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Anaerobic Bacteria-Cystine Sulfide Solution

Anaerobic Cystine Sulide

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#1 SunsetSatine

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Posted 13 October 2012 - 06:38 PM

HI!

I'm working with Anaerobic Bacteria and I add Cystine-Sulfide Solution as a reducer. For some reaon, though, it keeps precipitating and I can't figure out why. I've attached the protocol that I've been using. Anyone else have this problem and can share with advice? I would greatly appreciate it. Thanks!

Attached Thumbnails

  • Cystine-Sulfide Protocol.png


#2 El Crazy Xabi

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Posted 14 October 2012 - 03:38 PM

From a culture media handbook (quantities of reducers are variable depending on the media to prepare):

Reducing Agent Solution:
Composition per 110.0mL:
L-Cysteine·HCl·H2O ..................................................................... 2.5g
Na2S·9H2O.................................................................................... 2.5g
Preparation of Reducing Agent Solution: Add 110.0mL of distilled/
deionized water to a 250.0mL flask. Boil under N2 gas for 1 min. Cool
to room temperature. Add L-cysteine·HCl·H2O and dissolve. Adjust to pH 9
with 5N NaOH. Add washed Na2S·9H2O and dissolve. Distribute in
10.0mL volumes into tubes. Autoclave for 10 min at 15 psi pressure–121°C.



Few others prepare both solutions separately

Na2S·9H2O Solution:
Composition per 10.0mL:
Na2S·9H2O.................................................................................... 0.5g
Preparation of Na2S·9H2O Solution: Add Na2S·9H2O to distilled/
deionized water and bring volume to 10.0mL. Sparge with N2.
Autoclave for 15 min at 15 psi pressure–121°C. Cool to 25°C. Store anaerobically.
Cysteine Solution:
Composition per 10.0mL:
L-Cysteine·HCl·H2O..................................................................... 0.5g
Preparation of Cysteine Solution: Add L-cysteine·HCl·H2O to
distilled/deionized water and bring volume to 10.0mL. Mix thoroughly.
Sparge with 100% N2. Autoclave for 15 min at 15 psi pressure–
121°C.


So, it seems you may need to raise the pH if you prepare them together

#3 SunsetSatine

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Posted 14 October 2012 - 04:14 PM

Hi! Thanks for your response. I have the solutions stored in balch tubes in 10 mL aliquots. I'm to prepare everything under my Coy Anaerobic
chamber. I'm not sure how to check the pH since the pH meter is outside the chamber. Posted Image I can't check the pH while I'm preparing either because of the same reason. Any advice?



#4 SunsetSatine

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Posted 14 October 2012 - 07:42 PM

HI!

I'm working with Anaerobic Bacteria and I add Cystine-Sulfide Solution as a reducer. For some reaon, though, it keeps precipitating and I can't figure out why. I've attached the protocol that I've been using. The solution comes out a pale yellow out of the autoclave and then it slowly starts to parcipitate. Anyone else have this problem and can share with advice? I would greatly appreciate it. Thanks!

Attached Thumbnails

  • Cystine-Sulfide Protocol.png


#5 El Crazy Xabi

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Posted 14 October 2012 - 08:15 PM

The reducing agent can be prepared outside teh chamber while it is sparged with N2. If not, you can try in the anaerobic chamber with pH strips. (I would sparge the NaOH solution, though)

#6 SunsetSatine

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Posted 15 October 2012 - 05:37 PM

Ah! Okay, I see. Thanks so much!

How do I know how much to raise the pH by? Just until I see the ppt dissolve? Since, they are in balch tubes, I'm thinking I can sparge a needle and then load with NaOH anerobically?

#7 El Crazy Xabi

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Posted 15 October 2012 - 07:57 PM

In the recipe I copy&pasted says up to pH 9.0

I haven't ever worked in anaerobiosis but if you are gonna add from a NaOH solution, I would say that is better to sparge the NaOH too, to avoid insert any air again. If you control the volume you are adding to the first stock you made, you may repeat it later without even control the pH (though it would be recommendable to measure the pH at the end as a quality control.

#8 SunsetSatine

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Posted 16 October 2012 - 01:30 PM

Okay! Thank you very much!

#9 SunsetSatine

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Posted 12 December 2012 - 07:45 AM

Would the concentration of that stock be considered 10% if I distrubuted ten mLs into tubes and then only take 0.1 mL into 9.9 mls of media?

#10 pito

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Posted 12 December 2012 - 10:06 AM

Would the concentration of that stock be considered 10% if I distrubuted ten mLs into tubes and then only take 0.1 mL into 9.9 mls of media?


if the stock is 100% and you take 0,1ml and add this into a total volume of 10ml, you diluted it not 10 times but 100 times...

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.






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