# 78b: CHOPPED MEAT MEDIUM (N2/CO2)


## Main sol. 78b

500	g	Ground beef (fat free)	
25	ml	NaOH (1 N)	
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	
1000	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 Casitone, yeast extract, hydrogen
phosphate and resazurin, then add water to a final volume of 700 ml. Sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, then add cysteine and adjust pH to 7.0.
Dispense medium under same gas atmosphere into anoxic Hungate-type tubes containing meat particles (use ca. 7 ml liquid medium for 1 - 2 g meat particles), then autoclave at 121°C for 20 min.
2. In some cases (as indicated in the catalogue) the addition of Haemin and Vitamin K1 or Vitamin K3 is necessary. Add to 1000 ml of medium after autoclaving:

10	ml	**Haemin solution** (optional)	
10	ml	**Vitamin K1 solution** (optional)	
1	ml	**Vitamin K3 solution** (optional)	


## Haemin solution

50	mg	Haemin	
1	ml	NaOH (1 N)	
100	ml	Distilled water	

1. Dissolve 50 mg haemin in 1 ml 1 N NaOH; make up to 100 ml with distilled water and filter sterilize. Store refrigerated.


## Vitamin K1 solution

0.1	ml	Vitamin K1	
20	ml	Ethanol (95 %)	

1. Dissolve 0.1 ml of vitamin K1 in 20 ml 95% ethanol and filter sterilize. Store refrigerated in a brown bottle.


## Vitamin K3 solution

5	mg	Vitamin K3	
1	ml	Ethanol (95%)	
100	ml	Water	

1. Dissolve 5 mg vitamin K3 in 1 ml 95% ethanol, dilute to 0.05 mg/ml in water and filter sterilize. Store refrigerated in a brown bottle.