Main sol. 29

Internal Solution-ID: S37

This solution is used in 2 media and was first defined in medium 29.

Compound	Amount	Unit	Conc. [g/L]	Conc. [mM]
Solution A	460	ml	-	-
1 Aliquot Solution A into 100 mL screw-cap bottles, filled with 46 mL each. Bubble with N₂/CO₂ and autoclave at 121°C for 15 min (as decribed below).
2 Prepare the following solutions (resazurin, bicarbonate and Pfennig's heterotrophic salts) and sterilize as given below.
Resazurin solution	450	ml	-	-
Bicarbonate solution	50	ml	-	-
Pfennig's heterotrophic salts solution B	26	ml	-	-
3 Add bicarbonate solution and Pfennig's heterotrophic salts to the resazurin (complete volumina, i.e. 50 mL bicarbonate solution and 26 mL Pfennig's heterotrophic salts solution). Bubble with CO₂ in an ice bath under sterile conditions.
4 Fill 50 ml of this mixture to each bottle of solution A (46 mL + 50 mL).
5 Before use, add 4 ml sulfide solution (1.5%) and 0.1 ml Vitamin B₁₂ solution to each 100 mL bottle.
Sulfide solution, 1.5%	40	ml/l	-	-
Vitamin B12 solution	1	ml/l	-	-
6 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).
7 Adjust pH to 6.8-7.1 with 1M Na₂CO₃ (filter-sterilized).

29	PFENNIG'S MEDIUM II
40	PFENNIG'S MEDIUM II WITH SALT