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- M. tuberculosis tends to grow in clumps even in the detergent-
containing 7H9, especially when a culture is incubated with-
out disturbance for more than 30 days. In unagitated cultures,
the clumps sink to the bottom of the container where they
form solid pellicles. This makes the direct and accurate mea-
surement of growth difficult and often the results are poorly
reproducible. For growth rate determination and the uniform
exposure of the bacilli to experimental treatment, it is very
important that the bacilli are evenly dispersed, preferably as
single cells with a minimum level of clumps. Larger clumps can
be broken by vortexing the culture with 1 mm glass beads. The
glass beads will be sterilized by autoclaving and added to the
Universal tubes before CFU counting. - Fine clumps can be broken by sonication. As sonication pro-
duces aerosols, caps of tubes containing M. tuberculosis will be
tightly screwed and sonicated in an ultrasonic water bath in the
Biosafety Cabinet. The time for sonication will not exceed
5 min because the bacterial cells will be killed with longer
sonication. - CFU counts are calculated as CFU/mL = colony counts × dilu-
tion factor × 10. For example, if 100 colonies are found on the
plate for the dilution of 10−^5 , viability will be calculated as 100
× 100,000 × 10 = 1 × 108 CFU/mL. - In order to keep M. tuberculosis virulence, the strain will be
grown in a mouse for 2 weeks. The lung will be collected and
CFU counts are performed. A single colony will be picked and
streaked on 7H11 agar plates which will be incubated at 37 °C
for 3 weeks. The bacteria grown on the agar plates will be col-
lected and added in 10 mL of PBS. The clumps will be broken
as Subheading 3.2, steps 5 and 6. The bacterial strain will be
stored at − 70 °C for subsequent animal inoculation. To deter-
mine the CFU counts prior to animal inoculation, viable
counting is performed prior to freezing and once again after
thawing. - Treatment must start not later than 3 weeks after infection as a
high does bacterial infection (10^5 CFU/mouse) will cause
mouse death if the mice are left untreated. - Rifampicin is administered 1 h before the other drugs to avoid
drug to drug interactions [ 16 ]. - At 16 weeks of treatment, organ CFU counts usually become
negative. Therefore entire organs or one-third of organs need
to be plate out to examine the number of bacterial cells in the
organs on agar plates. - This step is to further confirm that there are no bacterial cells
in the organs as CFU count negative bacilli may grow in liquid
medium.
Yanmin Hu and Anthony Coates