# 193a: PHB/PYRUVATE MEDIUM


## Main sol. 193a

942	ml	**Solution A**	
30	ml	**Solution B**	
20	ml	**Solution C**	
1	ml	**Solution D**	
10	ml	**Solution E**	

1. Solution A is sparged with 80% H2 and 20% CO2 gas mixture to reach a pH below 6 (at least 30 min), then distributed under the same gas atmosphere in anoxic Hungate-type tubes or serum vials to 30% of their volume and autoclaved. Solution B is autoclaved separately under 80% N2 and 20% CO2 gas atmosphere. Solutions C and D are prepared under 100% N2 gas atmosphere and sterilized by filtration. Solution E is autoclaved separately under 100% N2 gas. To complete the medium appropriate amounts of solutions B to E are added to the sterile solution A in the sequence as indicated. Final pH of the medium should be 6.8 - 7.0.
2. After inoculation, pressurize vessels with 80% H2 and 20% CO2 gas mixture to 1 bar overpressure.
3. Note: Addition of 10 - 20 mg sodium dithionite per liter (e.g. from 5% (w/v) solution, freshly prepared under N2 and filter-sterilized) may stimulate growth at the beginning. For transfers use 5 - 10% inoculum.


## Solution A

3	g	Na2SO4	
0.2	g	KH2PO4	
0.3	g	NH4Cl	
7	g	NaCl	
1.3	g	MgCl2 x 6 H2O	
0.5	g	KCl	
0.15	g	CaCl2 x 2 H2O	
1	ml	**Selenite-tungstate solution**	
1	ml	**Trace element solution SL-10**	
1	g	Poly[(R)-3-hydroxybutyric acid] (SIGMA)	
0.5	ml	Sodium resazurin (0.1% w/v)	
940	ml	Distilled water	


## Solution B

1.5	g	Na2CO3	
30	ml	Distilled water	


## Solution C

5	g	Na-pyruvate	
20	ml	Distilled water	


## Solution D

1	ml	**Wolin's vitamin solution (10x)**	


## Solution E

0.4	g	Na2S x 9 H2O	
10	ml	Distilled water	


## Selenite-tungstate solution

0.5	g	NaOH	
3	mg	Na2SeO3 x 5 H2O	
4	mg	Na2WO4 x 2 H2O	
1000	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.


## Wolin's vitamin solution (10x)

20	mg	Biotin	
20	mg	Folic acid	
100	mg	Pyridoxine hydrochloride	
50	mg	Thiamine HCl	
50	mg	Riboflavin	
50	mg	Nicotinic acid	
50	mg	Calcium D-(+)-pantothenate	
1	mg	Vitamin B12	
50	mg	p-Aminobenzoic acid	
50	mg	(DL)-alpha-Lipoic acid	
1000	ml	Distilled water