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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.
Materials and methods
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.
|A. aegerita||P-5||2018-03-01||–||Heavy mold|
|G. sessile HW||P-1||2018-03-01||–||Heavy mold|
|G. frondosa NH||P-1||2018-03-01||–||Heavy mold|
|I. obliquus||P-1||2018-03-01||+||Strong growth|
|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|
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.
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.
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 ↩︎
Paul Stamets. Growing Gourmet and Medicinal Mushrooms, chapter 12, pages 110–121. Ten Speed Press, 3rd edition, 2000. ↩︎