# 110: CHOPPED MEAT MEDIUM WITH CARBOHYDRATES


## Main sol. 110

500	g	Ground beef (fat free)	
1000	ml	Distilled water	
25	ml	NaOH (1 N)	

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 ml, and then add: 

30	g	Casitone	
5	g	Yeast extract	
5	g	K2HPO4	
0.5	ml	Sodium resazurin (0.1% w/v)	
4	g	D-Glucose	
1	g	Cellobiose	
1	g	Maltose	
1	g	Starch (soluble)	
0.5	g	L-Cysteine HCl, add to make medium anoxic	
15	g	Agar, if required	

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:  and either  or 

10	ml	**Haemin solution**	
10	ml	**Vitamin K1 solution**, alternative	
10	ml	**Vitamin K3 solution**	

3. To make medium anoxic boil it, cool under 100% N2 gas atmosphere, add 0.5 g/l L-cysteine hydrochloride and adjust pH to 7.0. Dispense under same gas atmosphere 7 ml medium 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 30 min. For agar slants use 15.0 g agar per 1000.0 ml medium.


## 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.