Hi Martin,

I do agree with the previous opinions that your medium is extremely rich. Yeast extract, formate, acetate, sludge fluid and fatty acid mix all together is more than your organisms probably need for growth. Our M. hungatei e.g. grows on inorganic medium with only 0,1 % acetate supplement. The more organic you use, the riskier it is to get issues with cross-contamination. Especially yeast extract is a dangerous source concerning spores, therefore your idea on a contamination with spore-formers like Clostridia is justified. Also the acidification of your medium hints in that direction.

Hi, Martin,

I did not get a question... What I could see was that you have a grown culture of Methanospirillum with some probably bacterial cells = contamination?

Indeed. The bacteria must be resistant to vankomycin. The question was, if somebody does not know a common contaminant or wasnt familiar with something similar to this. I am unable to run PCR or sequenate the samples due to financies (I am just a bachelor degree student) so I am in dead end. I will probably try the agar media but I am sceptic that it will help since the growth rates are really low.

1) If you want to get rid of bacteria you may try also chloramphenicol !

2) You need to come through your medium preparation protocol - to see where you re getting a contamination:

A. It could be during medium preparation - you need to have a control bottle - uninoculated!

B. Methanospirillum culture is contaminated.

Remark. You are using quite "rich" medium - you have to be very precise in your work with medium and cultures.

First of all, thank you very much for your help and suggestions:

Ad 1) Chloramfenicol is affecting methanogens in such way, that I am unable to use it. That was a minor experiment during assessing the proper antibiotics for purification.

Ad 2) That was the first what I did. I even started writing special laboratory book, just for this case. But nothing really helped and I weren´t able to find the error, even with the help of my associate professor.

I had some hot spot, such as a cylinder that might not be sterilized properly, or for example glove/hand during manipulation. But I tried many ways how to change it. I even prepared the medium completely in anaerobic chamber, but the effect was still same.

I had an idea, that it might be done by some sporulating bacteria like Clostridia, but I couldn´t find any, or perhaps something from the Ruminal fluid, even if it was clarified and sterilized.

I will asses another round of sterility test. As well, the medium itself appear to be sterile.

Thank you very much.

Dear Martin Cerny,

What about the gas mixture you use for preparing anaerobic conditions? By instruction from DSMZ " Sparge medium with 80% H2 and 20% CO2 gas mixture for 30 – 45 min to make it anoxic." Do you use any bacteriostatic filter in the gas system?

Also, some bacterial spores can survive even high temperatures of autoclaving, e.g. doi:10.1099/00207713-50-5-1741. Therefore a control bottle - uninoculated, only with sterilized media as proposed earlier would be of great help.

With best wishes,

Anne Menert.

Dear Anna Menert,

Because of safety reasons, we are unable to work with the gas micture with such high percentage of hydrogen. So I use 80:20 N2/CO2 instead, and I finalize it with sparging by 5% H2 and 95% CO2 which is lately used for a cultivation.

I will definitely talk with my supervisor about the possible use of bacterial filter for the gas dispension, as well for a oxygen scrubber.

Thank you for the paper, I wil try to read it to get as much form it as possible.

Have a nice day,

Martin Černý

Hi Martin,

I do agree with the previous opinions that your medium is extremely rich. Yeast extract, formate, acetate, sludge fluid and fatty acid mix all together is more than your organisms probably need for growth. Our M. hungatei e.g. grows on inorganic medium with only 0,1 % acetate supplement. The more organic you use, the riskier it is to get issues with cross-contamination. Especially yeast extract is a dangerous source concerning spores, therefore your idea on a contamination with spore-formers like Clostridia is justified. Also the acidification of your medium hints in that direction.

Are you trying to culture a specific species? I agree with Anne that the methanogen likely requires 80% H2/ 20% CO2 in the headspace. In my graduate work, we cultivated four species using 80/20 H2/CO2. Some methanogens can use acetate or methanol instead. The methanogen also may need warmer temperatures - many are at least mesophiles.

As for contamination, when in your process are you autoclaving? Previously, I have made media on the lab bench, put into an anaerobic glove box to deoxygenate, poured into bottles and sealed the bottles. Then I remove the bottles from the chamber and autoclave them. Then I add methanogens to the medium and pressurize with 2 bar H2.

Hi Martin,

I see that you are very thoroughly working and thinking!

One point for the anoxic hood. In my very long experience of working with anoxic hoods I have never met "sterile hood". From another side I have known the very many people facing contamination of various anoxic cultures. That was just because they poured plates or dispensed medium or just open media bottles in anoxic hoods.

I hope that your protocol does not have such steps!

When we were working on a lab bench with opening "anoxic" bottles we sparge the bottle with gases we using "gassing syringe" for filter gas sterilisation . Such a syringe is described in

Article Starting Up Microbial Enhanced Oil Recovery

Have a good day, I am sorry that I didn´t respond for so long period of time.

I have managed to track the source of this contamination of failed L-cystein solution which was dispensed to various media as a stock solution. Via anaerobic test and sporulation test I was able to obtain samples, from which I isolated DNA and which I was observing under microscope. Indeed, the contamination likely rose from laboratory environment and is a mixture of sporulating rods.

Dear Linda Dengler,

I tried to change the media and hopefully I was able to retrieve culture of M. hungatei. I have used medium from Anna Kraft that our department obtained as a help from your university. Thank you very much for your help. I wanted to ask, if I could contact you for further information about your university, if you don´t mind.

Dear Rebecka L. Mickol,

I am dispensing prepared media on the laboratory table, because I am unable to use dispenser, which I would be able to bring into the anaerobic hood. The media I prepare contain volatile are heat-unstable compounds, such as sodium bicarbonate, cystein, many vitamins, etc. (The medium for Methanobrevibacter smithii). I managed to reduce my media on organic, such as Yeast extract, and they are currently working as well.

After a preparation of basal medium, I boil on the stove and during the process I gas it by mixture of CO2/N2. After this, I autoclave it, the I add resazurin and volatile compounds via syringes and check the pH and redox potential of the media. Then I dispense it and gas it by H2/CO2, however I am not able to use the desired 80/20 gas mixture, so I have only 5/95.

Dear Alexander Galushko,

Thank you very much, I realized that we are using the same device, as you have described in the paper. Also, it was really useful for cross-check, because we use a similar trace elements and vitamin solution stocks.

I also spoke with my laboratory manager, and on his behalf I wanted to ask, what is a mean value of real humidity in the anaerobic hood. Just for orientation.

I will update results of my contamination identification, if somebody was interested, what may occur in anaerobic cultivation of methanogens.

Thank you all very much for you help and valuable advises.

Dear Martin, I am happy that you could solve the problems. Of course you are very welcome to contact me personally for further information.