Hey Farah,

I wouldn't worry about transferring the bacteria to new media containing glycerol, as most bacteria are very tough and will last a very long time at -80C if they aren't thawed and frozen man, many times. To avoid the possibility of losing bacterial stocks, I would make multiple stocks, that way you could just freeze/thaw one stock at a time whenever you need the bacteria while the others stay at a consistent -80C. So, one of two things will happen, that one stock will keep producing healthy bacterial cultures until it is depleted or it will eventually not produce viable cells; and at that point you can discard it and move onto your next stock. 

There is no specific threshold for freeze/thaw cycles as each bacterium is different, but if you make multiple stocks it should never be an issue. I know I've frozen/thawed the same vial 10+ times in the past and didn't see a decline in cell viability over time.  

Just keep in mind that bacterial stocks are extremely tough MOST of the time, so I wouldn't worry too much about losing your stocks. 

Best, 

David

Hey Farah,

I would agree with David.  Obviously different strains of bacteria fare differently in cryogenic conditions, but for the most part, bacteria in glycerol at -80C should be good for multiple years. 

Deep freezing strains for a long period of time may end up reducing the population diversity, but I am assuming your library consists of multiple clones each with a knockout/insertion/mutation.  Thus, I don't think that this particular problem should concern you.  Since it's a library, freeze-thaw cycles just to sustain the library seems unnecessary. 

Good luck!

Tanush

Dear David VanDerway and Tanush Jagdish

Thank you very much for your answers. 

Actually, I have a big metagenomic library (100.000 clones) which is used every time for screening. As I am using 384-well plates for storing, the volume for each stock is around 80uL only so every time we do the inoculation for screening the glycerol stock is melted very quickly so this is why I am bit worried. 

For more details about my strain, It is EPI300T1R from Epicentre with pCC1FOS fosmid. 

Hi,

I have stocks of Escherichia coli that are frozen at -60 to -80 since 25 years without problems. If I understand you right, you are dealing with a library. If so, in-between amplification for generation of a new stock could impact the representativeness of your library. In this case, I would certainly advise against preparing new stocks once in a while. Better, as pointed out earlier, prepare several stocks in parallel (aliquots) of your very first library.

Best, Ralf

Dear Farah,

i would lime to suggest you that to place these strains separately, and not in 96 well plate, as you have frequent work with them its better to separate the this procedure may be longer or it takes time but it is good for your stains as you are doing meta genomic studies and you should prevent bacterial death or even mutations. Another thing is that there is a short storage procedure for -20 degree with glycerol you can use it for frequent use.

all the best.   

Dear Dr Ralf Koebnik

Thanks for you answer. I am happy to hear that E.coli may resist for 25 years old so that may reassure me. So I think I have to follow your advice, Thank you. 

Dear Dr Mohammed Alsaiqali 

Thank you sir for answering. 

In my opinion due to the big number of my library (100.000) and which is increasing day after day, using 384-well plates is better than using 96 one which is also better than separate in 100.000 tubes independently. It is an issue of screening as well, because in the moment of screening it is more practical to screen 384 clones in one time rather than screen clone by clone. 

Cordially!

Hi Farah

I have some stock that were still active since15 years ago. If we need these bacteria, just put-out from the Deep Freeze, directly swab with cotton then sub-culture in Blood agar then soonly the stock tube put back to deep freeze. During sub-culture do not wait the stock in cryo-tube melt/thaw. 

My colleague from Netherlands has an experience that one of the mecA of their MRSA stocks lost after storage for years (I am forget the long of storage). [Only very small number]

Best for you

Kun

Dear prof Kuntaman Kuntaman

Thank you very much for your answer. I am using Lb agar for my E.coli, has the use of blood agar an advantage for E.coli growth? 

Cordially! 

it may depend on the bacteria you store. Anyway, so far I have not seen any unculturable stored glycerol broth, even some 80s staph isolates which were at least 30 yrs old. 

You do not need to any sub-culture for any storage in minus 80, for the next storage; You just remind that if you need the isolate sub-culture for anything else, do not wait the stock thaw for sub-culture; just directly swab the surface of freezed bacterial stock, then plating (BA).

good.....

Thank you Dr Wei Gao for you answer. 

Yes Prof Kuntaman Kuntaman, I have got your idea. Thank you 

it's better to do twice stock cultures for each isolated bacterium and duration of stoage is more than 10 years at -70c and if tou need to used it just subculture it in blood agar

 Thank you Marwa Mohamed for answering. 

If The frozer temperature  is stable, strains will be kept for many years. Some fastidious bacteria need to be sucultured time to time like: Haemophilus, Neiseeria gonorrhoae.

I agree with Marwa to do twice stock cultures especially for fastidious bacteria.

 Thank you prof Nadjia Ramdani-Bouguessa for you answer.

Cordially !

 It's better to do two subculture, so with each thawing most of the bacteria will become inactive and after taking the sample we should put the sample in a liquid nitrogen again and keep it in -80. 

 Dear Dr Srwa Ali

Thanks a lot for your answer. May I ask why should we put sample in liquid nitrogen before storage, is it to avoid thermal shock? Or there is another reason? 

Dear Farah, you can keep your samples for many years but remember to do several stocks in order to used it once after you thaw them. I have kept many of my strains for over twenty years. Good lock

Dear Teresita Sainz 

Thanks for the information. 

Very Cordially !