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- Clips, tubing—30 mm (VWR International, Lutterworth,
UK). - Clips, tubing—40 mm (VWR International, Lutterworth,
UK). - Keck ramp clamp tubing clamps—3/8′′ (Cole-Parmer,
Hanwell, London, UK). - Keck ramp clamp tubing clamps—1/4′′ (Cole-Parmer,
Hanwell, London, UK). - Bulb hand-pump (blowing ball with reservoir) (VWR
International, Lutterworth, UK). - Nitrogen gas (BOC Medical, Worsley, UK).
- Air pump (Brighton Systems, New Haven, UK).
- Middlebrook 7H10 agar: The ingredients required for 1 L of
Middlebrook 7H10 agar are: (15.0 g), Na 2 HPO 4 (1.5 g),
KH 2 PO 4 (1.5 g), (NH4) 2 SO 4 (0.5 g), l-glutamic acid (0.5 g),
sodium citrate (0.4 g), ferric ammonium citrate (0.04 g),
MgSO 4 ·7H 2 O (0.025 g), ZnSO 4 ·7H 2 O (1.0 mg), pyridoxine
(1.0 mg), biotin (0.5 mg), CaCl 2 ·2H 2 O (0.5 mg), malachite
green (0.25 mg), OADC enrichment (100.0 mL), glycerol
(5.0 mL). Add the glycerol to 900 mL of distilled water and
add the remaining components, except for the OADC enrich-
ment. Mix thoroughly. Gently heat and bring to the boil.
Autoclave the mixture for 15 min at 121 °C. Cool to 50 °C
and aseptically add 100 mL of OADC enrichment. Mix thor-
oughly and pour 25 mL into each petri dish. The ingredients
required for 100 mL of OADC enrichment are: bovine serum
albumin fraction V (5.0 g), glucose (2.0 g), NaCl (0.85 g),
oleic acid (0.05 g), catalase (4.0 mg). Prepare OADC by add-
ing all the components to the distilled water and bring the
volume to 100 mL. Mix thoroughly and then filter-sterilize the
solution using a 0.2 μm filter. OADC enrichment is also avail-
able as a premixed powder from BBL Microbiology Systems
and Difco Laboratories. - CAMR Mycobacterium Medium (CMM Mod2): the ingredi-
ents required for 1 L of CMM Mod2 are ACES buffer (10.0 g),
KH 2 PO 4 (0.22 g), distilled water (500 mL),CMYCO solution 1
(10 mL),CMYCO solution 2 (10 mL), CMYCO solution 3
(100 mL), CMYCO solution 4 (10 mL), biotin (10 μg/mL
solution) (10 mL), NaHCO 3 (0.042 g), glycerol (0.75 g),
CMYCO solution 5 (10 mL), and Tween 80 (2 mL). Add the
first two ingredients to the first volume of distilled water
(500 mL). Add the remaining ingredients and solutions in the
order listed. Stir the solution to dissolve all the ingredients.
Adjust the pH to 6.5 with 20% KOH. Filter-sterilize the medium
Charlotte L. Hendon-Dunn et al.