# 212a: TREPONEMA RECTALE MEDIUM


## Main sol. 212a

917	ml	**Solution A**	
30	ml	**Solution B**	
4	ml	**Solution C**	
4	ml	**Solution D**	
30	ml	**Solution E**	
1	ml	**Solution F**	
2	ml	**Solution G**	
10	ml	**Solution H**	
10	ml	**Solution I**	

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

0.5	g	KH2PO4	
0.33	g	MgCl2 x 6 H2O	
0.4	g	NaCl	
0.4	g	NH4Cl	
0.05	g	CaCl2 x 2 H2O	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
50	ml	**Clarified rumen fluid**	
1	g	Trypticase peptone (BD BBL)	
2.8	g	Na2SO4	
1	ml	**Vitamin K1 solution**	
3.1	ml	**Volatile fatty acid mixture**	
0.5	ml	Sodium resazurin (0.1% w/v)	
860	ml	Distilled water	


## Solution B

1.5	g	Na2CO3	
30	ml	Distilled water	


## Solution C

0.4	g	Starch (soluble)	
4	ml	Distilled water	


## Solution D

0.8	g	D-Glucose	
4	ml	Distilled water	


## Solution E

30	ml	**Sugar mix**	


## Solution F

1	ml	**Seven vitamins solution**	


## Solution G

0.5	g	Na-pyruvate	
2	ml	Distilled water	


## Solution H

0.3	g	L-Cysteine HCl x H2O	
10	ml	Distilled water	


## Solution I

0.4	g	DL-Dithiothreitol (DTT)	
10	ml	Distilled water	


## Trace element solution SL-10

10	ml	HCl (25%)	
1.5	g	FeCl2 x 4 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
6	mg	H3BO3	
190	mg	CoCl2 x 6 H2O	
2	mg	CuCl2 x 2 H2O	
24	mg	NiCl2 x 6 H2O	
36	mg	Na2MoO4 x 2 H2O	
990	ml	Distilled water	

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

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	ml	Distilled water	


## Clarified rumen fluid

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

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.


## Volatile fatty acid mixture

548.5	ml	Acetic acid	
193.5	ml	Propionic acid	
129	ml	Butyric acid	
32.25	ml	n-Valeric acid	
32.25	ml	iso-Butyric acid	
32.25	ml	DL-2-Methylbutyric acid	
32.25	ml	iso-Valeric acid	


## Sugar mix

56.7	g	Cellobiose	
56.7	g	Sucrose	
60	g	Maltose x H2O	
63.3	g	Trehalose	
25	g	D-Xylose	
1000	ml	Distilled water	


## Seven vitamins solution

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