Radik, Melanie 1-2

Desulfonamide Tiedjei (DTB) Media

1 liter

Ingredient / 1 L
Milli-Q H2O / 800 ml
PIPES (Na or K salt) / 1.5 g
Na2SO4 / 1.42 g
Yeast Extract / 1.0 g
Mineral Solution (See below) / 20 ml
DL Minerals (for Trace Metal Solution) / 10 ml
Complete volume with Milli-Q H2O to / 1000 ml

Use iron-free beaker, stir bar, and canulas

1.  Put 800 ml milli-Q water into a beaker with a stir bar

2.  Add all ingredients

3.  Allow to mix and dissolve completely

  1. Adjust pH of the solution to 7.3

4.  Once the solution is at a sufficient pH add the remaining ingredients:

NaHCO3 / 3.0 g
0.1 % Resazurin / 1 ml

5.  Aliquot 10ml per pressure tube or 100ml per 156ml serum bottle

6.  Bubble out media with 80:20 of N2:CO2

  1. 10ml in Pressure Tubes:

·  6 minutes open with canulas in liquid

·  6 minutes with stopper on and canulas in liquid

·  3 minutes with stopper on and canulas in headspace above liquid

  1. 100mL in 156ml Serum Bottles:

·  30 minutes open with canulas in liquid

·  15 minutes with stopper on and canulas in headspace above liquid

7.  Crimp to close tightly

8.  Label tubes DTB.

9.  Autoclave on a fast/wrapped cycle for 30 minutes

10.  Add the following:

  1. Vitamin Solution 10.0ml/L
  2. Sodium Pyruvate 40mM
  3. 50mM Na2S2O4 (fresh) 10.0ml

Mineral Solution

1 L

Ingredient / 1 L
Milli-Q H2O / 800 ml
NaCl / 40.0 g
NH4Cl / 50.0 g
KCl / 5.0 g
KH2PO4 / 5.0 g
MgCl2 * 2 H2O / 8.3 g
CaCl2 * 2 H2O / 1.0 g
Complete volume with Milli-Q H2O to / 1000 ml

DL Mineral Mix

1 Liter

Ingredient / 1 L
Milli-Q H2O / 800 ml
NTA Trisodium Salt (Free acid) / 1.5 g
MgSO4 / 3.0 g
MnSO4 * H2O / 0.5 g
NaCl / 1 g
FeSO4 * 7 H2O / 0.1 g
CaCl2 * 2 H2O / 0.1 g
CoCl2* 6 H2O / 0.1 g
ZnCl2 / 0.13 g
CuSO4* 5 H2O / 0.01 g
AlK(SO4)2 * 12 H2O / 0.01 g
H3BO3 / 0.01 g
Na2MoO4 * 2 H2O / 0.025 g
NiCl2 * 6 H2O / 0.024 g
Na2WO4 * 2 H2O / 0.025 g
Complete volume with Milli-Q H2O to / 1000 ml

Use iron-free beaker and stir bar

  1. Put 800 ml milli-Q water into a beaker with a stir bar
  2. Add all ingredients
  3. Allow to mix and dissolve completely
  4. Complete volume with milli-Q water in a graduated cylinder
  5. Store at 4oC