# 830d: R2A MEDIUM (MICROAEROBIC)


## Main sol. 830d

0.5	g	Yeast extract	
0.5	g	Proteose peptone (BD DIFCO no. 3)	
0.5	g	Casamino acids	
0.5	g	Glucose	
0.5	g	Starch, soluble	
0.3	g	Na-pyruvate	
0.3	g	K2HPO4	
0.05	g	MgSO4 x 7 H2O	
1000	ml	Distilled water	

1. Dissolve ingredients, then sparge solution with 80% N2 and 20% CO2 gas mixture for at least 30 - 45 min to remove dissolved oxygen and to saturate the solution with CO2. Dispense solution in vials suitable for anaerobic cultures (e.g., Balch-type tubes or serum vials) to 30% volume (e.g., 5 ml medium in a 16 ml Hungate-type tube) under the 80% N2 and 20% CO2 gas atmosphere. Close vials with butyl rubber septa to prevent the free exchange of oxygen with the external atmosphere and autoclave them. Adjust pH of the autoclaved medium to 5.0 - 5.5 using a sterile stock solution of sodium carbonate (5% w/v), prepared under 80% N2 and 20% CO2 gas mixture.
2. Before inoculation, add sterile air to obtain 5% O2 in headspace gas atmosphere (e.g., add 2 ml air to a Hungate-type tube).