Bacterial cell culture media Clostridium difficile

Bacterial culture is a process of letting bacteria multiply in a controlled fashion (temperature, humidity, oxygen content or shaking), in a predetermined culture medium (antibiotic resistance to obtain homogenous clones). It is an important step, especially during cloning, as a single cell can be grown homogeneously (on semi-solid or in liquid conditions) to obtain colonies. As mentioned, bacteria can be cultured in broth cultures (Luria broth or LB) or Petri dishes (Agar plates). A specific antibiotic can be added to the broth or agar plates in order to grow bacteria which have the gene insert conferring its resistance to that antibiotic. Following points are necessary to consider for optimal growth conditions: 1. In general, most bacteria grow well at 37C, but there are some strains which require growth temperatures between 25-30C. 2. It is ideal in broth cultures to fill the flask to ⅓ or less of the total flask volume for optimal aerobic growth. 3. Shaking speeds between 140-180 rpm are appropriate to ensure aeration and that the cells are surrounded by fresh media, and do not settle.

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Chromogenic Culture Media

Biomerieux

Upstream tips	Protocol tips	Downstream tips
	CDIF media were supplied as pre-poured ready to use plates by bioMérieux (Craponne, France); each contained meat peptone (porcine) 8.0 g, taurocholate (bovine) 1.0 g, yeast extract 3.5 g, sodium chloride 6.0 g, selective mixture 0.27 g, chromogenic mixture 0.3 g, agar 13 g and purified water. TCCFA plates were supplied by PathWest Laboratory Medicine Excel Media and followed the original formulation of CCFA ( George et al., 1979 ) apart from reducing the antibiotic concentrations by 50 % (cycloserine 250 µg ml −1 and cefoxitin 8 µg ml −1) and including egg yolk at 60 ml l −1, plus the addition of 0.1 % synthetic sodium taurocholate (Sigma Cat # T4009), ρ-hydroxyphenylacetic acid at 1 g l −1 and agar at 13 g l −1. All plates were stored at 4 °C until used.

Protocol tips
CDIF media were supplied as pre-poured ready to use plates by bioMérieux (Craponne, France); each contained meat peptone (porcine) 8.0 g, taurocholate (bovine) 1.0 g, yeast extract 3.5 g, sodium chloride 6.0 g, selective mixture 0.27 g, chromogenic mixture 0.3 g, agar 13 g and purified water. TCCFA plates were supplied by PathWest Laboratory Medicine Excel Media and followed the original formulation of CCFA ( George et al., 1979 ) apart from reducing the antibiotic concentrations by 50 % (cycloserine 250 µg ml −1 and cefoxitin 8 µg ml −1) and including egg yolk at 60 ml l −1, plus the addition of 0.1 % synthetic sodium taurocholate (Sigma Cat # T4009), ρ-hydroxyphenylacetic acid at 1 g l −1 and agar at 13 g l −1. All plates were stored at 4 °C until used.

Protocol tips

CDIF media were supplied as pre-poured ready to use plates by bioMérieux (Craponne, France); each contained meat peptone (porcine) 8.0 g, taurocholate (bovine) 1.0 g, yeast extract 3.5 g, sodium chloride 6.0 g, selective mixture 0.27 g, chromogenic mixture 0.3 g, agar 13 g and purified water. TCCFA plates were supplied by PathWest Laboratory Medicine Excel Media and followed the original formulation of CCFA ( George et al., 1979 ) apart from reducing the antibiotic concentrations by 50 % (cycloserine 250 µg ml −1 and cefoxitin 8 µg ml −1) and including egg yolk at 60 ml l −1, plus the addition of 0.1 % synthetic sodium taurocholate (Sigma Cat # T4009), ρ-hydroxyphenylacetic acid at 1 g l −1 and agar at 13 g l −1. All plates were stored at 4 °C until used.

Manufacturer protocol Publication protocol 1 Relevant paper

Difco™ Brain Heart Infusion Agar, BD

VWR

Upstream tips	Protocol tips	Downstream tips
	supplemented with either trehalose or glucose as indicated. Anhydrous tetracycline was used at 500 ng/ml to induce expression of ptsT or treA from ectopic expression vectors.

Protocol tips
supplemented with either trehalose or glucose as indicated. Anhydrous tetracycline was used at 500 ng/ml to induce expression of ptsT or treA from ectopic expression vectors.

Manufacturer protocol Publication protocol 1 Relevant paper

COLUMBIA BLOOD AGAR BASE

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	-pH 7.3 ± 0.2 @ 25°C -Cool to 50°C and add 5% sterile defibrinated blood.

Protocol tips
-pH 7.3 ± 0.2 @ 25°C -Cool to 50°C and add 5% sterile defibrinated blood.

Manufacturer protocol Publication protocol 1 Relevant paper

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