# 843: HELMUTKOENIGIA MEDIUM


## Main sol. 843

1	g	NaCl	
0.5	g	KCl	
0.4	g	MgCl2 x 6 H2O	
0.1	g	CaCl2 x 2 H2O	
0.3	g	NH4Cl	
0.2	g	KH2PO4	
0.15	g	Na2SO4	
1	ml	**Trace element solution SL-10**	
1	ml	**Selenite-tungstate solution**	
1	g	Trypticase peptone (BD BBL)	
0.5	ml	Sodium resazurin (0.1% w/v)	
1.5	g	Na2CO3	
30	ml	**Sugar mix**	
50	ml	**Yeast autolysate**	
1	ml	**Seven vitamins solution**	
20	ml	**Co-factors solution**	
0.4	g	DL-Dithiothreitol (DTT)	
900	ml	Distilled water	

1. Dissolve ingredients (except carbonate, sugar mix, yeast autolysate, vitamins, co-factors, and DTT), sparge medium with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic. Distribute under same gas atmosphere into anoxic Hungate-type tubes or serum vials to 30% of their volume (e.g., 4.5 ml per Hungate-type tube) and autoclave. After sterilization add carbonate from a sterile anoxic stock solution prepared under 80% N2 and 20% CO2 gas atmosphere and sugar mix, yeast autolysate, vitamins, co-factors and DTT from sterile anoxic stock solutions prepared under 100% N2 gas atmosphere and sterilized by filtration.  Check pH of complete medium and adjust to 7.2 - 7.4, if necessary.
2. Note: Basal medium and yeast autolysate have to be prepared freshly and cannot be stored for longer periods.


## 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	


## Yeast autolysate

280	g	Yeast extract (dry baker's yeast, SIGMA YSC-1)	
1000	ml	Distilled water	

1. Suspend dry baker's yeast (SIGMA YSC-1) in distilled water and allow cells to autolyse for 48 h at 55°C. Thereafter, cell debris are removed by centrifugation (3.700 x g for 30 min) and the amber-colored supernatant is stored in aliquots at -20°C.


## Co-factors solution

2.5	g	Thiamine pyrophosphate	
0.25	g	Pyridoxal hydrochloride	
0.25	g	Pyridoxal phosphate	
0.05	g	Ca-folinate	
0.05	g	ß-NAD	
0.05	g	Coenzyme A	
0.05	g	FAD	
65	mg	Haemin (dissolved separately in 10 mM NaOH)	
25	mg	Nicotinamide	
2.5	mg	Folic acid	
0.5	mg	Riboflavin	
1000	ml	Distilled water	

1. Adjust pH to 7.0 with 1 N NaOH against indicator paper, filter sterilize and store 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	


## 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