Hello Li,

Can you explain me how you measure the dissolved oxygen in your solution ? Did you flush the headspace of your serum bottle with N2/CO2 or the solution ?

I think the better way to make this kind of solution is inside an anaerobic chamber with N2 flushed water. Be sure that the solution is never exposed to the ambient air.

I hope that I've helped you and other contributions/experiences are welcome.

It is not clear for what purposes your FeCl2 will be used. There exists chemical way by addition of Na2SO3 . It works when excess concentration of Na2SO3 is employed. But I do not know if Na2SO3 addition is conveniet for your further testing.

Best regards

Vit

您好：

厌氧氯化亚铁溶液是什么意思？ 是防止亚铁离子被氧化吗？可以用煮沸的去离子水作为溶剂，瓶子上方可以充满氮气创造厌氧条件。

It is for our experiment bacteria, they reduce Fe3+ to Fe2+ and we want to see if they will still be alive without O2 present. We made the solution the normal way and put N2 gas to flush 20 minutes for the 100 mL serum bottle to try to create anaerobic environment, but after autoclave, it shows red PPT on the bottle.

we will try to get the water in the anaerobic chamber and flush the gas in there to get the air out, then make the solution there.

Flushing N2 CO2 through water (medium) pH ~ 7 & serum bottle is not really sufficient to remove the rest of dissolved O2.

I would suggest following protocol:

1) boil water (medium) ~ several minutes

2) put boiled still hot water into flushed with N2 serum bottle using syringe-flush techniques

3) wait till temperature comes to room temperature (under N2 atmosphere)

4) "drop" FeCl2 into serum bottle (during this stop flushing)

5) close serum bottle with black rubber stopper (that is used for Bach tubes and stated to be gas "impermeable" :-)

6) autoclave it (mild conditions).

7) store at +4C

8) make sure that if you take an aliquote of Fe-solution with the syringe you need to add also anoxic gase into the serum bottle to maintain silghtly higher pressure inside.

However, my experience is that this also do not prevent slight Fe2+ oxidation by remaining O2.

Some remarks.

1) If you do not care about some hydrogen in the Fe2+ solution you may add a bit of H2 into gas phase of serum bottle + palladium catalizator before autoclave.

2) Please, check if your FeCl2 is not a bit oxidised in a bottle. It is quite stable when dry. If it sucks a bit moisture from air - then chemical oxidation may occur.

3) Several other protocols could be also fine. For ex. I used also following:

a) put FeCl2 (dry!) into serum bottle (no moisture inside !!!)

b) flushed it with anoxic gase

c) close and sealed it with rubber stopper.

d) autoclave it (mild conditions)

e) separately prepare anoxic water in another serum bottle (sterilise it in autoclave or using filter sterilisation when you add with syringe the reguired volume of water into serum bottle with FeCl2.

f) store at +4C

As you may see that is also tedious procedure and still slight Fe2+ oxidation may occur.

It may work very good if you add some of reducing agents! For ex. as it was recommended above!

Another efficient and proven way to remove traces of oxygen is to use FeS.

Amorphous ferrous sulfide can prepared by a procedure descried described by Brock and O’Dea (1977). Equimolar quantities of ferrous am­monium sulfate (39.169 g) and sodium sulfide (24.018 g of Na2S · 9H20) are brought to boiling, and mixed in a glass-stoppered bottle (filled to the top). The copious black precipitate of ferrous sulfide is allowed to settle, the clear water is decanted, and the precipitate is washed four or five times with boiling distilled water. The final suspension can be transferred while hot to screw-capped bottles or tubes and gassed with 02-free N2. The preparation is not difficult to make and, once prepared, can be stored for weeks.

REFERENCE (slightly modified from:)

Thomas D. Brock and Kathleen O'Dea 1977 Amorphous ferrous sulfide as a reducing agent for culture of anaerobes. Applied and Environmental Microbiology 33: 254-256

I work with anaerobes and this is my way of preparing anoxic solution.

1) Place your solution in a serum bottle, seal it with butyl rubber septum and aluminum cap.

2) Use a 21ga needle to pierce through the butyl rubber septum (this serves as a gas outlet).

3) Connect your gas supply to another 21ga needle and pierce the needle through the butyl rubber septum. Ensure that the needle is inside the liquid phase. Turn on the gas (I use 500mL/min of gas at 40psi).

4) After 10 mins, remove both needles simultaneously.

5) Autoclave your sample.