161- Place the culture in an ultrasonic water bath (Branson
Ultrasonic B. V.) for 5 min in order to obtain uniformly dis-
persed single cell suspension (see Note 7). - Serials of tenfold dilutions of the cultures are made in 7H9
broth with 0.05% (v/v) Tween 80 but without ADC. - 100 μL of samples are added to one-third segments of the agar
plates in duplicate. - The inocula are allowed to dry into the agar and the plates are
incubated in double polythene bags for 3 weeks at 37 °C. - Viability is expressed as colony forming units (CFU) per mil-
liliter (see Note 8). - Antibiotics at different concentrations are added into log-phase
and stationary-phase cultures in the hypoxia model (see
Subheading 3.2). - The cultures are incubated at 37 °C without disturbance.
- At different time point, the cultures are washed with phosphate
buffered saline (PBS) for three times to remove the antibiotics. - Viability is determined using CFU counting (see Subheading
3.2) or broth counting (see Subheading 3.6). - BALB/c mice are infected intravenously via the tail vein with
1.2 × 105 CFU of mouse-passaged M. tuberculosis strain H37Rv
per mouse [ 13 , 14 ] (see Note 9). - Mice are randomly allocated into experimental groups and
control group. - Control group consists of infected and untreated mice.
- Four of these are sacrificed at 2 h after infection to monitor
initial bacterial loading in lungs and spleens of mice. - Four are killed at the beginning of treatment, 3 weeks after
infection (see Note 10). - The treatment groups are administrated with a combination of
rifampicin (R), isoniazid (H), and pyrazinamide (Z) for
16 weeks (see Note 11). - Treatment is given by daily gavage (0.2 mL) for 5 days per
week at the dosages of R 10 mg/kg, H 25 mg/kg, and Z
150 mg/kg. - The drug suspension is prepared freshly for the daily dosage.
- Immediately after termination of 16 weeks of chemotherapy,
the remaining mice are administered 0.5 mg/mouse of hydro-
cortisone acetate by daily oral administration for 8 weeks to
suppress host immune response.
3.3 Antibiotic
Exposure In Vitro
3.4 Cornell mouse
Model
A Method to Evaluate Persistent Mycobacterium tuberculosis In Vitro and in the...