# 816: SPOROTOMACULUM MEDIUM


## Main sol. 816

920	ml	**Solution A**	
1	ml	**Solution B**	
1	ml	**Solution C**	
50	ml	**Solution D**	
10	ml	**Solution E**	
5	ml	**Solution F**	
5	ml	**Solution G**	
10	ml	**Solution H**	

1. Sparge solution A with 80% N2 and 20% CO2 gas mixture for 30 - 45 min to make it anoxic, dispense under same gas atmosphere into anoxic Hungate-type tubes or serum vials and autoclave. Solutions B, C, F, G, and H are autoclaved separately under 100% N2 gas. Solution D is autoclaved under 80% N2 and 20% CO2 gas atmosphere and autoclaved. Solution E is prepared under 100% N2 gas atmosphere and sterilized by filtration. To complete the medium appropriate amounts of solutions B to H are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be checked and adjusted to 7.6 with a sterile anoxic stock solution of Na2CO3 (5% w/v), if necessary.


## Solution A

0.2	g	KH2PO4	
0.3	g	NH4Cl	
0.5	g	KCl	
1	g	NaCl	
0.15	g	CaCl2 x 2 H2O	
0.4	g	MgCl2 x 6 H2O	
0.5	ml	Sodium resazurin (0.1% w/v)	
920	ml	Distilled water	


## Solution B

1	ml	**Trace element solution SL-10**	


## Solution C

1	ml	**Selenite-tungstate solution**	


## Solution D

2.5	g	Na2CO3	
50	ml	Distilled water	


## Solution E

2.8	g	3-Hydroxybenzoic acid	
10	ml	Distilled water	

1. Neutralized with NaOH.


## Solution F

0.5	g	Yeast extract	
5	ml	Distilled water	


## Solution G

0.5	g	Trypticase (BD BBL)	
5	ml	Distilled water	


## Solution H

0.3	g	Na2S x 9 H2O	
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