110
- After 18–20 h, calculate the CFU number for the resistant
strain in question, the anaerobic Gram-negative flora and the
aerobic gram-positive flora in each sample from the number of
colonies on the plates.
From the selective plates calculate the LOG CFU number per
0.5 g of stools for the three bacterial populations for each antibi-
otic and the control groups. Lower detection limit is LOG(50) per
0.5 g of feces (see Note 16). - On day 1, 2, 4, and 8 pick several randomly selected E. coli
colonies from plates used for CFU calculations. Streak the cho-
sen colonies on to blood agar, incubate overnight and freeze a
loopful (5 μL inoculation needle) in broth containing 15%
glycerol at − 80 °C. This is done for plates representing each
cage. - Crude Extract DNA lysates are made by taking 1 μL (1 μL
inoculation needle) of bacteria from a plate and transfer it into
300 μL of DNase-free water (Invitrogen) and incubate for
10 min at 95 °C. Centrifuge at 5000 × g for 10 min to pellet
cell remnants. Transfer the supernatant to a new tube or avoid
touching the pellet when using the DNA. Store at − 20 °C. - Dilute the primers to 20 μM in DNase-free water (see Note
17 ). - PCR mastermix: Two different PCR mastermixes are created,
each containing one of the two primers. For each sample mix
12.5 μL of multiplex PCR kit without Q-solution (Qiagen),
7.5 μL of DNase free water, and 2.5 μL of 20 μM primer (1247
or 1283). Create a batch mix when testing more samples and
distribute into PCR tubes afterward. Then, add 2.5 μL of tem-
plate DNA (or DNase-free water as negative control) to each
tube and start the appropriate reaction in the thermal cycler:
(a) 95 °C for 15 min.
(b) 35 cycles of 94 °C for 1 min, 38 °C (primer 1247)/36 °C
(Primer 1283) for 1 min, 72 °C for 2 min.
(c) 10 min elongation step at 72 °C. - Electrophoresis:
(a) The E-Gel® is placed in the holder of the E-Gel® electro-
phoresis system.
(b) Load 15 μL of DNase-free water and 5 μL of PCR product
for each sample.
(c) The marker is loaded as 1 μL of marker to 9 μL of DNase-
free water.
(d) The gel is run for 34 min at 50 V.
3.3 Storage
of Isolates and RAPD
Frederik Boëtius Hertz et al.