# 110a: CMC MEDIUM (N2/CO2)


## Main sol. 110a

30	g	Casitone	
5	g	Yeast extract	
5	g	K2HPO4	
0.5	ml	Sodium resazurin (0.1% w/v)	
0.5	g	L-Cysteine HCl x H2O	
10	ml	**Sugar mix**	
1.5	g	Na2CO3	
1000	ml	**Meat filtrate**	

1. Dissolve ingredients, except cysteine, sugar mix, and carbonate, boil medium for 1 min, then cool to room temperature under 80% N2 and 20% CO2 gas atmosphere, add 0.5 g/l L-cysteine-HCl x H2O and dispense under same gas atmosphere into Hungate-type tubes (for strains demanding meat particles put those first into the tube, use 1 part meat particles to 4 or 5 parts fluid). Autoclave at 121°C for 20 min. After autoclaving add the sugar mix from a sterile anoxic stock solution prepared under 100% N2 gas and carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas mixture. Adjust the pH of the medium to pH 7.0, if necessary.


## Meat filtrate

500	g	Ground beef (fat free)	
25	ml	NaOH (1 N)	
975	ml	Distilled water	

1. Use lean beef or horse meat. Remove fat and connective tissue before grinding. Mix meat, water and NaOH, then boil for 15 min with stirring. Cool to room temperature, skim fat off surface, and filter, retaining both meat particles and filtrate. To the filtrate add water to a final volume of 1000.0 ml.


## Sugar mix

4	g	D-Glucose	
1	g	Cellobiose	
1	g	D-Maltose	
1	g	Starch (soluble)	
10	ml	Distilled water