78d . MEDIUM FOR TREPONEMA PARVUM

Equipment needed: Autoclave Filter Gassing station Hungate tubes

Final gas composition: 80 % N2 20 % CO2 read more

Final pH: 7.0

Description: Not in use anymore!

Select a strain (optional):

	Compound	Amount	Unit
	Medium 78	1000	ml
1 To medium 78 add per liter:
	Calf serum (newborn or fetal)	10	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	5	ml
	Glutathione solution 14%(w/v)	10	ml
	Sugar solution	10	ml
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 #

1000

	Compound	Amount	Unit
	Vitamin B₁₂	50	mg
	Pantothenic acid	50	mg
	Riboflavin	50	mg
	Pyridoxamine hydrochloride	10	mg
	Biotin	20	mg
	Folic acid	20	mg
	Nicotinic acid	25	mg
	Nicotine amide	25	mg
	alpha-lipoic acid	50	mg
	p-Aminobenzoic acid	50	mg
	Thiamine-HCl x 2 H₂O	50	mg
	Distilled water	1000	ml
1 Stir for some hours, filter sterilize the solution.

Amino acid solution #

	Compound	Amount	Unit
1 Amino acid solution per 50 ml:
	L-Histidin	0.6	g
	L-Serin	0.5	g
	L-Glutamine	0.7	g

Hemin solution #

	Compound	Amount	Unit
	Hemin	5	mg
	NaOH (1 N)	1	ml
	Distilled water	50	ml
1 Dissolve 5 mg hemin in 1 ml 1 M NaOH, fill up to 50 ml with dist. water.

Peptone mixture #

	Compound	Amount	Unit
1 Peptone mixture per 50 ml:
	Tryptone peptone (Difco 0123-17)	1.6	g
	Casamino acids (Difco 0230-17)	1.6	g
	Yeast extract	1.6	g

Ascorbic acid solution 2%(w/v) #

	Compound	Amount	Unit
	Ascorbic acid	1	g
	Distilled water	50	ml
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 #

	Compound	Amount	Unit
1 N-Acetylglucosamin and pyruvate solution per 50 ml :
	N-Acetylglucosamine	5	g
	Na-pyruvate	5	g

Glutathione solution 14%(w/v) #

	Compound	Amount	Unit
	Glutathione	7	g
	Distilled water	50	ml
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 #

1025

	Compound	Amount	Unit
	Ground beef (fat free)	500.0	g
	Distilled water	1000.0	ml
	NaOH (1 N)	25.0	ml
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:
	Casitone	30.0	g
	Yeast extract	5.0	g
	K₂HPO₄	5.0	g
	Resazurin	1.0	mg
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.
	L-Cysteine HCl	0.5	g
	Agar , for solid medium	15.0	g
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:
	Haemin solution optional	10.0	ml
	Vitamin K1 solution , alternative optional	10.0	ml
	Vitamin K3 solution , alternative optional	10.0	ml

Haemin solution #

100

	Compound	Amount	Unit
	Haemin	50	mg
	NaOH (1 N)	1	ml
	Distilled water	100	ml
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 #

100

	Compound	Amount	Unit
	Vitamin K₃	5	mg
	Ethanol (95%)	1	ml
	Water	100	ml
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 #

	Compound	Amount	Unit
	Vitamin K₁	0.1	ml
	Ethanol (95 %)	20.0	ml
1 Dissolve 0.1 ml of vitamin K₁ in 20 ml 95% ethanol and filter sterilize. Store refrigerated in a brown bottle.

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