Hi Cristina,

It is a really usefully and easy technique. I am trying to keep it simple - after O.N. cultivation, I put 0.7 ml of the culture in new sterile 1.5 ml tube and add 0.3ml sterile glycerol (brief vortexing). It works and still I do not have problems with my cells. Wish You the same!

P.S. look here also

https://www.researchgate.net/post/Concentration_of_glycerol_used_for_storage_of_bacteria

Dear Cristina,

the conservation success depends upon the nature and viability of the bacterial cells, mostly. If you aim to store for a long time (>6 months), at -20, most of the bacteria will survive. I take a few colonies and resuspend them in the cryo-beads broth, centrifuge at 2000-3000 rpm, take out the maximum amount of glycerol broth (its composition is provided by the manufacturer, you do not have to make it, but it is generally 20-25% glycerol in tripticase soy broth) and store. The aim is to reduce the metabolism, the oxygen needs of the organism and to make the adhere to the surface of the beads.

Check your cultures periodically.

Success!

Hi Cristina,

Interesting question. I have never thought of replacing the culture media before storage and it makes complete sense what you are saying. However, centrifuging and resuspending large bacterial stocks will be very laborious and tedious.

With my method, I place my bacterial stock directly form it's culture media into a cryo vial containing glycerol (final concentration of 20 % v/v). I vortex before storing. I have been able to maintain my cells at - 80 degrees for up to year.

Good luck,

Regards.

No no replacement of the broth culture is needed. Add the final 20% glycerol to the fermented borth. Stir briefly and let it stand to equilibrate for 20-40 min ...before freezing.

Always best to grow in liquid as colonies on plates are often made up of a number of dead cells. Use a liquid culture that is going through early to mid exponential phase. Some of your cells will die in the thawing process and some will die during the freezing, therefore you want them cells to be as fresh as possible prior to long term storage. Sorry if this is a repeat of other advice. All the best

Hi, as other said, I simply mix the saturated overnight culture with a sterile 60% glycerol solution in a 2:1 ration, ending with final concentration of 20%. Doing this, my bacteria survived for >20 years (E.coli) and >12 years (Xanthomonas) at -80 degree-Celsius and they even survived moving from one country to another one in Europe (i.e. thawing and re-freezing).

Cheers!

NO i do not centrifuge the cell and replace it with new broth. I have a stock of 50% glycerol in LB ready. So when ever i need to make a glycerol stock i usually get an aliquot of the cell in log phase (A595 = 0.6 - 0.8) and add equal amount of the sterile 50% glycerol stock LB media and freeze it in liquid nitrogen. By this i ensure that some new LB is added as well as the final concentration of glycerol is 25%.

The advantage of having a higher glycerol concentration, as described in Regina-Michaela's answer, is that the culture remains at least semi-liquid at –20°C. This can make getting a sample a lot more rapid, which will aid preserving the stock. However, high glycerol concentrations in general are not particularly liked at least by E. coli, so it's a bit of a trade-off. I agree that they are certainly usable for at least 5 years, which depends on the genotype as well.

I have used freshly grown from plate as well as broth. in case of broth final concentration of more than 15 % was good without changing media or without any centrifugation, and had -20 for storage. Plates are also good for technique.

When I store non-spore formers I use a fresh sample of the same broth in log phase, but never centrifuge it, just vortex. When storing spore-formers I grow the strains in a suitable solid medium and check microscopically. When more than 80% of the population sporulate I make the stocks in the appropriate medium plus glycerol 20% final concentration. When storing at - 80C my bacteria survive at least 10 years.

It is always good to go with broth for glycerol stock preparation. Its better to make glycerol stock from mid exponential phase cells with 20% of glycerol. Mix well and then can keep it in -80 degree. This should work well as many of researchers said already.

First of all do not replace the culture broth with fresh medium. Discard the culture broth and suspend the organism in an inert medium like skimmed milk or glycerol solution. This is maintenance of the culture and not growing the culture, where you would like to retain the properties of the culture and at the same time do not want to kill it. Atleast this is the way I sometime preserve the cultures.

I think it is not necessary to replace the culture broth by fresh medium. You will put fresh medium when you re-growth the bacteria after frezze. I only centrifugate bacteria when need a very highg concentreation of bacteria to ensure the survival after the process as is the case of cyanobacteira. In this case a need a culture of 50 ml, Then centrifugate it to obtain a final volume of 1 ml resuspended in 15 % DMSO. In al the other cases I add concentrated glicerol directly over an overnight culture to a final concentrationof 15-20 % as explain the other comments

For species which grow well, grow bacterial culture overnight, obviously longer for slow growers. Dilute c. 1/4 with fresh medium (pre-warmed) next morning, grow another couple of hours, use directly to make glycerols. Add 1/3 volume of sterile 60% glycerol, mix, freeze and store in liquid nitrogen. Putting glycerols directly to -80C also works for standard E. coli strains.

Bacteria are often robust and E. coli will usually survive as a glycerol if prepared from liquid culture at any stage, even if stationary for days. (Although you run the risk of selecting for adaptive mutants if the culture has been excessively stressed in any way).

I do both. If the density of cells in the culture is good enough to make a stock with a lot of cells, I simply add 50% glycerol to make 20% stock. If the culture gives less numbers of cells, I usually collect cells from 5 mL culture and resuspend in 1-2 mL 20% glycerol. Both work just fine. Good luck.

I usual use bacterial culture at log phage (A600

I have made bacterial stocks in two ways. For E. coli cultures that our lab uses to insert cloned amplicons from the environment, we grow up our cultures in a LB + glycerol freezing media (7% glycerol) overnight and then directly freeze them. I have also grown up cultures on agar plates and resuspended pure cultures in the equivalent broth medium (minus agar) with 20% glycerol added.

I'm not sure if I would recommend centrifugation in case you destroy any of the cells. In my mind, once you revive a glycerol stock you will inoculate it into fresh media anyways, so you shouldn't worry about losing any nutrients after it has grown overnight. There's also a chance you could contaminate your stock when adding fresh media, so it's best to just let it grow overnight and freeze it.

I agree with Rongfeng Li. Before storing at -20oC or at -80oC, hand shaking or mix by a spectrafuge mini is good enough.

Vo Thi Chi Mai

University of Medicine and Pharmacy in Ho Chi Minh City

Hi Cristina,

For B. subtilis, I use over night culture, centrifuge to obtain biomass then add glycerol 20%, mixe, transfere to cryotube, and let standing at RT for about 20min before congel. For yeast, I put directly glycerol 15% on 48h plate. I find that higer concentration of glycerol change my yeast's morphology when cultured in plate, but still good if cultured in liquid. Hope that this is useful for you.

Hi, I always use bacteria from agar plate to avoid an accidental contamination. 15% glycerol is fine. Anyway, you can find optimal method for one model strain, but when you work with collection of isolates form different species or genera it is almost impossible to find a method equally good for every bactera.

Thank you everyone for your helpful answers!!