24
- Defrost an aliquot of each strain on ice.
- Resuspend each aliquot in ice-cold PBS such that its final con-
centration is 1 × 108 CFU/mL. - Mix together an appropriate amount of each aliquot on ice to
create a suspension of bacteria at the ratio required by your
experiment. Vortex well, both before and after mixing. - Prepare and infect mice according to local procedures and
experimental plan. - Immediately after infection, prepare a decimal serial dilution of
the mixed inoculum and plate 100 μL of dilutions 10−^4 , 10−^5
and 10−^6 onto BHI agar plates containing each of the relevant
antibiotics individually (see Note 16). Incubate overnight at
37 °C and record growth to verify the dose and strain ratio
used in the infection. - After sacrificing animals according to local procedures and
experimental plan, extract organs using sterile tools and place
into sterile 7 mL homogenizer tubes containing 2.8 mm
ceramic beads (see Note 17). Organs may be frozen at − 20 °C
until homogenized. - Add a known volume of sterile PBS (e.g., 1 mL) to each tube
containing an organ. Homogenize according to machine man-
ufacturer’s guidelines. - Prepare a decimal serial dilution of the organ homogenate and
plate 100 μL aliquots of each dilution onto BHI agar contain-
ing relevant antibiotics. - Incubate overnight at 37 °C and record growth to calculate
the bacterial load per strain per organ. Remember to take into
account the addition of PBS prior to homogenization. - For comparisons between two CFU counts, a two-tailed,
unpaired Student’s t-test may be used. For comparisons of
bacterial strain ratios between two groups (e.g., treated and
nontreated), a nonparametric test such as Mann-Whitney U or
Kruskall–Wallis is preferable. Common statistical analysis
programs such as GraphPad Prism are capable of performing
these tests. See Fig. 2 for an example of the output from these
experiments.
4 Notes
- Design primers to provide approximately 1 kb of homology to
the insertion site. This region will be duplicated in the final
merodiploid construct, with the plasmid backbone (and antibi-
otic resistance cassette) inserted between the two matching
regions. Since it is the goal of these experiments to not alter
3.5 In Vivo Studies:
Mouse Infection
and Quantification
of Bacterial Load
Gareth McVicker et al.