5. When the desired volume has been delivered, turn off the
syringe pump and close the drug port on the CR stopcock,
simultaneously opening the primary flow path. Turn on the
peristaltic pump to reestablish the medium input.
3.5 Sampling from
the CR (See Note 3)
1. In the incubator, wipe the sampling microclave attached to the
3-way stopcock with an alcohol swab and let the alcohol
evaporate.
2. Turn off the peristaltic pump and turn the stopcock to open the
port secured by the microclave.
3. Inside the BSC, remove a 1 mL Luer-lock syringe from the
packaging and draw sterile air into the barrel. Attach the
syringe to the microclave and push the air into CR to purge
the stainless-steel tube of medium.
4. Aspirate a sample of medium (~100μL) from the CR into the
syringe. This will be used to determine the current circulating
level of drug(s) in the cartridge system. Once the sample has
been taken, turn back the stopcock to open the primary flow
path, and restart the peristaltic pump.
5. Freeze the sample at À80
C or process for LC-MS/MS
analysis.
3.6 Growth of Seed
Culture
1. Inoculate a small volume (50 or 100μL) from a frozen glycerol
stock of M. tuberculosis H37Rv (or H37Ra) into 10 mL of
Middlebrook 7H9-OADC medium.
2. Grow for 4–5 days at 37
C until the culture reaches mid to late
log phase (OD 600nm between 0.7 and 1.2). M. tuberculosis
H37Ra may require an additional 2–3 days to reach the same
optical density as the H37Rv strain.
3. Use an appropriate dilution of this culture as the seed to
inoculate the HF bioreactor(s).
3.7 Bioreactor
Inoculation
1. Before bacteria are introduced into the HF cartridge, establish
and maintain medium flow through the system for at least
2 days to equilibrate the system and ensure that there are no
defects in the flow path generating leaks or blockages. Monitor
fluid level in the CR as a primary indicator of stable, leak-free
flow and kink-free tubing.
2. Run the system at the flow rate that will be used to establish the
PK parameters of the experiment for at least 24 h prior to
bacterial inoculation.
3. Inside the BSC, dilute the bacterial seed culture to the density
desired for inoculation (see Note 14).
722
A. Brett Mason and Ve ´ ronique Dartois
syringe pump and close the drug port on the CR stopcock,
simultaneously opening the primary flow path. Turn on the
peristaltic pump to reestablish the medium input.
3.5 Sampling from
the CR (See Note 3)
1. In the incubator, wipe the sampling microclave attached to the
3-way stopcock with an alcohol swab and let the alcohol
evaporate.
2. Turn off the peristaltic pump and turn the stopcock to open the
port secured by the microclave.
3. Inside the BSC, remove a 1 mL Luer-lock syringe from the
packaging and draw sterile air into the barrel. Attach the
syringe to the microclave and push the air into CR to purge
the stainless-steel tube of medium.
4. Aspirate a sample of medium (~100μL) from the CR into the
syringe. This will be used to determine the current circulating
level of drug(s) in the cartridge system. Once the sample has
been taken, turn back the stopcock to open the primary flow
path, and restart the peristaltic pump.
5. Freeze the sample at À80
C or process for LC-MS/MS
analysis.
3.6 Growth of Seed
Culture
1. Inoculate a small volume (50 or 100μL) from a frozen glycerol
stock of M. tuberculosis H37Rv (or H37Ra) into 10 mL of
Middlebrook 7H9-OADC medium.
2. Grow for 4–5 days at 37
C until the culture reaches mid to late
log phase (OD 600nm between 0.7 and 1.2). M. tuberculosis
H37Ra may require an additional 2–3 days to reach the same
optical density as the H37Rv strain.
3. Use an appropriate dilution of this culture as the seed to
inoculate the HF bioreactor(s).
3.7 Bioreactor
Inoculation
1. Before bacteria are introduced into the HF cartridge, establish
and maintain medium flow through the system for at least
2 days to equilibrate the system and ensure that there are no
defects in the flow path generating leaks or blockages. Monitor
fluid level in the CR as a primary indicator of stable, leak-free
flow and kink-free tubing.
2. Run the system at the flow rate that will be used to establish the
PK parameters of the experiment for at least 24 h prior to
bacterial inoculation.
3. Inside the BSC, dilute the bacterial seed culture to the density
desired for inoculation (see Note 14).
722
A. Brett Mason and Ve ´ ronique Dartois