66
- Return all probes to the chemostat and ensure that all con-
nections through the head plate are tightened. Pressure-test
the vessel prior to autoclaving to check for air leaks.
Submerge the vessel in a container of water with the probe
fittings just under the water level. Vent filters should be
above the water line so that they do not get wet (see Note
4 ). Leave all clamps in place apart from the air inlet and air
outlet clamps, which should be removed. Extend the air
outlet tubing past the vent filter using an additional length
of tubing, with a thumb-wheel clip attached, and place the
end of the tubing below the water surface. Place a 50 mL
syringe onto the air inlet line and depress the plunger to
push air in. Air bubbles should be seen coming out of the air
outlet. Close the thumb-wheel clip on the air outlet exten-
sion and continue to push more air into the system. Observe
whether air bubbles rise out of the vessel, particularly from
the probe fittings on the top plate. Repair and retest any
leaks that appear. Release the clamp on the air outlet and
reclamp the air inlets before autoclaving. - Autoclave the vessel at 121 °C for 30 min to achieve sterility.
- Ensure that waste, medium, and acid bottles are connected to
the chemostat. Insert tubing into the peristaltic pumps.
Connect all probes, stirrer, and CO 2 analyzer (optional) to the
correct channel on the Eycoferm controller. - Calibrate the oxygen probe by warming up the vessel to 37 °C
while stirring, and pump in nitrogen and air alternately until
calibrated between 0% and 100% DAT, which is equivalent to
between 0% and 20% dissolved oxygen tension (DOT) (see
Note 8). - Switch off the heater and the stirrer. Drain the water from the
chemostat. - Fill the vessel with 400 mL of CMM medium via the medium
line. Warm the medium to 37 °C. Ensure that the stirrer
unit is responding to the DAT setting on the Eycoferm con-
troller by observing an increase in stirrer speed as the DOT
level in culture decreases. Set maximum and minimum stir-
rer speeds. - Make up the inoculum by taking three confluent plates of myco-
bacterial colonies, which have been incubated for 2–3 weeks (see
Note 9), and scrape them into 10 mL of autoclaved, distilled
water. Add the inoculum through the sample port. - Leave in batch mode for approximately 48 h. For the first 24 h
the air inlet should be closed at the cabinet connection to allow
the DAT set point of 50% DAT to be reached (see Note 10).
Following this, open the air inlet as the culture will now require
additional oxygen.
3.2 Establishing
Steady-State Growth
Charlotte L. Hendon-Dunn et al.