60
continuous culture the bacteria are in equilibrium with their envi-
ronment and growing at a constant generation time. While the
organisms are in this state, individual growth parameters can be
varied independently so that the direct effect of a single stimulus
(such as antibiotic action) on the viability and molecular genetics
of an organism can be investigated. Antibiotic activity in M. tuber-
culosis can be assessed under a range of different environmental
stimuli in continuous culture. Details of these growth conditions,
which include low pH, low oxygen, and slow growth rates, have
been described previously [ 2 , 3 , 6 – 8 ].
Here, bacilli replicating at the different growth rates are
assessed for their responses to static concentrations of antibiotic
[ 6 , 7 ]. Continuous cultures can be sampled throughout a time-
course (in the presence of the antibiotic) for a variety of measure-
ments that include viability, (colony-forming units) mutation
rate, changes in population genetics (whole genome sequencing),
and phenotypic analyses (microarray, RNAseq, metabolomics)
[ 2 , 3 , 5 – 9 ].2 Materials
(see Note 1; specific equipment used by the authors)- 1 L Glass Vessel (DASGIP) (Eppendorf, Stevenage, UK).
- pH probe (220 mm) plus leads (Mettler Toledo—gel filled)
(Brighton Systems, New Haven, UK). - Buffer, reference standard, pH 4.0 ± 0.01 at 25 °C.
- Buffer, reference standard, pH 7.0 ± 0.01 at 25 °C.
- Broadley James Dissolved Oxygen (DO) probe (220 mm) plus
leads (Brighton Systems, New Haven, UK). - Broadley James membrane kit (for DO probe) (Brighton
Systems, New Haven, UK). - Broadley James filling solution (DO probe electrolyte)
(Brighton Systems, New Haven, UK). - DOT signal amplifier (Brighton Systems, New Haven, UK).
- Dual-wire PFA sheathed stainless steel RT probe plus leads
(temperature probe) (Brighton Systems, New Haven, UK). - Portex silicone rubber tubing—6 × 2 mm (SLS, Nottingham,
UK). - Sterilin silicone tubing—4 × 1.6 mm (Bore × wall) (SLS,
Nottingham, UK). - Esco silicone tubing—1 × 2 mm (Bore × wall) (SLS,
Nottingham, UK). - Ty-Fast cable ties—186 mm (RS components Ltd., Corby,
UK).
2.1 Assembly
and Maintenance
of the Chemostat
Charlotte L. Hendon-Dunn et al.