# 339a: WILKINS-CHALGREN ANAEROBE BROTH (N2/CO2)


## Main sol. 339a

33	g	dehydrated Wilkins-Chalgren medium (Oxoid CM0643)	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.3	g	L-Cysteine HCl	
5	g	D-Glucose	
1	g	Na2CO3	
1000	ml	Distilled water	

1. Dissolve 33.0 g/l dehydrated Wilkins-Chalgren medium (Oxoid CM0643) in distilled water and add 0.5 ml/l Na-resazurin solution (0.1% w/v), bring to the boil and cool to room temperature while sparging with 80% N2 and 20% CO2 gas mixture. Add L-cysteine-HCL x H2O (0.3 g/l), dispense the medium into anoxic Hungate-type tubes or serum vials and autoclave. After autoclaving supplement medium with 5.0 g/l D-glucose added from a sterile anoxic stock solution sterilized by filtration. Adjust pH of medium to 6.8 with a sterile anoxic stock solution of Na2CO3 (5% w/v) prepared under 80% N2 and 20% CO2 gas atmosphere.
2. Note: In case resazurin is not colourless addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) can be used to completely reduce the medium.