64
- Phosphate buffered saline (PBS) pH 7.4 (Severn Biotech Ltd.,
Kidderminster, UK). - CMM Mod2 medium (see Subheading 2.1 for the recipe).
- Middlebrook 7H10 agar plates with OADC enrichment (see
Subheading 2.1 for the recipe). - CMM medium (see Subheading 2.1 described for the recipe).
- 40% v/v Formaldehyde (SLS, Nottingham, UK).
- Columbia blood agar plates (Biomerieux, Basingstoke, UK).
- Tryptone soya agar plates (VWR, Lutterworth, UK).
- Middlebrook 7H10 agar plates with OADC enrichment (see
Subheading 2.1 for the recipe).
3 Methods
Assemble the chemostat as shown in Fig. 1.- Fill a clean 1 L glass vessel with deionized water (approximately
500 mL) and add a magnetic stirrer bar. - Fit the titanium top-plate by means of the plastic threaded col-
lar ensuring that the O-ring is in place and intact. - Insert oxygen probes and pH probes through the top plate by
means of the titanium threaded compression ports ensuring
the threaded collars are tightened to provide an airtight seal.
Probes should be checked first for any damage and to ensure
they are clean and that the oxygen probe contains electrolyte
(see Note 2). - Assemble effluent and medium lines using silicon tubing and
connect them to the vessel (see Note 3). - Assemble and connect the air inlet, the off-gas condensate bot-
tle (Duran bottle), the acid addition lines (optional; for con-
trolling pH), media addition lines, and sample port to the
vessel. Add vent filters (0.2 μm) to air inlets, air outlets, sample
port side arms, waste bottles, collection bottles and medium
bottles to maintain sterility and to prevent the buildup of pres-
sure in the vessel (see Note 4). - Prepare 1 M HCL in a 1 L duran bottle and autoclave it
(optional; for pH control). - Place essential equipment in the safety cabinet, which will
house the chemostat. This includes the electronic stirrer, two
peristaltic pumps (medium and effluent), the CO 2 analyzer
(optional), two autoclaved waste bottles, the medium bottle,
the DOT amplifier, and all leads required for connection
through the cabinet bulkhead.
2.5 Sampling
and Plating M.
tuberculosis
to Determine Viability
2.6 Monitoring
Chemostat Parameters
3.1 Assembling
the Chemostat
Charlotte L. Hendon-Dunn et al.