Kashefi, Kazem 1-2
Geoglobis ahangari Modified Marine Medium
1L
Ingredient
Milli-Q H2O
MgCl2 * 6H2O
NaCl
CaCl2 * 2H2O
KCl
KH2PO4
(NH4)2SO4
NaBr
SrCl2* 6H2O
MgCl2 * 7H2O
1mM Na2SeO4
NaHCO3
DL Vitamins
Wolfe’s minerals
Poorly crystalline Fe(III)
Complete volume with Milli-Q H2O to
1L
700 ml
9.00 g
19.0 g
0.30 g
0.50 g
0.50 g
0.30 g
0.05 g
0.02 g
0.15 g
1 ml
2.81 g
10 ml
10 ml
100 mmol
1000 ml
Use iron-free beaker, stir bar, and canulas
1. Put 700 ml milli-Q water into a beaker with a stir bar
2. Dissolve all minerals (except NaHCO3) in milliQ water
3. Add trace minerals, vitamins, and Poorly crystalline Fe(III).
4. Adjust pH to 6.0 with HCl
5. Add NaHCO3.
6. Dispense media into serum bottles (45 ml) or pressure tubes (9 ml).
7. Gas with appropriate gas phase (80% N2: 20%CO2 or 80% H2: 20% CO2)
i. 4 minutes open with canulas in liquid
ii. 8 minutes with stopper on and canulas in liquid
8. Crimp to close tightly
9. Autoclave on a fast/wrapped cycle for 30 minutes
10. When inoculating, add electron donor and cysteine-HCl (0.5ml of 50mM for 10
ml of media) in addition to 1 ml of inoculum.
11. Pressurize the bottles to 1 bar overpressure and incubate.
Wolfe’s Mineral Solution:
Available from ATCC as a sterile ready-to-use liquid (Trace Mineral Supplement)
Catalog no. MD-TMS
Ingredient
1L
Milli-Q H2O
500 ml
Nitrilotriacetic Acid
1.5 g
MgSO4 * 7 H2O
3.0 g
MnSO4 * H2O
0.5 g
Kashefi, Kazem 2-2
1.
2.
3.
4.
5.
6.
NaCl
1.0 g
Fe SO4 * 7 H2O
0.1 g
CoCl2 * 6 H2O
0.1 g
CaCl2
0.1 g
ZnSO4 * 7 H2O
0.1 g
CuSO4 * 5 H2O
0.01 g
AlK(SO4 )2 * 12 H2O
0.01 g
Na2MnO4 * 2 H2O
0.01 g
Complete volume with Milli-Q H2O to
1000 ml
Put 500 ml milli-Q water into a beaker with a stir bar
Add nitrilotriacetic acid
Wait for the NTA to dissolve fully.
Adjust pH of the solution to 6.5 with KOH
a. When the pH approaches 5.0 use a lower normality base such as 5N or
even 2N.
Once the solution is at a sufficient pH add the rest of the ingredients one at a time.
Complete volume with milli-Q water in a graduated cylinder
DL Vitamin Solution
1 Liter
Ingredient
Milli-Q H2O
In Fridge
Biotin
Pantothenic Acid
B-12
p-aminobenzoic acid
Vitamin Box
Thioctic Acid (alpha lipoic)
Nicotinic Acid
Thiamine
Riboflavin
Pyridoxine HCl
Folic Acid
Complete volume with Milli-Q H2O to
1L
800 ml
0.002 g
0.005 g
0.0001 g
0.005 g
0.005 g
0.005 g
0.005 g
0.005 g
0.01 g
0.002 g
1000 ml
Use iron-free beaker and stir bar
Protect solution from light
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 in a dark bottle