Dear Isfaqul Hussain,

I normally prepared anoxic stock solution of cysteine (with conc 0,25 - 0,5 M) and added into the anoxic medium that was already in cultivation flasks I never added it into the medium before autoclaving. If you add cysteine into anoxic broth without contact with air (autoclave medium in closed bottle under anoxic gas) you may autoclave this medium under very mild conditions (lower temperature, shorter time).

ANoxic sotck solution was prepared by flitration of cysteine into sterile serum bottle sealed with rubber stopper and aluminium cap. Then I was making solution anoxic by vacuum and saturation of the solution with anoxic N2. THe solution was stored at 4C.

I hope that helps, Alexander.

Dear Alexander

Thank you very much for the answer.

Dear Marine,

1) Normally anoxic stock solution of cysteine could be well conserved at +4C for a very long time (an year or so). I took it out of +4C just for taking the aliquot and then immediately put it back! If cysteine is oxidized then you should see much of white precipitate! Please, remember also that under strict anoxic conditions the solution is quite stable to work with it at room temperature for several hours.

2) No, normally check the pH, adjust it if necessary to 7-7,2 and then filter sterilize to a sterile serum bottle that  is connected to lines to a vacuum pump and to N2 then I make several times cycles - vacuum - N2 saturation.

Dear Alexander,

Could you explain why do you have to store the solution at 4C and why is the solution "quite stable to work with at RT" only for several hours under anoxic conditions? What happens to Cys? If the environment is strictly anoxic, there should be no worries that Cysteine could racemise into cystine, right?

Thank you in advance!

Dear Kaspar,

That was just practical experience. In order to keep the solution ALAP (as long as possible) good for usage caution was taken to slow down AMAP (as much as possible) oxidation of cysteine. Bottles with anoxic solutions normally are closed with black butyl rubber stoppers & sealed with aluminium caps. Air is slowly diffusing through the rubber stoppers. If many samples were taken from the bottles with syringes then the stoppers have many holes that facilitate air diffusion inside the bottle To slow down the air diffusion AMAP it is advisable to keep the bottle at lower temperature. Also the rate of cysteine oxidation by oxygen is lower at lower temperature. That were major points of my recommendation.

With best regards, Alexander.

Dear Alexander,

Thanks for the quick reply!

Have you seen problems also with cysteine solutions stored inside an anaerobic chamber at room temperature? Cause this is what I have been doing - prepare the stock exactly as you described and store it in an anaerobic chamber - and I still see that after a couple of weeks, the stock is not reducing my medium as it should be (though I see no precipitate).

Thanks again!

Dear Kaspar,

First of all it is not so easy to keep "anaerobic" chamber very "anoxic". If you have a chamber-type then it is easier than a tent. If you have a tent-type - then it is somehow dynamic equilibrium between diffusion of oxygen through the tent material and oxygen removal by catalisator. If bottle with cysteine solution is open - then it is also working as oxygen scavenger.

However, since you do not observe turbidity in cysteine solution then there is no strong oxidation of cysteine. In such a case I would recommend to increase conc of cysteine in stock solution (1) and if your microorganism can stand  higher conc of cysteine I would recommend to increase conc of cysteine in solution (2) and (3) after addition of cysteine into the medium I would recommend immediate closing bottle = isolation of gas phase of the medium from the gas phase of the chamber-tent.

That is all what came to my mind while thinking about trouble. I hope that might help somehow.

With best wishes for good reduction! Alexander.

Thanks Alexander!

I operate inside a chamber, so there should be very little oxygen there. Also I store my Cys stock in a serum bottle with butyl septum, so there should not be a lot of oxygen transfer from the anaerobic chamber's atmosphere inside the bottle.

As I cannot understand why the stock is not working if its perfectly clear and stored in a serum bottle inside an anaerobic chamber, I will just continue preparing a fresh stock each time.

What concentration of cysteine is in your stock solution and final conc in the medium?

Stock is 228mM and final in growth medium ~2mM.

Dear Alexander,

I am creating a cysteine stock for an anaerobic mercury bioreporter. If you can, I would appreciate your critique on my method. I created a 40 mM cysteine stock. The steps I took were;

1. made phosphate buffer (67 mM) from phosphoric acid and Milli-Q water and titrated to pH of 2 with NaOH (this is to reduce the pH and not affect mercury speciation, also the bioreporters work in 67mM phosphate buffer).

2. Bubbled with argon

3. placed in anaerobic chamber with 0% oxygen due to a palladium catalytyst and excess hydrogen.

4. L-Cysteine was measured inside the chamber and dissolved in buffer to 40mM concentration and wrapped in aluminum.5. performed assay with the cysteine

My concern is whether the low pH and phosphate species might interact with the cysteine.

Ben

can methylene blue work as oxygen indicator as resazurin or not ?

Dear Ben,

I apologize that I respond only now - I was busy with summer and early autumn issues.

IN general I do not have much experience to work with cysteine at low pH. I hope that your protocol worked fine! Alexander

Hello, Isfaqul Hussain 

Late response but I hope this can help..

Yes, you may add L-Cysteine Hydrochloride (powder) to the medium before autoclaving. It won't affect its structure. I add mine at 0.5g/L. I use PBBM for isolation of acetogens. 

NRB

Thanks Nicole

Dear Alexander,

I added cysteine soon to my media after I autoclaved it. I adjusted my pH before autoclaving. Does the cysteine affect my pH? Do I have to readjust it? 

I assume you are using cysteine-HCl, which would drop your pH a lot if your medium is not buffered enough. You can add cysteine before autoclaving as well, assuming your medium is anoxic and properly sealed in serum bottles.

I am using L-Cysteine in powder form and prepare the solution by adding DI water. Do I have to add a buffer soon to my media? I did not want to add cysteine before autoclaving cause I was concerned the oxidized product of cysteine may harm the bacteria

I cannot give you very detailed advice here as I do not know the details of your experiments. However, most bugs equally like cysteine and cystine, so generation of cystine should not harm them. I assume you work with acetogens, thus you make your media anoxic before autoclaving anyways, so there is no oxidation happening.

On another note, I wonder where do you get pure cysteine for a reasonable price to be used for media? Secondly, bear in mind that if you do not work inside an anaerobic chamber throughout your media prep, your cysteine is going to oxidise before autoclaving - that is why people mostly use cysteine-HCl, so it oxideses less.

Good luck!

to Azadeh Farzaneh

When I was working with cystein as a reducing agent - I added a cysteine reagent to water and quickly adjust pH!!! to 7 - 7,2 with NaOH and then immediatly made it anoxic via several vacuuming - saturating N2 procedures and then soft autoclaving/ IN this case I still have a bit of cystine formed. Very often I used the following - after adjusting pH I filter sterilised it with the syringe help into sterile serum bottle that is connected to the vacuum pump - then several cycles vacuum - N2. Then storage at +4C. In this case cystein solution was stable for quite long!

Alexander,

How do you do the vacuum-N2? Last time I put my bottle of media in water bath and put a hose with filter in media to bubble nitrogen in it (exactly like this youtube video https://www.youtube.com/watch?v=jTRS7OundUw&t=325s). But after bubbling for 3 hours the reassuring was still pink. I added the filtered 1% cysteine solution and let it sit over night. It became colorless finally but there was a very thin pinkish layer on surface of media. It became totally colorless after I put it in the anoxic chamber. But I still don't know how can I prepare totally anoxic media in the lab. the hard part is the empty head space. 

Hi Dear Kaspar Valgepea and all,

I have used cysteine in aerobic fermentation media and autoclaved at 121c for 20 minutes, after several hours of fermentation the media become white precipitated and bad smelly also the yeast growth was very low? have you people answer why this happened?

Hi Rasool,

When you autoclave cysteine, it oxidizes. you should add cysteine after the autoclave. for sterilization, you need to filter sterilize the cysteine solution.

Hi Rasool,

L-cysteine will precipitate if it is in the environment that has oxygen present. Thus, why would you add it to the aerobic fermentation? (maybe you misspelled?).

I have always prepared L-cysteine hydrochloride in the anaerobic environment:

1) flush di water with N2:CO2 (80:20) for 10 min (time varies depending on the volume)

2) add solid cysteine hydrochloride to flushed water

3) flush headspace (NOT the water) for additional 2 min (again, depends on how much water you use) with N2:CO2 (80:20) 

4) seal with rubber stopper and aluminum seal

5) autoclave at 121C for 15 min.

Keep the autoclaved solution in the fridge. If not - it will precipitate over time.

Thank you Azadeh Farzaneh, I will add cysteine by syringe filter.

Thank you Anna Doloman for your detailed answer. I am doing aerobic fermentation not anaerobic. So I cannot avoid oxygen, I just want the way to utilize cysteine without precipitation in aerobic fermentation. Thank you again