# 683: TISSIERELLA (AMO.1) MEDIUM


## Main sol. 683

5.8	g	NaCl	
0.4	g	MgCl2 x 6 H2O	
0.3	g	KCl	
0.15	g	CaCl2 x 2 H2O	
0.27	g	NH4Cl	
0.2	g	KH2PO4	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
10	g	Yeast extract (OXOID)	
0.5	g	Trypticase peptone (BD BBL)	
2	g	L-Serine	
2	g	L-Threonine	
5.64	g	N-methylhydantoin (SIGMA)	
0.5	ml	Sodium resazurin (0.1% w/v)	
4.5	g	NaHCO3	
1	ml	**Wolin's vitamin solution (10x)**	
0.5	g	L-Cysteine HCl x H2O	
1000	ml	Distilled water	

1. Dissolve ingredients (except bicarbonate, cysteine and vitamins) and sparge medium with 100% N2 gas for 30 - 45 min to make it anoxic. Adjust pH to 8.3 with 10 N NaOH, add bicarbonate and cysteine and dissolve. Switch gas phase to 80% N2 and 20% CO2 gas mixture and flush only the head space of the medium vessel. Dispense medium under 80% N2 and 20% CO2 gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave 15 min at 121°C. Add vitamins to the autoclaved medium from an anoxic stock solution prepared under 100% N2 gas atmosphere and sterilized by filtration. Adjust pH of complete medium to 8.3, if necessary.
2. Note: If creatinine is used as the substrate replacing N-methylhydantoin, prepare separately an anoxic, filter sterilized stock solution of creatinine under 100% N2 gas atmosphere.


## 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	


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water