29 . PFENNIG'S MEDIUM II

Equipment needed: Autoclave Filter Ice bath

Final pH: 6.8 - 7.1

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	Compound	Amount	Unit
	Solution A	460	ml
	Solution B	40	ml
	Solution C	50	ml
	Solution D	450	ml
	Solution E	26	ml
	Solution F	10	ml
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 #

460

	Compound	Amount	Unit
	CaCl₂ x 2 H₂O	0.25	g
	Yeast extract	0.25	g
	Distilled water	460.00	ml
1 Fill 10x 46ml in 100 ml screw-cap bottles. Bubble with N₂/CO₂ and autoclave 121°C 15 min.

Solution B #

135

	Compound	Amount	Unit
	Distilled water	135	ml
1 Prepare in a screw-cap bottle, bubble with N₂ to replace air, close tightly and autoclave.

Solution C #

	Compound	Amount	Unit
	NaHCO₃	1.5	g
	H₂O	50.0	ml
1 Bubble with CO₂ and filter sterilize into sterile, gas-tight, 100 ml screw-cap bottle.

Solution D #

450

	Compound	Amount	Unit
	Resazurin (0,1%)	0.5	ml
	Distilled water	450.0	ml
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 #

	Compound	Amount	Unit
	Ammonium chloride	0.35	g
	Ammonium acetate	0.25	g
	Pyruvic acid sodium salt	0.25	g
	Dextrose	0.25	g
	MgSO₄ x 7 H₂O	0.50	g
	KCl	0.35	g
	KH₂PO₄	0.35	g
	Trace element solution SL-10 B	1.00	ml
	Distilled water	25.00	ml
1 Filter sterilize into sterile, gas-tight, 100 ml screw-cap bottle.

Solution F #

100

	Compound	Amount	Unit
	Vitamin B₁₂	0.01	g
	Distilled water	100.00	ml
1 Filter sterilized

Neutralized sulfide solution 3% (w/v) #

100

	Compound	Amount	Unit
	Na₂S x 9 H₂O	3	g
	Distilled water	100	ml
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

Compound	Amount	Unit
Distilled water	1000.0	ml
HCl (25%)	7.7	ml
FeSO₄ x 7 H₂O	1.5	g
ZnCl₂	70.0	mg
MnCl₂ x 4 H₂O	100.0	mg
H₃BO₃	300.0	mg
CoCl₂ x 6 H₂O	190.0	mg
CuCl₂ x 2 H₂O	2.0	mg
NiCl₂ x 6 H₂O	24.0	mg
Na₂MoO₄ x 2 H₂O	36.0	mg

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