# 78a: CHOPPED MEAT MEDIUM FOR TREPONEMA SP.


## Main sol. 78a

1. To the ready medium, add 50 ml/l amino acid solution and 5 ml/l vitamin solution. These solutions must not be prepared under anaerobic conditions.

1000	ml	**Main sol. 78**	
5	ml	**Vitamin solution**	
50	ml	**Amino acid solution (filter sterilized)**	


## Amino acid solution (filter sterilized)

0.6	g	L-Histidine	
0.5	g	L-Serine	
0.7	g	L-Glutamine	
50	ml	Distilled water	


## Vitamin solution

50	mg	Vitamin B12	
50	mg	Pantothenic acid	
50	mg	Riboflavin	
10	mg	Pyridoxamine hydrochloride	
20	mg	Biotin	
20	mg	Folic acid	
25	mg	Nicotinic acid	
25	mg	Nicotine amide	
50	mg	alpha-lipoic acid	
50	mg	p-Aminobenzoic acid	
50	mg	Thiamine-HCl x 2 H2O	
1000	ml	Distilled water	

1. Stir for some hours, filter sterilize the solution.


## Main sol. 78

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	
1	mg	Resazurin	

2. To make medium anoxic bring it to a boil, cool under 100% N2 gas atmosphere, add 0.5 g/l L-cysteine hydrochloride and adjust pH to 7.0. Dispense under 100% N2 gas atmosphere by filling 7 ml medium into anoxic 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.

0.5	g	L-Cysteine HCl	
15	g	Agar, for solid medium	

3. 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**, alternative (optional)	
10	ml	**Vitamin K3 solution**, alternative (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 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.


## 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.