Radik, Melanie 1-5
Fresh Water Fe Gel Media with Selenate and Acetate
1 liter
Milli-Q H
2
O
DL Vitamin
DL Mineral
NaHCO
3
NH
4
Cl
Ingredient
NaH
2
PO
4
* H
2
O
1 mM Na
2
SeO
4
Sodium acetate (for 10mM)
700 ml
10 ml
10 ml
2.5 g
0.25 g
0.6 g
1 ml
1.36 g
1 L
KCl
Fe Gel
0.1 g
X ml (amount necessary for final concentration of
100 -150 mM)
1000 ml Complete volume with Milli-Q H
2
O to
If making tubes:
Make in small batches – big batches don’t keep
Use iron contaminated beaker, stir bar, canulas, and disposable pipettes
Stir iron gel for at least 15 minutes before adding
Protect media from light
1.
Put 800 ml milli-Q water into a beaker with a stir bar
2.
Add all ingredients a.
Final concentration of iron gel in media should be 100 – 150mM
(example: 500ml of media should have ~50ml of a 1M FeGel stock)
3.
Allow to mix and dissolve completely
4.
Complete volume with milli-Q water in a graduated cylinder
5.
Aliquot 10ml per pressure tube a.
Media should be kept stirring constantly while aliquoting.
6.
Bubble out media with 80:20 of N
2
:CO
2 a.
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
7.
Crimp to close tightly
8.
Autoclave on a fast/wrapped cycle for 30 minutes
9.
Do not expose this media to direct sunlight
10.
Before inoculating cells add an electron donor and an electron acceptor.
11.
Before using each 10 ml tube, add 0.25 ml of 0.4% CaCl
2

2H
2
O.
12.
Transfer 10% cultures, starting from Fe(III) citrate.
13.
Do not add NTA: it should not be necessary and diminishes the amount of magnetite produced.
If making 100ml bottles:

Radik, Melanie 2-5
Make in small batches – big batches don’t keep
Use iron contaminated beaker, stir bar, canulas, and disposable pipettes
Stir iron gel for at least 15 minutes before adding
Protect media from light
14.
Put 800 ml milli-Q water into a beaker with a stir bar
15.
Add all ingredients except the iron gel
16.
Allow to mix and dissolve completely
17.
Complete the volume with milli-Q water in a graduated cylinder to 1000 ml minus the volume for the Iron Gel added later.
18.
Pipette Iron Gel into each 156ml serum bottle to make a final concentration of
100 – 150mM Iron Gel a.
Example: 500ml of media should have ~50ml of a 1M FeGel stock
19.
Aliquot media into each 156ml serum bottle to make the final volume 100ml
20.
Bubble out media with 80:20 of N
2
:CO
2 a.
100mL in 156ml Serum Bottles:

30 minutes open with canulas in liquid

15 minutes with stopper on and canulas in headspace above liquid
21.
Crimp to close tightly
22.
Autoclave on a fast/wrapped cycle for 30 minutes
23.
Do not expose this media to direct sunlight
24.
Before inoculating cells add an electron donor and an electron acceptor.
DL Mineral Mix
1 Liter
Ingredient
Milli-Q H
2
O
NTA Trisodium Salt (Free acid)
1 L
800 ml
1.5 g
MgSO
4
MnSO
4
* H
2
O
NaCl
FeSO
4
* 7 H
2
O
CaCl
2
* 2 H
2
O
CoCl
2
* 6 H
2
O
ZnCl
2
CuSO
4
* 5 H
2
O
AlK(SO
4
)
2
* 12 H
2
O
3.0 g
0.5 g
1 g
0.1 g
0.1 g
0.1 g
0.13 g
0.01 g
0.01 g
H
3
BO
3
Na
2
MoO
4
* 2 H
2
O
NiCl
2
* 6 H
2
O
Na
2
WO
4
* 2 H
2
O
0.01 g
0.025 g
0.024 g
0.025 g
Complete volume with Milli-Q H
2
O 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

Radik, Melanie 3-5
3.
Allow to mix and dissolve completely
4.
Complete volume with milli-Q water in a graduated cylinder
5.
Store at 4 o C
DL Vitamin Solution
1 Liter
Milli-Q H
2
O
In Fridge
Ingredient
Biotin
Pantothenic Acid
B-12 p-aminobenzoic acid
Vitamin Box
Thioctic Acid (alpha lipoic)
Nicotinic Acid
Thiamine
Riboflavin
Pyridoxine HCl
Folic Acid
1 L
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
Complete volume with Milli-Q H
2
O to 1000 ml
Use iron-free beaker and stir bar
Protect solution from light
6.
Put 800 ml milli-Q water into a beaker with a stir bar
7.
Add all ingredients
8.
Allow to mix and dissolve completely
9.
Complete volume with milli-Q water in a graduated cylinder
10.
Store at 4 o C in a dark bottle
Iron Gel Solution
Use iron contaminated beaker, stir bar, centrifuge bottles, and disposable pipettes.
1.
Place 600 ml of Milli-Q H
2
O in a 2L beaker and place on a stir plate with a large stir bar.
2.
Weigh out 108g of Fe Chloride.
3.
Add Fe Chloride to the water.
4.
Once the Fe Chloride is fully dissolved remove approximately 50ml of this solution and set aside.
5.
Calibrate your pH meter and place the electrode in the large beaker of Fe
Chloride.
6.
pH the solution to 7.0 using 10N NaOH. a.
It should take a lot of NaOH, but do not add the sodium hydroxide too quickly. b.
It is best if you add it a few drops at a time with a pasteur pipette.

Radik, Melanie 4-5 c.
The solution is going to look very clumpy, wash clumps off electrode periodically. d.
When the pH reaches about 5.0 the sodium hydroxide should finally get mixed in and the solution will become very thick. e.
Make sure the solution is still mixing f.
Switch to a lower normality NaOH g.
Continue to slowly add NaOH until you reach 7.0. h.
If pH goes over 7.00, use set aside pure Fe Chloride solution to bring it back down. i.
Use a pasteur pipette dropwise, doesn’t take much. i.
Once you have reached pH 7.0 allow the solution to sit and spin for 30 minutes. j.
After 30 minutes recheck the pH of your solution i.
If necessary adjust accordingly so it is at pH 7.0. ii.
It is not unusual for it to have dropped below 6.5.
7.
Distribute your solution evenly into six 500ml centrifuge bottles.
8.
Add milli-Q water to each bottle to bring up the volume of the solution to approximately 2/3 the volume of the vessel.
9.
Balance the bottles with milli-Q water.
10.
Centrifuge them at 5000 rpm for 20 minutes.
11.
Decant supernatant from each bottle.
12.
Scrap the gel from the sides and add milli-Q water so you can resuspend it. a.
The best way to do this is to fill the bottle with about 375ml of water, or
2/3 of the way full and then just put the cover on and shake it well.
13.
Once you have re-suspended all the bottles repeat steps 8-12 at least 4 or 5 times.
14.
The gel is complete when the water left in the bottle after being centrifuged is no longer clear but yellow/red.
15.
When you are confident that the gel is finished a.
Scrape all the gel into one bottle b.
Resuspend by adding about 375ml of Milli-Q and shaking vigorously. c.
A stir bar should be added to this bottle. d.
Store the Iron Gel in the 4 degree walk-in refrigerator.
16.
Using hydroxylamine check the total Fe(III). a.
Ideally it should be around 1M.
17.
Always check the final Fe(III) concentration in the media prepared.
To measure Fe concentration:
This step can be done either just after the above or at any time thereafter.
1.
Shake bottle vigorously and place on stir plate.
2.
Gel should be stirring well (with the little whirlpool in the middle) for at least 15 minutes before pipetting. a.
If the bottle cannot be made to stir well, shake vigorously for 3-5 minutes by hand and pipet quickly.
3.
Using a yellow tip that has been cut at the end, add 100ul gel to 4.7mL 0.5N HCl and 200uL fresh 6.25N hydroxylamine.
4.
Mix and close vial.
5.
Allow mixture to digest overnight in the dark.
6.
Add 100uL of digest to 4.9mL 0.5N HCl.

Radik, Melanie 5-5
7.
Add 100uL of dilution to 4.9mL ferrozine solution (found in 4 o
C walk-in).
8.
Measure absorption at 562nm against a blank with 0mM Fe on the standard curve saved as FeTrev.
9.
Multiply by 2500 for the dilutions made.
10.
The result should be between 1-1.2M for a good concentration.