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DSMZ

DSMZ 78a . CHOPPED MEAT MEDIUM FOR TREPONEMA SP.

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* Medium for Treponema brennaborense DSM 12168

Plus glucose (0.4%) plus bicarbonate plus 0.5 bar hydrogen in the gas space

Growth conditions: 3-7 days anaerobic 37 °C pH 7.00
Equipment needed: Anaerobic workbench Autoclave Filter Gassing station Hungate tubes
Final pH: n.d.
Select a strain (optional):

ml
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.
Compound Amount Unit
Medium 78 1000 ml
Vitamin solution 5 ml
Amino acid solution (filter sterilized) 50 ml
Glucose 4 g/l

Amino acid solution (filter sterilized) #

50
Compound Amount Unit
L-Histidine 0.6 g
L-Serine 0.5 g
L-Glutamine 0.7 g
Distilled water 50.0 ml

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.

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 #

20
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.