflurane gas.
2. The whole animal is imaged (dorsal and ventral) for a maximum of 5 min at
various times during the experiment using a highly sensitive optical imaging
system, such as an IVIS
®
Imaging System.
3. Quantify total photon emission from defined regions of interest (e.g., bladder,
kidney, flanks, abdomen, or heart) within the images of each animal (e.g., using
Xenogen's LivingImage
®
software).
4. Following imaging, return mice to their cages until the next imaging session.
4. Notes
1. pUT is a conjugative suicide vector that is only maintained in strains of
E.
coli
expressing the protein, such as S17-1
pir
and CC118
pir
(22,23)
.
Thus, recipient bacteria that do not express the protein can only become stably
bioluminescent and tetracycline resistant if the mini-Tn5
luxCDABE
Tc
R
cassette
transposes onto their chromosome. Furthermore, as the
lux
operon does not have
its own promoter, the strength of bioluminescence in the recipient bacterium is
dependent upon the endogenous promoter directly upstream of the site at which
the
lux
transposon integrates. Derivatives of this donor strain carrying alternative
antibiotic markers are also available (e.g., chloramphenicol, kanamycin, and strep-
tomycin).
2. The promoterless
luxABCDE km
R
operon is optimized from Gram-positive
expression and is silent on pXen-5. When transposition occurs, the strength of
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