# 78d: MEDIUM FOR TREPONEMA PARVUM


## Main sol. 78d

1000	ml	**Main sol. 78**	

1. To medium 78 add per liter: 

10	ml	Calf serum (newborn or fetal)	
5	ml	**Amino acid solution**	
5	ml	**Vitamin solution**	
5	ml	**Hemin solution**	
5	ml	**Peptone mixture**	
5	ml	**Ascorbic acid solution 2%(w/v)**	
5	ml	KCl (100 g/l)	
5	ml	**N-acetylglucosamin- and pyruvate solution**	
10	ml	**Glutathione solution 14%(w/v)**	
10	ml	**Sugar solution**	

2. Prepare medium anaerobically under N2:CO2 (80:20). After inoculation of the tubes, pressurize them with hydrogen gas to 150 kP (1.5 bar).
3. All following solutions were filter sterilized


## 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.


## Amino acid solution

1. Amino acid solution per 50 ml: 

0.6	g	L-Histidin	
0.5	g	L-Serin	
0.7	g	L-Glutamine	


## Hemin solution

5	mg	Hemin	
1	ml	NaOH (1 N)	
50	ml	Distilled water	

1. Dissolve 5 mg hemin in 1 ml 1 M NaOH, fill up to 50 ml with dist. water.


## Peptone mixture

1. Peptone mixture per 50 ml: 

1.6	g	Tryptone peptone (Difco 0123-17)	
1.6	g	Casamino acids (Difco 0230-17)	
1.6	g	Yeast extract	


## Ascorbic acid solution 2%(w/v)

1	g	Ascorbic acid	
50	ml	Distilled water	

1. Dissolve 1 g ascorbic acid in 40 ml dist. water, adjust pH to 7.0. fill up to 50 ml.


## N-acetylglucosamin- and pyruvate solution

1. N-Acetylglucosamin and pyruvate solution per 50 ml : 

5	g	N-Acetylglucosamine	
5	g	Na-pyruvate	


## Glutathione solution 14%(w/v)

7	g	Glutathione	
50	ml	Distilled water	

1. Dissolve 7 g glutathione in 40 ml dist. Water, adjust pH to 7.0 and fill up to 50 ml.


## Sugar solution

1. Depending on organism use different modification .


## 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.