11. Dispense the sample into a sterile screw-cap tube and discard
the syringe into a sharps box containing 20% bleach.
12. Clean the HFC ECS ports and external surfaces of the cartridge and tubing with a disinfectant wipe before returning the
cartridge cassette to the duet pump in the incubator.
13. Estimate the number of viable bacteria (cfu) present in the ECS
by serial dilution of the sample, followed by plating known
volumes of each dilution, in duplicate, on 7H11 agar plates (see
Note 16). Figure 4 includes several examples where such viable
counts were used to enumerate M. tuberculosis H37Rv in cultures grown either without (see Fig. 4a), or with a course of
drug treatment (see Fig. 4b). If necessary, a small volume of
ECS sample may be extracted using organic solvents in preparation for drug quantitation by LC-MS/MS.
Fig. 4 Examples of M. tuberculosis H37Rv grown in HF bioreactors with/without
drug treatment. (a) Bacterial growth curves in HFCs seeded with identical
volumes of culture at two different densities (gray circles, A 600 ¼ 0.01; blue
squares, A 600 ¼ 0.001). Each data point is an average of duplicate CFU
determinations. (b) M. tuberculosis H37Rv viability after treatment with isoniazid
(1.16 mg/day) (blue) or isoniazid (1.16 mg/day) plus rifampicin (14.6 mg/day)
(red). Drugs were infused over 60 min at a flow rate of 34 mL/h on a daily basis
from day 0. Each data point is an average of duplicate CFU determinations
Mycobacterium Tuberculosis in a Hollow Fiber System
725
the syringe into a sharps box containing 20% bleach.
12. Clean the HFC ECS ports and external surfaces of the cartridge and tubing with a disinfectant wipe before returning the
cartridge cassette to the duet pump in the incubator.
13. Estimate the number of viable bacteria (cfu) present in the ECS
by serial dilution of the sample, followed by plating known
volumes of each dilution, in duplicate, on 7H11 agar plates (see
Note 16). Figure 4 includes several examples where such viable
counts were used to enumerate M. tuberculosis H37Rv in cultures grown either without (see Fig. 4a), or with a course of
drug treatment (see Fig. 4b). If necessary, a small volume of
ECS sample may be extracted using organic solvents in preparation for drug quantitation by LC-MS/MS.
Fig. 4 Examples of M. tuberculosis H37Rv grown in HF bioreactors with/without
drug treatment. (a) Bacterial growth curves in HFCs seeded with identical
volumes of culture at two different densities (gray circles, A 600 ¼ 0.01; blue
squares, A 600 ¼ 0.001). Each data point is an average of duplicate CFU
determinations. (b) M. tuberculosis H37Rv viability after treatment with isoniazid
(1.16 mg/day) (blue) or isoniazid (1.16 mg/day) plus rifampicin (14.6 mg/day)
(red). Drugs were infused over 60 min at a flow rate of 34 mL/h on a daily basis
from day 0. Each data point is an average of duplicate CFU determinations
Mycobacterium Tuberculosis in a Hollow Fiber System
725