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DSMZ

DSMZ 212a . TREPONEMA RECTALE MEDIUM

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Equipment needed: Autoclave Centrifuge Filter Gassing station Hungate tubes
Final gas composition: 80 % N2 20 % CO2 read more
Final pH: 7.0
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ml
Compound Amount Unit
Solution A 917 ml
Solution B 30 ml
Solution C 4 ml
Solution D 4 ml
Solution E 30 ml
Solution F 1 ml
Solution G 2 ml
Solution H 10 ml
Solution I 10 ml
1 Add and dissolve ingredients of solution A, adjust pH to 7.0, and sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Dispense medium under the same gas atmosphere into anoxic Hungate-type tubes to 30% of their volume and autoclave. Solution B is prepared under 80% N2 and 20% CO2 gas atmosphere and autoclaved. Solutions C, D, and H are autoclaved under a 100% N2 gas atmosphere. Solutions E, F, G, and I are prepared under 100% N2 gas and sterilized by filtration. To complete the medium appropriate amounts of solutions B to I are added to the sterile solution A in the sequence as indicated.
2 Note: Some cultures are shipped in semi-solid medium which stimulates growth at the beginning. For agar stabs 3.00 g/l agar are added to the complete medium from a sterile anoxic stock solution (2% w/v). Upon receipt add anoxically 1 - 2 ml of the recommended freshly prepared liquid medium to the agar tube and incubate for 3 - 5 days . After incubation transfer 0.5 ml of the resulting cell suspension in the liquid phase to tubes with liquid medium.

Solution A #

917
Compound Amount Unit
KH2PO4 0.50 g
MgCl2 x 6 H2O 0.33 g
NaCl 0.40 g
NH4Cl 0.40 g
CaCl2 x 2 H2O 0.05 g
Trace element solution SL-10 1.00 ml
Selenite-tungstate solution 1.00 ml
Clarified rumen fluid 50.00 ml
Trypticase peptone
(BD BBL) 		1.00 g
Na2SO4 2.80 g
Vitamin K1 solution 1.00 ml
Volatile fatty acid mixture 3.10 ml
Sodium resazurin
(0.1% w/v) 		0.50 ml
Distilled water 860.00 ml

Solution B #

30
Compound Amount Unit
Na2CO3 1.5 g
Distilled water 30.0 ml

Solution C #

4
Compound Amount Unit
Starch
(soluble) 		0.4 g
Distilled water 4.0 ml

Solution D #

4
Compound Amount Unit
D-Glucose 0.8 g
Distilled water 4.0 ml

Solution E #

30
Compound Amount Unit
Sugar mix 30 ml

Solution F #

1
Compound Amount Unit
Seven vitamins solution 1 ml

Solution G #

2
Compound Amount Unit
Na-pyruvate 0.5 g
Distilled water 2.0 ml

Solution H #

10
Compound Amount Unit
L-Cysteine HCl x H2O 0.3 g
Distilled water 10.0 ml

Solution I #

10
Compound Amount Unit
DL-Dithiothreitol
(DTT) 		0.4 g
Distilled water 10.0 ml

Trace element solution SL-10 #

1000
Compound Amount Unit
HCl
(25%) 		10.0 ml
FeCl2 x 4 H2O 1.5 g
ZnCl2 70.0 mg
MnCl2 x 4 H2O 100.0 mg
H3BO3 6.0 mg
CoCl2 x 6 H2O 190.0 mg
CuCl2 x 2 H2O 2.0 mg
NiCl2 x 6 H2O 24.0 mg
Na2MoO4 x 2 H2O 36.0 mg
Distilled water 990.0 ml
1 First dissolve FeCl2 in the HCl, then dilute in water, add and dissolve the other salts. Finally make up to 1000.00 ml.

Selenite-tungstate solution #

1000
Compound Amount Unit
NaOH 0.5 g
Na2SeO3 x 5 H2O 3.0 mg
Na2WO4 x 2 H2O 4.0 mg
Distilled water 1000.0 ml

Clarified rumen fluid #

1000
1 Rumen fluid from cow or sheep (obtained from fistulated animals or abattoir refuse) is filtered through muslin, autoclaved at 121°C for 15 min and then centrifuged at 27,000 g for 20 min. The supernatant is made anoxic by sparging with 100% N2 gas for 15 min, dispensed under same gas atmosphere into anoxic serum vials to 30% of volume and then stored frozen at -20°C.

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.

Volatile fatty acid mixture #

1000
Compound Amount Unit
Acetic acid 548.50 ml
Propionic acid 193.50 ml
Butyric acid 129.00 ml
n-Valeric acid 32.25 ml
iso-Butyric acid 32.25 ml
DL-2-Methylbutyric acid 32.25 ml
iso-Valeric acid 32.25 ml

Sugar mix #

1000
Compound Amount Unit
Cellobiose 56.7 g
Sucrose 56.7 g
Maltose x H2O 60.0 g
Trehalose 63.3 g
D-Xylose 25.0 g
Distilled water 1000.0 ml

Seven vitamins solution #

1000
Compound Amount Unit
Vitamin B12 100 mg
p-Aminobenzoic acid 80 mg
D-(+)-biotin 20 mg
Nicotinic acid 200 mg
Calcium pantothenate 100 mg
Pyridoxine hydrochloride 300 mg
Thiamine-HCl x 2 H2O 200 mg
Distilled water 1000 ml