Anything over 10% should be ok. The point of the glycerol is prevent freezing damage, such as ice crystal formation.

At lower glycerol concentrations like 10-20% the frozen stock will be fairly solid at -80C, while at higher concentrations (30-50%) it may remain partially liquid. Either is fine but over time (years) the cells will settle down at bottom if still liquid.

use the 30% glycerol, add fresh culture to final concentration, mixed well and freeze quickly by liquid N2 and then keep it in -80 freezer.

Be careful of contamination and keep it more than 1 year.

Good luck

I use 20ml of glycerol and 30ml of bacterial culture for working seed store at -80°C

Glycerol by volume/volume

You can use 5ml of 88% glycerol and 5 ml of bacterial culture, and preferably freeze quickly by liquid nitrogen and transfer to -80 freezer.

I think the combination of glycerol and DM water (containing washed bacterial cells) mixed together to decrease the freezing point than alone use of glycerol (Its freezing-point depression). In that case 20% - 40 % v/v glycerol may be enough to lower freezing point of stock to store. Needlessly more glycerol can be used but solution become more viscous may create problems in pipetting. In my case I have used 20% glycerol to prepare bacterial stocks.

I use 15% glycerol and -80°C storage, directly on overnight culture (w/o spinning).

I recently had to grow bacteria from 1997 (frozen this way, 16 years ago), and they grew fine!

I always scrape the surface of the frozen stock and seed on plate or medium, so that the stock lasts longer (never thawed).

15% and v/v

I use 20-30% glycerol in TSB and -80°C storage. The bacteria come from a ON culture on TSA plates.

600 µl medium and 400 µl glycerol (80%, v/v), finally consantration 32%.

20% glycerol (v/v) is good for short term storage in -20 or -80 but 50% glycerol is better for long term storage at -80oC.

Hi, a good way to do it is to make a mix of your media and add 40% glycerol and autoclave for sterilisation. You can then pellet your cells and add 300 microlitres of this 40% glycerol and 300 microlitres of fresh medium to create a final concentration of 20% glycerol. This work well for my bugs (marine bacteria and E. coli). Hope this helps.

As you can see, everything works just fine, so throw some glycerol on your bacteria and freeze them ;)

Hi Sophia, generally people use or 50% Bacteria and 50% glycerol or 70% bacteria and 30% glycerol. Good Luck.

Based on my experience, -20 is not good to preserve bacterial and fungal cultures in 15-20% glycerol because I have lost clones that kept in -20 after few months. -80 is much safer

I use 20% works fine. However, glycerol is very viscous so I usuall make a 50% stock and filter sterlise.

I always use 25% glycerol (v/v).

I simply add 800 ul of stationary phase (overnight) bacterial culture to 800 ul sterile 50% glycerol. Snap-freeze in liquid nitrogen and store at -80C. I have known people to keep their glycerol stocks at -20C but I prefer to trust -80C.

Recover bacteria by scooping out a small chunk of frozen stock with a pipette tip and using that to inoculate a liquid culture.

Hi! I use 20% glycerol concentration to conserve reference bacteria and bacteriophages and it's working (at -80°C). Of course you have to sterilize glycerol before using it and mix it with bacteria culture. The problem is when you have transformed bacteria then you have to check your stock once a while. Good luck!

Hi! I use 50% glycerol (500 uL of cell culture and 500 uL of sterile glycerol). I put it directely to -80°C and when I need it to start a new culture, it works!

I made a 60% glycerol solution sterile and then i mix 0.5ml of this glycerol with 1ml of my bacteria culture. I keep them few years at -80C.

I honestly suggest you should read at least one of "现代分子生物学实验技术，卢圣栋，中国协和医科大学出版社" or other similar books. I visited your city, which is a very nice place. Regards, JB.

hi sophia.. in my lab, we are using 25% v/v and directly put in -80°C. you may go for storing multiple vials just to avoid any discomfort..

My lab uses 25% sterile Glycerol

Based on the my own research experiences, 10 to 15% glycerin used for marine actinomcetes, at the same time, when i have stored in marine bacterial strains is 15% v/v in deep freezer (4C), Ok good luck

We use 40% (v/v) glycerol (stored in 4°C) and it works perfectly. Generally, I make stock by mixing 500 uL of 40% glycerol with 500 uL of overnight bacterial culture in a pre-chilled eppendorf tube on ice, and then immediately store it at -50°C. But, as many people shared their experience, -80°C is preferred compared to -50°C. To increase the viability, it is advised to not to thaw the vial; but simply scrape the frozen surface of the culture to make a spread plate. To ensure any loss and unwanted tedious effort you can make multiple stocks of any important bacteria/ clone(s).

I think 20% ethylene glycol-propylene glycol (1:1) v/v is ideal solution for you problem, that allows preservation in freezing temperatures for a prolonged period of time at -20 C for at least 2 month.

i generally use 150 µl of sterile glycerol (40%) and transfer 850 µl of the bacterial culture. if to store in -80°C freezer, you should first snap-freeze the bacterial stock by dropping it in a container of liquid nitrogen or if in -20°C freezer, the bacterial stock can be placed there with no further treatment.

We switched over from glycerol to DMSO for bacterial stocks a while ago. e testet it side by side for a while and have discovered no differences.

DMSO is easier to pipett and there is no need to autoclave it.

To generate bacterial stocks with it, add 75ul of DMSO to 1ml of culture in an appropriate tube. Close the tube, mix well and put it straight into the -80.

Hi, I have always used 35% (v/v) glycerol for lactic acid bacteria and it worked perfectly. Make sure to vortex the suspension very well before storage at -80C. In order to avoid thawing of the stock culture every time you want to plate the cells, you can use trays for eppendorf tubes that can hold temperature to -20 C.

Hope this helps,

Alice

It depends on the cell strains you used. For example, in the case of the BL21 and its derivatives (commonly used in recombinant protein expression), the manufacturers suggest a lower glycerol concentration for storage of the cells.

Since glycerol concentrations (> 10%) may lead to plasmid instability.

I transfert 1ml of fresh bacterial culture in a cryotube and add 1ml of a sterile (by filtration) solution of 50% glycérol (the final concentration of glycerol is 25%). Then I stock diretcly in a -80°C freezer.

We are using 80 % (v/v) sterile glycerol. Stock is prepared in the following way: to 0.5 mL of cell culture we add 0.5 mL of sterile 80 % glycerol, vortex immediately to evenly distribute cells and put to -80 C fridge.

When you need to use them, just touch the frozen stock with a tip and seed to the plate or culture. Do not thaw the stock.

I must warn you though, that if you need to use bacteria to express proteins at high rate (for purification), never use glycerol stock, because the yield of protein may drop dramatically. Instead, use freshly transformed bacteria.

I have always been storing E.coli strains at -80 degree C at 10% final glycerol concentration (v/v). Briefly, 750 ul of overnight grown E.coli cultures were quickly mixed with 250 ul of 40% sterile glycerol (pre-aliquoted) and kept on ice-bath until quickly I placed the tubes at -80 degree C. This protocol has been successfully used in labs ( that I know ) for about 20 years now.

First, I usually add glycerol to fresh liquid media 1:1 (v/v) then mix. This reduces the viscosity of the glycerol and provides fresh nutrients. I grow the bacteria to log-phase then add a volume of cells to the glycerol/media 1:1 (v/v) giving a final concentration of 50% cells: 25% fresh media; 25% glycerol. It is best to snap freeze in liquid nitrogen but a lot of cells will survive the slow freeze by adding to a freezer if liquid nitrogen is unavailable.

The beauty of using glycerol is that it is not that important how much you use, as long as it is enough. I always used a pasteur pipette for adding sterile glycerol: a few drops in an eppendorf tube for instance. You can judge by eye to have at least 10%. The glycerol prevents the forming of ice needles and you have to give the cells a few minutes to take it up. If submerged in glycerol it is possible to slowly freeze in stead of snap freezing.

Anything over 10% should be ok. The point of the glycerol is prevent freezing damage, such as ice crystal formation.

At lower glycerol concentrations like 10-20% the frozen stock will be fairly solid at -80C, while at higher concentrations (30-50%) it may remain partially liquid. Either is fine but over time (years) the cells will settle down at bottom if still liquid.

When it was working with E.coli BL-21, to preserve them to-80ºC it was using glycerol. The final concentration that I still had was a third, 33 %.

I used 20% glycerol, and I put the bacteria culture a -80°C freezer. The glycerol solution mut be well dispersed.

For Bl21 bacteria, we are using 60% glycerol for -80°C.

In our lab, we use bacteria in LB medium : 80% glycerol=3:1(V/V), so the final concentration of glycerol is 20%. After mixing them thoroughly, put them into a fridge at-80, I didn't met with pellet formation

We routinely conserve bacteria in 20% glycerol (v/v) at -80°C and put the samples from RT directly in the -80°C fridge. This way we keep samples for several years without noticeable bacterial decay if the tubes have not been used too much for bacteria samplings. Tht is one of the reasons why we have our bacterial collection in duplicate and prepare additional samples for strains that are used very often. Concerning bacterial pellets, we didn't notice this problem except when the samples have not been adequately homogeneized.

Are these transformed or competent cells? Sometimes there are specified concentration of glycerol based on the particular strain of bacteria, so i would check up on this. However in our lab we would grow a 5ml overnight culture (either LB or 2x YT media), take 400ul of this, add 600ul of sterile 50% glycerol (final conc. of 30%) and store at -80C immediately (ensure that the solution is throughrouly mixed - best to pipette rather than vortex). It is best to store them at -80C for a week before placing them in nitrogen. Or you can flash freeze the culture in dry ice or in liquid nitrogen, and then transfer them to -80. With this i have glycerol stocks that are years old and retain a high transformation efficiency

We have been using for 20 years glycerol 30-70%. Routinely we mix thoroughly 500 ul bacterial culture with 5-10 drops (by Pasteur pipette) of sterile 100% glycerol (autoclaved). The key is the vortexing after that , since it has to end up in a homogeneous solution, no rests of unmixed glycerol. Then, we put the tubes at -80C (you can even storage at -20C without problems after that). We haven’t seen bacterial pellets. I agree with all of the colleagues that advice the scratching method to take a sample of the stock.

Good luck!

i do 3x volume... it works best for me

In our Lab with most of the bacteria cell I use to preserve it at the 20% sterilized glycerol and preserve at -20 degree celcious.

I have used 20% stril glyserol (autoclaved) in fresh LB medium (for example: 4 ml of bacteri in 1ml of stril glyserol) then storage in -80C

You should use final concentration between 20-30% of sterile glycerol. Hopefully you wouldn't find any crytal pellets at -80C.

20% glycerol (of course autoclaved) in sterile LB media (or any special media that you might be using) is more than enough..good luck...and store at -80..:)

20% Glycerol would be the best for -20 storage. Add 200ul of sterile glycerol to 800ul of your broth culture and store it at -20.

In our lab, we make 50 % glycerol, and for making glycerol stock we use 500ul of bacterial culture and 500ul of 50% glycerol. Final conc will be 25% glycerol. Dip in liq nitrogen and store at -80 degree.

you should add 500 ul of glycerol to 2ml screwcap tubes and autoclave it. then add to it 500 ul of your broth culture and vortex it. then keep it in cryo box at -80 C. It will surely work. tried and tested.

best of luck in your research.

in the diagnostic lab I am in right now, we use skim milk for storage from -20 to -80c range.

From the textbook 10th edition, manual of clinical microbiology ASM press:

gram negative bacterial: freezing or ultra freeze,:use sucrose, lactose, or glycerol

hope this help

30% for stocks (long term), 10% for competent cells

50% of glycerol fonal conc. alows to pick a bit of the culture without thawing the vial. They last forever.

150 uL glyserol in 800 uL fresh bateria-LB medium then storage in -80C

There are two issues that you should consider. First, the cryoprotectant (e.g., glycerol) and second, the state of the culture you are attempting to preserve. The best way to freeze a culture for intermediate to long term storage is to prepare an overnight broth culture and to dilute 1:1 with sterile glycerol (20% to yield 10% final concentration, v/v).

Another point you should also take into consideration is the storage container. While there are a number of different vials, the most reliable are borosilicate glass (e.g. 10 dram) with lined screw caps that cover the opening rather than plug-type vials. This is especially true when preserving in LN2.

An alternative approach to freezing is to cut plugs from agar plates and preserve the plugs in 10% glycerol. The culture should be freshly prepared and in good health at the time plugs are cut.

I use in routine 30% vol/vol sterile gycerol or DMSO, and after vorting, samples are frozen in liquid nitrogen and stored in -80. I also avoid to thaw the sample and streak some "iced bacteria" directely on the appropriate plate+antibio.

%15 for bacteria, %50 for Candida species....

In my lab use the cryo-instant. Very easy and can stored in refrigerator but only for a month

For Freezing bacteria generally I prepare a solution of 50% glycerol and add in 1:1 with media for my stocks.

For BL21, BL21star and BL21 RIPL cells I have actually been able to freeze these as well. I will transform the plasmid into the BL21 RIPL cell (typically now), pick a single colony for an overnight culture. The next day I will do a large scale over-expression of my protein for 4hours. Then take 1mL of the overnight culture and add 1mL of 50% glycerol for a frozen stock. I can prep a couple tubes this way. Then the next time I need to express, I will use the frozen stock (all of the 2mL) for the overnight. This works very well and can save a lot of time. These frozen stocks have worked well even after 5 years frozen in -80C.

For Yeast you freeze in final concentration of 15% glycerol.

I also wanted to add that the 2mL tubes (1mL media:1mL 50%glycerol) I add directly to -80C freezer. I used to first freeze in Ethanol with dry ice or liquid nitrogen, but I observed no difference. So I skip that step. I says that this works for me with my RIPL cells and my pET2a expression constructs. Others mileage may vary.

Normally I also use 50% Glycerol and add it 1:1 to my cultures. After mixing them thoroughly they go immediately to -80°C. For recovery just scratch some culture from the surface and inoculate your fresh media. Alternatively you can also grow the cultures in medium that contains 8% glycerol. They are not as happily growing then without the glycerol but the recovery rate is - at least for E.coli - fine.

I use 20-80 % sterile glycerol and mix it 1:1 (v/v) with bacteria for gluconobacter sp. And keep it at -20 ( or first generation cultures after lyophilized form at -80 )

500uL of 100% glycerol with 500uL of LB broth+bacteria.

Glycerol storage of bacterial cultures

1. To 0.85 ml [3 ml] of bacterial culture, add 0.15 ml [0.53 ml] of sterile glycerol (sterilized by autoclave, final glycerol concentration : 15%, v/v).

2. Vortex the culture to ensure that the glycerol is evenly dispersed.

3. Transfer the culture to a labeled storage tube equipped with a screw cap.

4. Freeze the culture in dry ice or in liquid nitrogen, and then transfer the tube to –70 C for long-term storage.

* To recover the bacteria :

1) Scrape the frozen surface of the culture with a sterile inoculating loop.

2) Immediately streak the bacteria that adhere to the loop onto the surface of an LB agar plate containing the appropriate antibiotics.

3) Return the frozen culture to storage at –70 C.

4) Incubate the plate overnight at 37 C.

We normally use 30% glycerol and store at -80.

I often use 10% glycerol, 5% horse serum in Brain heart in culture media for transformed E.coli BL21 and XL1 blue with horse and freezing at-86°C.

For BL21 we usually add 15% of glycerol to the overnight culture grown on LB-medium without sodium chloride and antibiotics. Then the cells could be stored at -70C without significant loss of viability for at least half of year. .

I use this for all my bacterial cultures:pick a single colony and grow a liquid culture to mid-log phase, harvest cells by low-speed centrifugation and resuspend cells in 1/10 volume fresh medium. Add 0.6 ml of this to 0.9 ml of sterile 20% glycerol (v/v) and freeze in dry ice/ethanol, then store at -80C. Liquid nitrogen may cause tubes to blow up, dependent on what quality is used. I have some cultures in storage since 1994 without loss of viability.

15% glycerol (i.e. 150 microliters sterile autoclaved glycerol and 850 microliters of bacterial culture) works fine. Mix by vortexing before snap-freezing in liquid nitrogen. Store at -80°C.

Ni Hao Sophia,

I follow the same procedures also Lars Petrasovits follows... But I lose, because my bacteria are safe and sound (though a little bit cold, at -80°C, of course!) only since 2003...

250microlitreglycerol(80%)+750microlitre broth culture OR, 500microlitreglycerol(50%)+500microlitre broth culture can be stored in -80 after vortexing if liq. N2 is not available. but availability of liq.N2 definitely increases the cell viability as liq.N2 instatntly freezes the viable cells.

Use only a 80% glycerol because its only a prevent the bacterial culture for long day preservation.

I have been in some different labs. Between 15 to 50 %. Usually I used and it works, 15% of glicerol with bacteria broth medium. You can made 30% glicerol and 2x concentrated medium, autoclave it, after mixed and store your bacteria! And keep between -65 to -80ºC. Enjoy it!

The usual concentration is 15% v/v, but for some species (i.e. Campylobacter spp.) I foundi that 10% (and N2 freezing) is better.

For E. coli, I usually use 250uL of sterile glycerol and 750uL of bacteria in medium LB (1mL final volume), after overnight incubation at 37C degrees. Mix the cryotube by vortex, make a fast freezing and keep in -80C or N2.... It works better...

My experience is: If -80 ºC freezing is available, 10% glicerol will do fine, however if you have only say, normal freezing ( -30 ºC) it is better use 20% glicerol as this may work better to keep a higher viability of your stock culture (ive seen this in different laboratories).

To recover the bacteria, scrapping the surface of your stock culture and seed in the appropriate medium is better to maintain a high viability of your stock.

Sophia,

In my experience, Michael Benedik suggestion above is a very good one. It is not that complicated. I would prefer the concentration around 15% - 25%, because the stock is solid yet still fairly easy to work with and can be kept for many years. Simply mix the glycerol in and directly put in a -80C freezer would be fine. For long term storage temperature above -80C is not good.

Hi Sophia, I worked with 15% to 25% glycerol conenration for freezing bacteria at -80oC, and it was fine. I never used liq N2. If you dont have- dont worry about that. as I was advised in the, before taking to -80oC, I used to leave the stockes at RT for 30 -50 min. Always make stocks of fresh, linear growing phase of bacteria. I alwyas make Glycerol 50% as w/v . Just weigh glycerol and add water to make 50% and autoclave it. And mix 1:1 ratio glycerol and bac culture for frozen stocks as Madan kumar is saying. It worked for me, and hope is going to work for you too. Good Luck!

Sophia,

Above 50% glycerol stock is all fine for long storage. I am not in agreement with Yanglong that -80 C is not good for storage. I generally store in -80C if I want to store it for long time and it working very fine for me.

We have had good viability after storage of many (enteric) bacterial cultures (including Campylobacter jejuni) in 15 - 25% glycerol for 10 years or more. Some other bacteria (Campylobacter other than C. jejuni, rarer Aeromonas) do not remain viable after freezing. You need to determine this empirically. In addition, we have found that medium plus glycerol thaws quite quickly and that, when some cultures of bacteria stored in medium with glycerol thaw completely and are subsequently refrozen, viability can be lost. This is especially relevant when recovering a number of cultures from the same freezer box at a time. The beads used for freezing can help, as can the use of 20% skim milk for freezing. Unfortunately there are benefits and drawbacks to each method of freezing and no method seems to work well for everything.

Sophia, I think you have a habit of throwing up your hands, when you can not come to a conclusion. Now coming to the question of glycerol concentration, you have to find out the right concentration and the factors that affect this are very simple ones: first of all what is the bacteria; secondly, what are your objective (specific) for storage in glycerol; next is the medium conditions and cultural conditions that you wish to have and finally the characteristics of the bacteria that you want to preserve. As pointed out by Micheal, start with 10% and go up the concentration scale till you have optimized it for your need. Good luck.

I use 25% glycerol for storing my cultures in -80 and it works fine.

15% vol/vol is what I use for storage at -80C. To streamline the procedures, I make 100 ml of 30% glycerol / 70% LB mix once, pipet 500 microliters in two hundred screw capped cryotubes and store those in -80C, ready for use. When I want to make a glycerol stock of a new construct, I always streak a selective plate and after overnight incubation I inoculate a 2mL liquid culture with a single colony, and when the culture is reasonably turbid (generally just after lunch), I just pipet 500 microlitres into one of those tubes with 500 microlitres of 30% glycerol waiting (after melting of course), in duplicate. One tubes goes into permanent stock never to be touched, the other goes into the working stock (which is taken out regularly for streaking if people need to work with the construct). It pays off, nobody ever has an excuse not to make a glycerol stock, because the 30% glycerol tubes are ready waiting in the -80C freezer.

:)

I also use 25% sterile glycerol for storing cultures at -80oC and have good results. Just add an equal volume of 50% glycerol to your culture to make it easy.

15-25% of glycerol works well for storing most bacteria at -80 .

An easy way to do this would be to mix 1 ml broth culture of the bacteria of interest to 50:50 TSB:Glycerol; invert the cryotube to mix the contents thoroughly and freeze immediately at -80. This method works for a wide range of bacteria, but there are always some that need optimization.

Frequent freezing and thawing is not good for the viability of frozen cultures; so you need to keep the boxes on dry ice when you take them out and scrape out just enough to plate.

Just wanted to add that you can make the sterile 50:50 TSB:Glycerol aliquots in autoclavable cryotubes ahead of time.