# 28b: THIORHODOCOCCUS MEDIUM II


## Main sol. 28b

1000	ml	**Main sol. 28**	
1.5	%	NaCl	
0.05	%	Na2S2O3 x 5 H2O	
0.25	%	MgCl2 x 6 H2O	

1. Adjust to pH 7.5, anaerobic incubation under nitrogen/carbon dioxide atmosphere; light (see medium 28).


## Main sol. 28

460	ml	**Solution A**	

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.

450	ml	**Resazurin solution**	
50	ml	**Bicarbonate solution**	
26	ml	**Pfennig's heterotrophic salts solution**	

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.

40	ml/l	**Sulfide solution, 1.5%**	
1	ml/l	**Vitamin B12 solution**	

6. Adjust the pH with filter-sterilised 1M Na2CO3 to 7.1-7.3.
7. If needed, aliquot into sterile, N<sub>2</sub> gassed screw-cap tubes under N<sub>2</sub> gas.
8. 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.
9. Feed the actively growing culture periodically with neutralized 3% solution of sodium sulfide (use 1 -3 mL/100 mL depending on strain and cultivation stage) to replenish sulfide and with other supplement solutions (see Ref. 3365).

10	ml	**Neutralized sulfide solution 3% (w/v)**	


## 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.