Uncontrolled stress test: basidiomycete mycelium in –80 °C perlite cryotubes
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Homolka's perlite protocol12 is a clean and simple method to store basidiomycetes. This paper describes my implementation of his soaked perlite method and its uncontrolled revival.

Please note that during this experiment, the –80 °C freezer experienced random failure for some months. Then an unknown person moved the samples to –20 °C where they stayed several more months. Finally we moved the entire lab and performed skilled transportation, losing minimum coldness.

I publish it as a testament to the perlite protocol's resilience: about 25% revival or 4/12 basidiomycetes variously strong and weak under random adverse conditions. The method is robust overall and I discuss the implementation details at length.

Homolka also surveyed basidiomycete storage techniques in 2013.3 I summarized the non-nitrogen methods in plain language in diybio/water-cultures.

This was meant to be a companion piece to diybio/fungi-barcodes but the new method in that role is diybio/structured-water.

Materials and methods

todo: Add the bill of materials here. This is my convention.

The media and inoculum

I made an antibiotic cryoprotectant medium with Lennox LB (5 g/L NaCl), 25 μg/L chloramphenicol, and 10% glycerol. I sterilized the media along with a jar of perlite crystals and small glass bottles.

Then I made 5 mL aliquots with 0.5g perlite each and inoculated them with pure tissue culture. I gently scraped mycelium from the surface of an MEA (malt extract agar) plate with a sterile toothpick.

To allow gas exchange during incubation, I loosened the lids and wrapped them with Parafilm. I aerobically fermented the bottles for 10 days in an Innova 4000 shaking incubator at 28 °C and 150 RPM.

Loading the cryotubes

I drained the fermentation vials of liquid and transferred the inoculated perlite to 2 mL cryotubes. Before discarding the LB and storing the perlite, I viewed representative samples under a microscope. G. sessile and P. nameko clearly showed hyphae and conidia at 40× magnification.

It's worth noting that perlite is extremely difficult and unpleasant to work with. Sharp edges, attracted to static, easily displaced by air, not biodegradable, etc. I believe experimenting with other high-porosity stones would yield useful results.

Anyway, I stored the cryotubes in a cardboard freezer box and chilled them. Gradual cooling, as slow as reasonable, is vitally important for sucess. I stepped down freezers to control the rate: –20 °C until frozen, then –80 °C.

The cryotubes after draining the broth and loading the perlite

Reviving and testing

After the chaos between this section and the last, I found the tubes while cleaning the freezer. Remembering the wasted result of diybio/mushroom-mead, I revived the tubes in a 30 °C incubator.

I tapped out several individual crystals of each sample, of the hundreds in the tube. A single crystal can revive a plate, but taking the best growth of several attempts ensures good stock.

I cultured the plates for one week at ambient temperature until growth became apparent. To my surprise a notable amount of tubes survived the abuse and neglect.

Results and discussion

I revived all the tubes on 2020-02-14 and incubated them at room temperature for a week. The noted contamination likely happened during revival given the carefree nature of my technique.

Tabulated results

Species P Value Date Viability Notes
A. aegerita P-5 2018-03-01 Heavy mold
G. lucidum P-1 2018-03-01
G. sessile HW P-1 2018-03-01 Heavy mold
G. frondosa NH P-1 2018-03-01 Heavy mold
H. abietis P-4 2018-03-01 Bacteria
H. americanum P-2 2018-03-01 Bacteria
I. obliquus P-1 2018-03-01 + Strong growth
I. resinosum P-1 2018-03-01
L. edodes P-4 2018-03-01 + Weak growth
P. roqueforti P-2 2018-03-01 + Strong growth
P. nameko JPN P-1 2018-03-01 + Weak growth
P. tuber-regium P-2 2018-03-01

Fine-tuning the medium

Successful cryopreservation is largely a function of freezing and revival times: slow to freeze, quick to revive.

Malt extract is the typical basal sugar used in mushroom cultivation because its low pH and high peptone content selectively favors fungi over bacteria. A simplified version of Czapek medium may be ideal for perlite protocol: 30 g/L malt extract, 10% glycerol, 6 g/L sea salt, and an optional antibiotic. Assuming negligible or no metabolism at –80 °C, using 50% more sugar (30 g/L) may speed up revival without risking growth or contamination in storage.

A similarly high sugar ratio wouldn't likely be suitable for agar slants that require gas exchange for a slower but active metabolism. Serial transfers with agar slants excel on less nutritious media (10 g/L) to prevent metabolism under refrigeration.4 Another possible option is no nutrition at all, or only enough to perform the most essential metabolic functions supercooled.

Future work

Following this documented failure, I learned that storage is only as cold as you can afford to maintain. For this reason, I'm developing a new supercooled saline gel medium designed for –20 °C and informed by mammal flesh storage. Please see diybio/structured-water for details.

  1. Homolka, L., Lisá, L., Eichlerová, I., & Nerud, F. (2001). "Cryopreservation of basidiomycete strains using perlite." Journal of Microbiological Methods, 47(3), 307–313. https://doi.org/10.1016/s0167-7012(01)00338-4 ↩︎

  2. Homolka, L., Lisá, L., & Nerud, F. (2006). "Basidiomycete cryopreservation on perlite: Evaluation of a new method." Cryobiology, 52(3), 446–453. https://doi.org/10.1016/j.cryobiol.2006.02.003 ↩︎

  3. Homolka, L. (2014). "Preservation of live cultures of basidiomycetes – Recent methods." Fungal Biology, 118(2), 107–125. https://doi.org/10.1016/j.funbio.2013.12.002 ↩︎

  4. Paul Stamets. Growing Gourmet and Medicinal Mushrooms, chapter 12, pages 110–121. Ten Speed Press, 3rd edition, 2000. ↩︎