I used various storage methods, one that worked for "yeast like" fungi, such as the yeast form of mycosphaerella, was putting liquid suspension on silica gel (rather fine mesh), then freezing at -80. To revive the culture it is enough to sprinkle some of the silica gel on a plate with appropriate medium.

Another method for filamentous growing fungi is letting them grow over 5mm paper disk on the agar, then harvesting the disks and storing them on silica gel at -80.

I use S. cerevisiae with 15 % DMSO for long time storage at -80°C. These cultures are now 15 months old and I have no growth problems.

This method is very fast, cheap and easy.

Francesca, your method sounds interensting. Can you explain it better, with cell and silica gel quantities? I want to try it. Monica

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Preparation of frozen stocks of yeast cells

Goal: Storage of viable yeast cells (H. polymorpha, P. pastoris, S. cerevisiae) cells at –80 C

Material: 1. Sterile 2ml screw-capped vials or 2.2 ml eppendorf cups

2. Stickers

Solutions: 1. Sterile 87% glycerol

2. YPD, YND or mineral medium (MM)

Procedure:

1. Grow yeast cells overnight on YPD or on selective YND/MM medium (for plasmid-containing strains)

2. Add 400l of 87% glycerol in the cryotube containing proper label*

3. Add 600l of the overnight culture

4. Vortex the sample to mix the cells with the glycerol

5. Place the tube immediately in the -80C freezer

6. DO NOT forget to add your sample to the -80C freezer database!!

*Note: Label should contain proper identification of your sample. It is wise to wrap the label with a transparent tape. A sticker on the top may help to identify samples faster.

Inoculation: Small amounts of the frozen stock can be scraped with a loop and streaked on YPD plates. Take samples very quickly !!. The stock should not thaw !! Because of the high concentration of glycerol this occurs very quickly !!

Preparation of Penicillium rice

Method:

1. Place a single rice grain containing P. chrysogenum spores on an R-agar plate and shake the plate for 1 min.

2. Incubate the plate for 4 days at 25°C

3. Scrape off the colonies using 2 ml tap water and transfer the suspension to 12.5 g autoclaved rice, together with 4 ml autoclaved tap water.

Note:

When mycelium does not properly grow, first grow mycelium on YGG plates before transferring them to an R-agar plate.

you can store your fungi in 15 % DMSO for long time periods at -80°C.

Hi

We routinely store our fungi at -70 degrees either in 50% glycerol or in Cryoprotectant beads. Hope that helps.

It all depends on what your purpose is for the preservation of cultures. Lyophilization is of course the best way to go due to the fact that your cultures will not be so sensitive to temperature fluctuations. It can be easily kept between 4-8 C, but even room temperature could be OK. For cryopreservation you have to keep the temperature between -70-80 C, which could be risky during electricity shortages or breakdown of the freezer. The recipe for cryopreservation we normally use is glycerol (20ml), potato dextrose broth (4.8g) and distilled water (180ml). In some cases we stored fungi for longer than 10 years without losing viability. However, there are some fungi such as Fusarium that seem to be more sensitive to cryopreservation. Especially the more important cultures I would recommend to be preserved in more than one way e.g. lyophilisation, cryopreservation and maybe liquid nitrogen. The last one is prepared in exactly the same way as cryopreservation, but it is just kept in a canister with liquid nitrogen at -196 C. For the latter you do not need electricity, which is a good back up system. Hope this make sense.

Mucorales(lower fungi) can not be preserved by lyophilization rather store them in sterilized disstilled water at room temperature for long.

Just put filamentous fungi on sterilized water. Have an amount of fungi, a loop from a 7-day culture. I have in my lab cultures stored for ten years, still viable. Store at room temperature, protected from dust.

Yeast may be preserved in this way (although no all yeasts survive for long) or in criovials containing glycerol 25% on Sabouraud broth. Put criovials at -80°c immediatelly.

5% glycerol water or 10-15% DMSO can work well. It is important to control the temperature decrease to avoid the formation of crystals in the solutions, which may rupture and kill viable fungal cells. I suggest the following: 1) transfer the viable cells into cryovials with 5% glycerol or 10-15% DMSO; 2) place the vials at 4C for 20 minutes; 3) transfer the vials to -20C and stored them for 20 minutes; 4) places the vials into a Styrofoam box, close the box, and store the box with the vials at -80C. The Styrofoam will function as a temperature controller.

Dear colleagues! Thanks a lot for your useful comments. Unfortunatelly we don't have a freezer for temperature -80C. What about storage of cryoprotective tubes (with beads) at 4-7C? Wrong idea or not? What is the function of the beads in Cryoinstant tubes?

This is an excellent topic. We store yeasts and filamentous fungi for years at -80°C in a rich medium complemented with DMSO and glycerol. unfortunately some filamentous fungi (Fusarium as previously said, but also Scedosporium or Alternaria) do not survive this treatment. In our experience, beads fit well for yeast but not for the cited filamentous fungi. Liquid nitrogen may be adequate but requires special care that are not suitable everywhere. Any alternative validated procedure is welcomed

Usually in our lab store fungi and yeasts at three different temperatures 4 ° C, -20 ° C and -80 ° C. For storage at 4 ° C use 2 ml tubes containing mycelium slant covered with a layer of sterile glycerol to prevent dehydration of the agar (viavility time of 10 years depending on the strain). For storage at -20 ° C use a final concentration of 20% glycerol (you can freeze portions of agar containing mycelium, spores or cells in suspension), to -80 ° C the procedure is similar but used 50% glycerol.

In our laboratory store fungi for tree ways: a) on sterilized water b) spore in paper c)5% glycerol water or 12% DMSO. All in vials into a Styrofoam box,and store the box with the vials at 4 ° C, -20.

Very useful comments again. But still is Cryoinstant tubes suitable for storage at 4C or 20C? Does anyone have experience of using Vacuum Tubes? I guess vacuum should prevent oxidation and thus extend the term of storage.

I agree with the paraffin oil method. It just take a while for the fungus to grow when you subculture it from the paraffin, but has proven to be a relatively cheap and easy option. It seems like people do not know whether Cryoinstant tubes could work at 4C or 20C. I would suggest to do a small experiment to see if it will work before rolling it out on a big scale. Revive the cultures after 1 week, 1 month and 6 months respectively and then take a decision. You can then share your results with us as this could be of great benefit to all of us. In the mean time, I suggest to keep at methods that have proven to be working as you cannot take chances with a culture collection. If you lose the viability of your cultures you are never going to get it back......it is a gamble.

We use 25-50% Glycerol in cryo-tubes stored at -20 or -80. I have also stored fungi in a "regular" freezer like this but have not tested that over 5years.

Well, lets make an experiment. I began to collect the cultures both in Cryoinstant and in Vacuette at 5C and store them in regular freezer. Six months later I'll check the viability and report the results.

We use lyophilization for yeast and spore forming fungi while fungi in vegetative mycelial forms give better preservation results in 25-50% Glycerol in cryo-tubes stored at -20 or -80° C.

Thank you, Ghanshyam! What is the longest term of storage in cryo-tubes at -20C?

I use glycerol at 10% in cryo-tubes at -80°C. By this way the fungi are stable during several years.

Thank you, Adelaide! We still have no freezer for -80 ((

You can use -20°C.

It would be useful to check other parameters in addition to viability. What about enzyme and metabolite production? Has the morphology chaged? Ultimately, what has happened to the DNA? All these need checking...