DRL Lab 1-4
Iron Gel Media with Selenate and No Chelator using Iron Gel Salt Solution
500 ml
Ingredients
Milli-Q H2O
KCl
NH4Cl
NaH2PO4 * H2O
DL Vitamins
Non-chelated Minerals
(non-chelated trace elements for sulfate reducer medium)
Fe Gel Salt solution
1 mM Na2SeO4
Iron gel
Complete volume with Milli-Q H2O to
500 ml
350 ml
0.05 g
0.125 g
0.3 g
5 ml
0.5 ml
5.0 ml
0.5 ml
X ml (amount necessary
for final concentration of
100 -150 mM)
500 ml
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 N2:CO2
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.
If making 100ml bottles:
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
DRL Lab 2-4
11. Put 800 ml milli-Q water into a beaker with a stir bar
12. Add all ingredients except the iron gel
13. Allow to mix and dissolve completely
14. Complete the volume with milli-Q water in a graduated cylinder to 1000 ml
minus the volume for the Iron Gel added later.
15. 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
16. Aliquot media into each 156ml serum bottle to make the final volume 100ml
17. Bubble out media with 80:20 of N2:CO2
a. 100mL in 156ml Serum Bottles:
 30 minutes open with canulas in liquid
 15 minutes with stopper on and canulas in headspace above
liquid
18. Crimp to close tightly
19. Autoclave on a fast/wrapped cycle for 30 minutes
20. Do not expose this media to direct sunlight
21. Crimp to close tightly
22. Autoclave on a fast/wrapped cycle for 30 minutes
Non-Chelated Trace Element Mixture
1 Liter
Ingredient
1L
Milli-Q H2O
800 ml
FeSO4 * 7 H2O
1.05g
H3BO3
0.015g
MnCl2 * 4 H2O
0.05g
CoCl2 * 6 H2O
0.08g
NiCl2 * 6 H2O
0.012g
CuCl2 * 2 H2O
0.001g
ZnSO4 * 7 H2O
0.072g
Na2MoO4 * 2 H2O
0.018g
Na2 EDTA
3.6g
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. pH to 6.0
5. Complete volume with milli-Q water in a graduated cylinder
6. Store at 4oC
DL Vitamin Solution
1 Liter
DRL Lab 3-4
Ingredient
1L
800 ml
Milli-Q H2O
In Fridge
Biotin
0.002 g
Pantothenic Acid
0.005 g
B-12
0.0001 g
p-aminobenzoic acid
0.005 g
Vitamin Box
Thioctic Acid (alpha lipoic)
0.005 g
Nicotinic Acid
0.005 g
Thiamine
0.005 g
Riboflavin
0.005 g
Pyridoxine HCl
0.01 g
Folic Acid
0.002 g
Complete volume with Milli-Q H2O to
1000 ml
Use iron-free beaker and stir bar
Protect solution from light
7. Put 800 ml milli-Q water into a beaker with a stir bar
8. Add all ingredients
9. Allow to mix and dissolve completely
10. Complete volume with milli-Q water in a graduated cylinder
11. Store at 4oC 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 H2O 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.
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.
DRL Lab 4-4
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.
7. Add 100uL of dilution to 4.9mL ferrozine solution (found in 4oC 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.