# 29: PFENNIG'S MEDIUM II


## Main sol. 29

460	ml	**Solution A**	
40	ml	**Solution B**	
50	ml	**Solution C**	
450	ml	**Solution D**	
26	ml	**Solution E**	
10	ml	**Solution F**	

1. Mix solution D, C and E. Bubble with CO2 in an ice bath under sterile conditions.
2. Fill 50 ml in each bottle of solution A. Before using add 4 ml solution B and 0.1 ml solution F.
3. Adjust the pH with filter-sterilised 1M Na2CO3 to 6.8 -7.1.
4. Fill in sterile, N2 gassed screw-cap tubes under N2 gas.
5. During the first 24 h, the iron of the medium precipitates in the form of black flocks. No other sediment should arise in the otherwise clear medium. Feed periodically with neutralized 3% solution of sodium sulfide to replenish sulfide and with other supplement solutions (see Ref. 3365).


## Solution A

0.25	g	CaCl2 x 2 H2O	
0.25	g	Yeast extract	
460	ml	Distilled water	

1. Fill 10x 46ml in 100 ml screw-cap bottles. Bubble with N2/CO2 and autoclave 121°C 15 min.


## Solution B

135	ml	Distilled water	

1. Prepare in a screw-cap bottle, bubble with N2 to replace air, close tightly and autoclave.


## Solution C

1.5	g	NaHCO3	
50	ml	H2O	

1. Bubble with CO2 and filter sterilize into sterile, gas-tight, 100 ml screw-cap bottle.


## Solution D

0.5	ml	Resazurin (0,1%)	
450	ml	Distilled water	

1. Autoclave in a cotton-stoppered Erlenmeyer flask with an outlet tube for medium, connected to a glass outlet at the bottom of the vessel and has, at the other end, a silicon rubber tube with a pinch cock and a bell for aseptic dispensing of the medium into bottles.
2. Cool to room temperature under an atmosphere of N2/CO2 in an ice bath.


## Solution E

0.35	g	Ammonium chloride	
0.25	g	Ammonium acetate	
0.25	g	Pyruvic acid sodium salt	
0.25	g	Dextrose	
0.5	g	MgSO4 x 7 H2O	
0.35	g	KCl	
0.35	g	KH2PO4	
1	ml	**Trace element solution SL-10 B**	
25	ml	Distilled water	

1. Filter sterilize into sterile, gas-tight, 100 ml screw-cap bottle.


## Solution F

0.01	g	Vitamin B12	
100	ml	Distilled water	

1. Filter sterilized


## Neutralized sulfide solution 3% (w/v)

3	g	Na2S x 9 H2O	
100	ml	Distilled water	

1. The sulfide solution is prepared in a 250 ml screw-capped bottle with a butyl rubber septum and a magnetic stirrer. The solution is bubbled with nitrogen gas, closed and autoclaved for 15 min. at 121°C. After cooling to room temperature the pH is adjusted to about 7.0 by adding of sterile 2 M H2SO4 drop-wise with a syringe without opening the bottle.


## Trace element solution SL-10 B

1000	ml	Distilled water	
7.7	ml	HCl (25%)	
1.5	g	FeSO4 x 7 H2O	
70	mg	ZnCl2	
100	mg	MnCl2 x 4 H2O	
300	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