67- Switch the culture to a fed-batch mode by starting the medium
pump at a flow rate of 5 mL/h. Set medium pump by calibrat-
ing it to the required flow rate. Keep the culture at 5 mL/h for
24 h to increase the culture volume to 500 mL (see Note 11). - Start the culture in continuous mode at a flow rate of 5 mL/h
by switching on the effluent pump at a speed that is higher than
the medium pump in order to maintain the culture volume at
500 mL. For a slow growth culture the flow rate should be
maintained at this level. For a fast growth culture this flow rate
is maintained for 2 days to establish the culture in continuous
mode prior to an increase in flow rate (see Notes 12 and 13 ). - For a fast growth culture increase the flow rate to 10 mL/h for
2 days, and then increase the flow rate to 15 mL/h and moni-
tor the culture daily (see Notes 13 and 14 ). - Monitor DOT levels, stirrer speed (rpm) and optical density
(OD); these parameters should be stable for at least 3–5 MGT
to confirm that the culture is in steady state. - Sample the culture for viability (cfu/mL) (see Subheading 3.4).
- Prepare antibiotics from working stocks in sterile water and
pass through a 0.2 μm syringe filter. - Split the prepared antibiotic dilution into 2 volumes that will
achieve the desired final concentration in the 500 mL chemo-
stat and a 2 L bottle of media. - Aseptically add the antibiotic to a fresh 2 L bottle of media.
- A “pre-antibiotic” culture sample should be taken at this point
and processed to determine viability (see Subheading 3.4). - Drain the chemostat medium line by removing the tubing
from the pump and applying a vacuum to the media bottle
using a vacuum pump. - Connect the new medium bottle, containing the antibiotic, to
the medium line and prime the line. - Clamp off the medium line just before the medium begins to
enter the chemostat via the anti-grow-back device, reposition
the tubing in the pump, and remove the clamp. - Add antibiotic directly to the chemostat via the sampling port,
switch on the medium pump. Take a “0 h” culture sample
immediately for viability analysis. - Check the flow rate after antibiotic addition has been com-
menced (see Note 12). - Remove a culture sample from the chemostat (see Note 15).
- Spin 1 mL of culture at 6000 rpm (2415 x g) for 10 min.
- Remove the supernatant and wash the pellet by resuspending it
in 1 mL of PBS and spinning at 6000 rpm (2415 x g) for 10 min.
3.3 Addition
of Antibiotics
to the Culture System
3.4 Sampling
and Plating
M. tuberculosis
to Determine Viability
Application of Continuous Culture for Assessing Antibiotic Activity Against...