I use a 40% final concentration of glycerol. For this I mix equal amounts of bacterial culture and 80% glycerol (giving a final 40%). You should keep the cell-glycerol mixture at room temperature for half hour to allow the cells to incorporate glycerol. This is an important step as glycerol will protect the cells only if it can enter the cells. After that I flash freeze them by dipping the tube in liquid nitrogen. For later use, as Sherry said, the idea is to keep the cells frozen. So I keep a benchtop cooler at -80 C and transport the glycerol stocks in that. It keeps the cells frozen while I streak them out on the plate.

20% is absolutely okay. Do doubt about the frozen stock. Everything is fine. Just plate something to check if the bacteria are still alive.

I always use 50% Glycerol and for long term storage this vials:

http://thumbs.dreamstime.com/z/cryogenic-vials-27353364.jpg

I use a 40% final concentration of glycerol. For this I mix equal amounts of bacterial culture and 80% glycerol (giving a final 40%). You should keep the cell-glycerol mixture at room temperature for half hour to allow the cells to incorporate glycerol. This is an important step as glycerol will protect the cells only if it can enter the cells. After that I flash freeze them by dipping the tube in liquid nitrogen. For later use, as Sherry said, the idea is to keep the cells frozen. So I keep a benchtop cooler at -80 C and transport the glycerol stocks in that. It keeps the cells frozen while I streak them out on the plate.

We use 20% glycerol in whatever broth we grow the organism in (eg LB, BHI broth, etc). I am surprise that a 20% glycerol solution froze in at 4C. Glycerol is used as a cryoprotectant. We freeze the stock cultures in the 20% glycerol in broth at -80C. Remove the cryovial from the -80C and scrape some of the frozen material from the top into a broth or on a plate to revive the culture.

20% is ideal. I normally use equal volume of culture and 50% glycerol to give a final of 25%. Did you mix it well, the frozen vial might have uneven mixing of culture and glycerol or perhaps the concentration of glycerol stock that you are using is not right.

We usually use 40% glycerine in our Lab, it is very good, Try it

I use Glycerol stocks (25 %) by adding 1 mL of the culture to 1 mL of 50% glycerol and store it at -85 C

I believe 30% glycerol

We generally use 25% Glycerol as the final concentration for bacteria. For freezing fungal spores we go up to 50%.

Freeze the stocks quickly in a -80°C freezer. Do not freeze them again once thawed.

Bacteria and spores handled this way will stay viable for many years.

For our popular strains we make lots of tubes so we are able to take out one when we need it. When we run out of tubes we make a new large batch.

It is wise to keep some of your "old' tubes till you are certain that your new ones are okay. We check the new ones after a few weeks in the freezer.

According to my opinion, 20% glycerol stock is preferable for bacteria, no viability lost even for year when you freeze your glycerol stock under -80 degree temperature. Avoid freezing and thawing.

Check viability every 2 month by direct streaking freezed culture over suitable nutrient plate. This definitely works.

A part from this information, you should use active, healthy bacterial culture for example grow E.coli in L.B broth until its O.D 600 nm reach between 0.5-0.7, then use this active culture with 20% glycerol concentration

Thanks a lot guys!!!

It works.

Take Care

Where can I buy glycerol from in India?

15-25% final concentration is optimal!! I normally go for 25%

TSB with 15% Glycerol (commercially available. lots clinical micro labs use them)

https://tools.thermofisher.com/content/sfs/manuals/IFU65024.pdf

40% would be ideal for freeze storing

30% final concentration of glycerol would be ideal for freeze storing of bacterial cultures. For fungal culture, you can increase the conc. to 40%.

Regards

I have used 30% for bacteria and it worked perfectly and 40% for filamentous fungi. Best wishes.

I usually do 25%

Some bacteria can be stored at -80C in 10% glycerol e.g E. coli. usually 20-25% is good for most bacterial. 50% sterile Glycerol can be prepared in broth and then equally added to bacterial culture. This will make a final of 25% glycerol in the stock that can be frozen in -80C for a long period of time. For anaerobic bacteria. broth and glycerol must be degassed, but it is good enough if processed via autoclave. After autoclave d, the cap is closed tighly when cooled down. the bottle is opened in anaerobic chamber to ensure no air to sneak in.

I use add 60% sterile glycerol to bacterial culture and final concentration would be 50% . Vortex the culture to ensure that glycerol is evenly dispersed and immediately frozen to –80°C for long-term storage.

Amazing variation in the amounts of glycerol being used! we use to flash freeze in liquid nitrogen in 15% glycerol, but lately not had liquid nitrogen. Can they be allowed to freeze slowly to -80C and if so is it better to have higher concentration of glycerol?

Thank you

@Sharmila Jayasena.

yes you can do it without liquid nitrogen. i did it several times and it has worked. no 60% is enough

Make 30% glycerol by adding 30ul glycerol in 70 ul sterilized distill water. Autoclave at 121 Celsius for 20 mins. Then in an autoclaved screw cap eppendorf add 500ul overnight culture and 500ul 30% glycerol. Mix and incubate for 30 min.

Freeze at - 80 C for long time preservation.

I usually do 25% final concentration of glycerol, by mixing 25 ul glycerol in 75 ul of distilled water. autoclave at 120°C/20mn. in eppendorf, ad 250 ul of this préparation and 250 ul of overnight culture. conserv at -80°C.

the recommended % by the ATCC is 10%. i suggest to read the manual from which the bacteria/fungi was obtained.

From the point of physical chemistry, the eutectic point of glycerol/water mixtures is 66.7% glycerol, which freezes at -46.5 °C (doi:10.1021/ie50189a017). Thus, you can store such a mixture at -20 °C and it will not freeze, avoiding freeze/thaw damage to your sample.

The biological preferences of your bugs need not be the same, however.

Since last 15 years i am using 15% final conc. of glycerol solution and able to revive and store my bacterial culture and clones.

Can I use this method with agar cultured bacteria (already incubated overnight) , not broth ?

Are there any references that talk about the storage methods for bacterial/fungal culture ?

There is actually a lot of valid and validated info on the web. For free. e.g.

https://www.addgene.org/protocols/create-glycerol-stock/

In our lab we used to make 10% (v/v) glycerol stocks and stored the vials on -80C. This worked well and revival of cells was also easy.