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Mycologia
ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/umyc20
Fungal cryopreservation across 61 genera:
Practical application and method evaluation
Travis Zalesky, Alexander J. Bradshaw, Zolton J. Bair, Kyle W. Meyer & Paul
Stamets
To cite this article: Travis Zalesky, Alexander J. Bradshaw, Zolton J. Bair, Kyle W. Meyer & Paul
Stamets (01 Jul 2024): Fungal cryopreservation across 61 genera: Practical application and
method evaluation, Mycologia, DOI: 10.1080/00275514.2024.2363135
To link to this article: https://doi.org/10.1080/00275514.2024.2363135
© 2024 The Author(s). Published with
license by Taylor & Francis Group, LLC.
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Fungal cryopreservation across 61 genera: Practical application and method
evaluation
Travis Zalesky
a
, Alexander J. Bradshaw
b
, Zolton J. Bair
c
, Kyle W. Meyer
c
, and Paul Stamets
c
a
School of Geography, Development and Environment, University of Arizona, 1200 E University Boulevard, Tucson, Arizona 85721;
b
School of
Biological Sciences, University of Utah, 201 Presidents Circle, Salt Lake City, Utah 84112;
c
Fungi Perfecti LLC, Olympia, Washington 98507
ABSTRACT
Fungi occupy important environmental, cultural, and socioeconomic roles. However, biological
research of this diverse kingdom has lagged behind that of other phylogenetic groups. This is
partially the result of the notorious diculty in culturing a diverse array of lamentous fungal
species due to their (i) often unpredictable growth, (ii) unknown preferences for culturing condi-
tions, and (iii) long incubation times compared with other microorganisms such as bacteria and
yeasts. Given the complexity associated with concurrently culturing diverse fungal species, devel-
oping practical methods for preserving as many species as possible for future research is vital. The
widely accepted best practice for preserving fungal tissue is the use of cryogenic biobanking at
−165 C, allowing for the preservation and documentation of stable genetic lineages, thus enabling
long-term diversity-centered research. Despite the extensive literature on fungal cryopreservation,
substantial barriers remain for implementation of cryogenic biobanks in smaller mycological
laboratories. In this work, we present practical considerations for the establishment of a fungal
culture biobank, as well as provide evidence for the viability of 61 fungal genera in cryogenic
storage. By providing a pragmatic methodology for cryogenically preserving and managing many
lamentous fungi, we show that creating a biobank can be economical for independently owned
and operated mycology laboratories, which can serve as a long-term resource for biodiversity,
conservation, and strain maintenance.
ARTICLE HISTORY
Received 2 June 2023
Accepted 30 May 2024
KEYWORDS
Ascomycota; Basidiomycota;
cryogenics; culture
maintenance; liquid nitrogen
(LN
2
)
INTRODUCTION
Within Eukaryota, the kingdom Fungi is thought to be
one of the most specious major groups of organisms
(Purvis and Hector 2000), containing an estimated 6.3
million species (Baldrian et al. 2022), of which fewer
than 150 000 have been described (Větrovský et al.
2020), with new species descriptions being made at a
rate of ~2000 per annum (Cheek et al. 2020). Multiple
studies have found that approximately one third of
identified fungal species belong to the monophyletic
group Basidiomycota (Baldrian et al. 2022; Taylor et al.
2015). Although Basidiomycota does include unicellular
fungi such as yeasts, many basidiomycetes are sporo-
carp-producing, multicellular filamentous species ser-
ving multiple functions within an ecosystem, including
formation of mutualistic partnerships with plants (ecto-
mycorrhiza) and decomposition of recalcitrant plant
tissues containing cellulose, hemicellulose, and lignin
(saprophytes).
Fungi are critical to environmental health, acting as a
primary force of decomposition by converting complex
biological materials into their base components to be
returned to their respective nutrient cycle (Bahram and
Netherway 2021; Mayer et al. 2021; McGee et al. 2019).
In addition to their important ecological roles, human-
kind has benefited from fungal diversity, as evidenced
by the discovery of antibiotics such as penicillin
(Fleming 1929, 1941; Lobanovska and Pilla 2017;
Raper et al. 1944) and in emerging trends of sustainable
engineering (Alemu et al. 2022; Chatterjee and Venkata
Mohan 2022; Hotz et al. 2023). Additionally, many
edible mushrooms have generated interest in terms of
potential beneficial health effects. Approximately 38%
of the global mushroom industry is medicinal in nature
(Ferraro et al. 2022; Royse et al. 2017). As a result,
functional mushroom products have created a global
market with an estimated value of $29.1 billion USD
in 2022, which is projected to grow to $65.8 billion USD
CONTACT Travis Zalesky travisz@arizona.edu
Supplemental data for this article can be accessed online at https://doi.org/10.1080/00275514.2024.2363135.
MYCOLOGIA
https://doi.org/10.1080/00275514.2024.2363135
© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted
Manuscript in a repository by the author(s) or with their consent.
Published online 01 Jul 2024
by 2030, according to a recent report by Research and
Markets (Functional Mushroom Market Size 2022).
Despite their importance to the global ecosystem and
human culture, the sheer diversity of Fungi and their
highly varied cryptic lifestyles and nutritional niches
make them notoriously difficult to curate living tissue
cultures long term (Rämä and Quandt 2021). Although
viable fungal tissue can be obtained from many species
through direct collection in the field and subsequent
cultivation, this solution is not pragmatic for long-
term studies or those requiring access to rare and under-
studied species. Furthermore, many fungal species are
difficult to locate and isolate, as they may be endemic to
certain regions, are often seasonal, and may only pro-
duce ephemeral macroscopic reproductive structures
under very specific conditions (Bradshaw et al. 2022;
Linde et al. 2012). These collections are difficult to
repeat and can be mired in the complexity of official
taxonomic description, which can sometimes result in
new discoveries becoming completely lost (Aime et al.
2021; Hawksworth and Rossman 1997). As such, it is
important to indefinitely preserve specimens as living
mycelial cultures for future study.
Several methods of preserving fungal cultures are well
understood and widely implemented by both casual and
professional mycologists, the most basic of which
requires periodic aseptic transfers of mycelium to fresh
growth medium, followed by an incubation period dur-
ing which the fungus is actively growing. Unfortunately,
this approach is time intensive and requires continual
monitoring (Jong and Birmingham 2001; Singh 2017;
Stamets 1993). Furthermore, perpetual transferring of
the culture promotes genetic drift over time, as successive
generations diverge from the original source tissue, con-
tinuously increasing cell divisions and associated muta-
tions (Maheshwari and Navaraj 2008). This
phenomenon, known as strain senescence, is a constant
challenge for cultivation when seeking to maintain con-
sistent production from clonal strains (Pérez et al. 2021).
Mitigating strain senescence is critical to avoiding a
reduction of fruitbody yield, or even a total loss of the
ability to produce fruitbodies entirely (Shu et al. 2021).
Another widely utilized method of fungal tissue pre-
servation is cold storage at or near freezing temperatures
(Stamets 1993). Cultures can easily be grown on standard
nutritive media, either in Petri dishes or slant tubes, and
master cultures can be preserved at 1 to 4 C, typically for
several years. Although this approach is highly practical
and easily accessible to virtually any mycological labora-
tory, this method suffers several disadvantages. Some
species, especially those naturally inhabiting tropical
environments, are difficult or perhaps impossible to reli-
ably store using this method (Colauto et al. 2011; Stamets
1993). Additionally, cold-tolerant species may continue
to slowly metabolize (and thus senesce) under near-freez-
ing conditions. Although many fungi may be successfully
recovered after years in cold storage, long-term exposure
to these conditions often results in culture failures. The
primary means of protecting against total culture loss
when using cold storage methods is to create many
redundant storage cultures as well as periodically reviving
cultures by cultivating mycelium on fresh medium at
optimal incubation temperatures before returning to
cold storage. This method effectively lengthens the time
between subcultures but ultimately is subject to the same
disadvantages as the periodic transfer method (Jong and
Birmingham 2001).
For microbes including fungi, cryogenic storage (cryo-
preservation) at or below −165 C has been recognized as
an ideal solution for long-term maintenance of culture
libraries (Homolka 2013). The creation and maintenance
of a long-term live culture cryogenic storage system
allows for stability and reproducibility of results by facil-
itating widespread access to material with well-main-
tained documentation on lineage-specific traits (e.g.,
Danell and Flygh 2002). Despite the widely accepted
success of cryopreservation for filamentous fungi, signif-
icant barriers to entry remain for modestly resourced
culture laboratories seeking to establish a cryogenic fun-
gal biobank, including the initial cost of equipment and
the ongoing cost of liquid nitrogen (LN
2
). Further, meth-
ods related to cryogenic storage are highly technical, with
a wide variety of application-specific methods that are
unlikely to be consistently applicable in every laboratory
(e.g., Colauto et al. 2011; Danell and Flygh 2002;
Eichlerová et al. 2015; Homolka 2013; Jong and
Birmingham 2001; Linde et al. 2018; Tian et al. 2020).
Herein we present practical considerations for the
construction and maintenance of a cryogenic biobank
dedicated to filamentous fungi. In the interest of
improving clarity and accessibility of these methods
for smaller mycology laboratories, we outline a simple
and broadly applicable process that uses familiar myco-
logical techniques (after Homolka 2013) along with
affordable materials and equipment currently obtain-
able on the market. We also present viability testing
and data management methods using simple, accessible
techniques. In addition, we report the cryogenic storage
viability of 61 genera and more than 100 species across a
wide range of freezing durations, from short term (1 to 3
days) to long term (greater than 3 years), and cross-
reference the species of our culture library against fun-
gal samples commercially available from American Type
Culture Collection (ATCC, https://www.atcc.org/;
TABLE 1). Finally, we provide a summary of the equip-
ment and infrastructure used to implement this
2ZALESKY ET AL.: FUNGAL CRYOPRESERVATION
Table 1. Summary of cryogenically preserved species at Fungi Perfect, with comparison with American Type Culture Collection (ATCC)
catolog, accessed online 9 May 2023.
Genus Species Strains Batches Samples ATCC results Notes
a,b
Agaricus augustus 1 2 15 2
Agaricus bisporus 1 1 7 351
Agaricus bitorquis 2 3 23 124
Agaricus brasiliensis 1 2 16 0 Species complex in flux. Alternately known as A. subrufescens, 4 results.
b
Agaricus brunnescens 2 3 22 0
Agaricus lilaceps 1 2 15 0
Agrocybe praecox 1 1 7 0
Agrocybe sp. 1 1 7 6 Results at genus level.
a
Armillaria mellea 1 1 7 61
Auricularia nigricans 1 2 18 0 Formerly known as Auricularia polytricha, 15 results.
b
Calocybe indica 1 1 7 0
Calvatia cyathiformis 1 1 7 2
Chlorophyllum rhacodes 2 2 15 0 Formerly known as Macrolepiota rhacodes, 2 results.
b
Clitocybe odora 1 1 8 0
Coprinopsis nivea 1 1 8 0
Coprinus comatus 5 6 43 3
Cordyceps militaris 3 4 27 3
Cryomyces antarcticus 1 1 8 1
Cyathus striatus 1 1 8 2
Cyclocybe aegerita 1 1 7 12
Cyclocybe parasitica 1 1 7 2 Formerly in genus Agrocybe. Also known as C. cylindracea, 2 results.
b
Fistulina hepatica 1 1 7 4
Flammulina filiformis 2 2 14 0
Flammulina populicola 2 3 22 0
Flammulina velutipes 2 3 20 24
Fomes fomentarius 6 6 43 4
Fomitiporia robusta 1 1 8 0
Fomitopsis betulina 7 8 52 0 Formerly known as Piptoporus betulinus, 2 results.
b
Fomitopsis ochracea 1 1 8 0
Fomitopsis pinicola 3 3 25 7
Ganoderma applanatum 5 5 39 11
Ganoderma lucidum 8 8 56 11
Ganoderma neojaponicum 1 1 8 2
Ganoderma oregonense 10 11 83 6
Ganoderma polychromum 2 2 16 0
Ganoderma tsugae 1 1 8 7
Grifola frondosa 12 12 94 12
Gymnopilus sp. 2 2 16 2 Results at genus level.
a
Hericium abietis 4 4 32 5
Hericium coralloides 1 2 11 25
Hericium erinaceus 11 17 121 10
Heterobasidion annosum 2 2 16 20
Hypholoma capnoides 9 9 70 2
Hypholoma lateritium 1 1 7 5
Hypsizygus marmoreus 2 2 16 1
Hypsizygus sp. 1 1 8 2 Results at genus level.
a
Hypsizygus tessulatus 7 7 55 0
Hypsizygus ulmarius 5 5 39 1
Inocutis dryophila 1 1 8 1
Inonotus obliquus 5 7 53 1
Irpex lacteus 2 2 16 8
Laetiporus conifericola 4 4 32 0
Laetiporus montanus 1 1 8 0
Laetiporus sulphureus 5 5 40 8
Laricifomes officinalis 87 104 748 2 syn. = Fomitopsis officinalis.
Lentinula edodes 20 26 195 107
Lepista nuda 2 3 23 6
Lepista sp. 1 1 8 8 Results at genus level.
a
Leratiomyces ceres 1 1 8 0
Lyophyllum decastes 1 1 8 10
Macrolepiota procera 2 2 16 4
Marasmiellus sp. 1 1 8 39 Results at genus level.
a
Metacordyceps chlamydosporia 1 1 7 0 Formerly known as Pochonia chlamydosporia, 1 result.
b
Morchella angusticeps 3 3 24 4
Morchella esculenta 2 3 23 6
Morchella sp. 2 3 23 48 Results at genus level.
a
Mycenaceae sp. 1 1 8 0 Results at genus level.
a
Omphalotus guepiniiformis 1 1 8 0 Formerly known as Lampteromyces japonicus, 0 results.
b
Omphalotus olearius 1 1 8 12
Ophiocordyceps sinensis 2 2 14 0 1 result for O. sp.
(Continued)
MYCOLOGIA 3
approach, as well as cost estimates for establishing a new
biobank (TABLE 2). This work provides insights into the
establishment of a small-scale cryogenic biobank and
empirical data on the cryopreservation viability of a
diverse set of fungi, with the goal of making long-term
preservation of fungal cultures a more transparent and
approachable task.
MATERIALS AND METHODS
Preparation of cryogenic media.—The cryogenic
medium utilized throughout this study was modified
from a formulation by Homolka (2013). Fungal cryogenic
media consisted of perlite suspended in cryoprotectant-
supplemented malt extract agar (MEA) mixed in 2-mL
Table 1. (Continued).
Genus Species Strains Batches Samples ATCC results Notes
a,b
Phellinus igniarius 1 1 8 4
Phellinus robiniae 1 1 8 2
Phellinus everhartii 1 1 7 1
Phlebia radiata 1 1 8 5
Pholiota adiposa 1 1 8 3
Pholiota nameko 1 1 6 5
Pholiota sp. 3 3 24 52 Results at genus level.
a
Pleurotus columbinus 2 2 15 3 Results given for P. ostreatus var. columbinus.
b
Pleurotus djamor 1 2 18 9
Pleurotus dryinus 1 1 8 11
Pleurotus eryngii 1 2 16 10
Pleurotus nebrodensis 5 5 40 0
Pleurotus ostreatus 19 20 158 114 3 additional results for P. ostreatus var. columbinus.
b
Pleurotus pulmonarius 4 5 36 40
Pleurotus sp. 2 2 16 304 Results at genus level.
a
Polyporus annulatus 1 1 8 0 Formerly known as Ganoderma annularis, 0 results.
b
Polyporus tuberaster 1 1 8 2
Polyporus umbellatus 3 3 24 6
Psathyrella ammophila 1 1 8 0
Psathyrella aquatica 2 2 16 1
Pseudoinonotus dryadeus 1 1 8 0 Formerly known as Inonotus dryadeus, 0 results.
b
Rhodofomes cajanderi 1 1 8 0 Formerly known as Fomitopsis cajanderi, 7 results.
b
Rigidoporus ulmarius 1 1 8 4
Sarcomyxa serotina 1 1 8 0 Formerly known as Panellus serotinus, 1 result.
b
Schizophyllum commune 1 2 13 41
Sparassis crispa 2 2 15 3
Sparassis radicata 1 1 8 0
Stropharia ambigua 3 3 24 0
Stropharia rugosoannulata 5 7 55 4
Taiwanofungus camphoratus 1 1 7 0
Tolypocladium capitatum 1 1 7 0
Tolypocladium inflatum 2 2 16 7
Trametes elegans 2 2 16 0
Trametes versicolor 7 7 51 49
Tropicoporus linteus 3 4 29 1
Volvariella volvacea 1 1 8 19
Wolfiporia cocos 2 2 16 0 Formerly known as Poria cocos, 4 results.
b
a
For instances where species-level identification is not available, ATCC results are given at the genus level.
b
Where taxonomy is disputed or recently updated, common aliases were queried as well and alternate results are also listed.
Table 2. Minimum cost estimate of establishing a new cryogenic biobank in small scale.
Product Cost
Cryogenic storage tank Starting at $150
−80 C freezer Starting at $1500
Controlled-rate cell freezing container $146 to $519
Micro reagent spatula $3 to $25 per set
Sieves Starting at $48 per sieve
Heavy-duty thermal gloves $5 to $30
Lab coat $10 to $50
Face shield $5 to $30
Forceps $5 to $10
Oxygen meter Starting at $100
Cryogenic sample vials $50 per pack (50 vials)
Perlite < $1 per lb
Glycerol $10 per L
Liquid nitrogen $0.10 to $2 per L plus delivery (dependent on location and quantity)
Minimum estimated startup cost $2500.00
4ZALESKY ET AL.: FUNGAL CRYOPRESERVATION
self-standing externally threaded cryogenic vials
(Corning, product no. 430659; Corning, New York).
Bulk organic perlite was sifted between 2 mm and
250 µm mesh (American Society for Testing and
Materials Test Sieves no. 10 and no. 60, respectively).
After discarding the sub-250-µm perlite fraction, roughly
0.5 to 1 mL of size-sorted, sub-2-mm, perlite was loosely
packed and added to the cryogenic vials, filling each vial
to ~1/3 total volume (FIG. 1). The MEA consists of 2.7%
malt extract (m/m) and 2.7% agar (m/m) mixed in warm
tap water and supplemented with 5% glycerol (v/v) as a
cryoprotectant (Homolka 2013). Approximately 1.5 mL
of glycerol-supplemented MEA was transferred to each of
the perlite-filled cryogenic vials, keeping ~1/4 of the total
volume remaining as open headspace to allow for inocu-
lation (FIG. 1). After preparation, cryogenic vials were
sealed in an upright orientation in autoclavable bags and
sterilized in an All American Electric Sterilizer (model
75X-120V; Hillsville, Virginia) at 15 psi (1 kg/cm
2
) for
60 min. Following sterilization, cryogenic vials were
allowed to cool to room temperature and stored at −20
C until needed.
Sample labeling, data curation, and data
management.—Immediately prior to inoculation, cryo-
genic vials were removed from freezer storage and
allowed to thaw to room temperature. Once thawed,
vials were given a unique cryogenic vial label, which was
also transcribed into a custom electronic “cryo-in” record
containing additional species information, including (i) a
unique accession number for every preserved strain, (ii)
inoculation date, (iii) transfer number (internally, “plate
value”), (iv) number of vials inoculated, (v) date of freez-
ing (recorded post incubation), and (iv) any additional
inoculation notes (SUPPLEMENTARY TABLE 1).
Additionally, an electronic “cryo-out” record docu-
menting sample removal from cryogenic storage was
maintained separately, and every removed sample was
recorded with (i) accession number, (ii) species and
strain information, (iii) date frozen, (iv) date thawed,
and (v) quantity thawed. Both the “cryo-in” and the
“cryo-out” files are complementary and can be joined
by the accession number and date frozen columns to
facilitate further calculations, such as total samples fro-
zen, samples remaining per batch, and duration of
freezing (SUPPLEMENTARY TABLE 2).
A third “biobank map” diagram was maintained in
the form of a custom, color-coded, spreadsheet file
corresponding to the physical structure of our cryogenic
storage system (CSS), which enabled technicians to
rapidly locate samples (FIG. 2). Once a shelf was filled,
the corresponding record was printed and filed into a
centralized and accessible location, physically accompa-
nying the CSS. As samples were removed, their corre-
sponding sample squares in hardcopy were updated and
the thawing dates were recorded.
Culture inoculation and evaluation.—Cultures for
each cryogenically stored fungal species were grown
initially in Petri dishes of MEA, at room temperature,
and allowed to fully colonize (~2 to 5 weeks, depending
on the taxon) before being used as a source of inoculum
for medium-filled, sterile, cryogenic vials. Inoculations
from fully colonized plates were conducted aseptically
with a heat-sterilized scalpel in front of a HEPA (high-
efficiency particulate air) laminar flow hood by cutting a
small (~3 to 5 mm
3
) cube of myceliated agar from the
MEA plate and transferring the inoculum into a sterile
cryogenic vial (FIG. 3). Caps were immediately replaced
following the inoculation, and, depending on the species
growth rate, samples were allowed to incubate at room
Figure 1. Cryogenic vial preparation plate. A. Bulk organic perlite sifted between American Society for Testing and Materials Test
Sieves no. 10 and no. 60 (2 mm and 250 µm mesh, respectively). The perlite captured by the no. 60 mesh was retained and loosely
packed into 2-mL externally threaded self-standing cryogenic vials. B. Addition of cryogenic medium (glycerol-supplemented MEA) to
cryogenic vials containing perlite, prior to sterilization.
MYCOLOGIA 5
temperature for 5 to 76 days (SUPPLEMENTARY
TABLE 1). Samples were designated as fully incubated
through visual confirmation of mycelial growth occupy-
ing at minimum ~1 cm
3
within the cryogenic vial, dis-
playing hyphal growth penetrating the medium under
8× magnification (FIG. 4). Any contaminated, uninocu-
lated, or relatively slow-growing (taxon-specific) sam-
ples were discarded prior to freezing.
Cryogenic storage.—Before entering into cryogenic sto-
rage, fully colonized cryogenic vials were first cooled at a
controlled rate of −1 C/min to a final temperature of −80 C
using either a Mr. Frosty freezing container (Thermo
Scientific, Waltham, Massachusetts) and/or a CoolCell
alcohol-free freezing container (Corning). Cryogenic vials
were loaded into the container according to manufacturer
specifications and placed into a −80 C freezer (Forma
Scientific, model 923; Waltham, Massachusetts) for a mini-
mum of 4 h. Samples were occasionally left in their freezing
container in the −80 C freezer for as long as 48 h with no
apparent negative effects. Although no direct comparison
of cell freezing containers was made, both performed satis-
factorily and ultimately yielded viable cultures. After step-
down freezing, the samples were transferred into the gas
phase of our selected CSS, a Forma Scientific Cryo 100
model 740 with 90 L capacity.
Post–cryogenic freezing viability testing.—Between
1 and 3 days after samples entered cryogenic storage, the
viability of each specimen was tested to determine
whether the freezing process was successful. For each
specimen batch, two randomly selected cryogenic vials
were removed from cryogenic storage and were con-
sumed for viability testing, leaving the remainder for
Figure 2. Biobank map. Cryogenic storage system layout “map” kept adjacent to the cryogenic storage tank permits technicians to
rapidly identify sample location.
Figure 3. Cryogenic vial inoculation. Inoculation of a sterile
cryogenic vial with cryogenic medium using a small (3 to 5
mm
2
) square of inoculum taken from a well-colonized MEA
Petri dish.
6ZALESKY ET AL.: FUNGAL CRYOPRESERVATION
long-term storage. Viability samples were processed
aseptically at a sterile workbench in the presence of
HEPA-filtered laminar air flow where they were most
often thawed at room temperature (approximately 20 to
30 min). Occasionally, rapid thawing was also per-
formed by placing cryogenic vials inside a vial float in
a warm water bath, avoiding direct contact with or
submergence of the cryogenic vial in the water.
After thawing, cryogenic vials were surface-sanitized
with 70% isopropanol prior to opening. A small piece of
inoculum, ~3 to 5 mm
3
, was removed aseptically from
each vial using a heat-sterilized microreagent spatula
and transferred to a fresh MEA plate (FIG. 5). Each
cryogenic vial used for viability testing was sampled in
triplicate, yielding a total of six plates for viability testing
of each batch. Test plates were then incubated at room
temperature for 2 to 6 weeks (depending on taxa being
evaluated), and individual plate viability was assessed
qualitatively on a pass/fail basis. To pass the individual
viability test, cultures had to be uncontaminated and
well established on the plate medium, attaining a mini-
mum diameter of ~3 cm. Batches of samples of a given
taxon were designated as “viable” if at least four of the
six test plates met pass conditions. Viability assessment
results were recorded in the “cryo-out” record
(SUPPLEMENTARY TABLE 2).
We required initial viability testing of two cryogenic
vials per unique accession number for every batch
frozen but also tested all subsequently thawed vials for
viability, with a minimum expansion rate of one cryo-
genic sample vial to three MEA tester plates and with no
fixed upper expansion limit. Viability test plates were
repurposed for general culture use after successful colo-
nization had been documented.
Biobank sample diversity.—Species identification has
primarily been conducted by field identification at the
time of tissue collection. Many of our cryogenically
banked cultures, including all Laricifomes officinalis
strains, have had species identity confirmed by whole
genome sequencing or genetic barcoding of nuc rDNA
internal transcribed spacer region ITS1-5.8S-ITS2 (ITS
barcode; Schoch et al. 2012). Historically, fungal taxon-
omy has undergone significant revisions, particularly in
recent years with next-generation sequencing efforts;
thus, our archival taxonomic records have been com-
pared with Index Fungorum (https://www.indexfun
gorum.org/) and MycoBank (https://www.mycobank.
org/) databases to determine currently accepted taxo-
nomic nomenclature and were updated as warranted.
Using this updated species list, we queried the ATCC
catalog for each unique species with search filters
“Fungi” and “Products” applied and recorded the num-
ber of results for each species (accessed 9 May 2023). In
cases where species-level identification is not available,
genus-level search results were recorded. For any spe-
cies with recently updated or disputed taxonomy, com-
mon aliases were also queried and results were recorded
in the “Notes” column in TABLE 1.
Figure 4. Inoculated cryogenic vials. A. An example well-inocu-
lated cryopreservation vial. B. The same vial in A under 8×
magnification. Bright white mycelium can be seen originating
from the inoculum square and penetrating down into the cryo-
genic medium.
Figure 5. Viability test plate inoculation. Inoculation of a MEA
Petri dish from a post-freezing cryogenic vial using a small (3 to
5 mm
2
) inoculum piece, carefully removed from the vial using a
sterilized micro reagent Scoopula.
MYCOLOGIA 7
RESULTS
Total strains and diversity tested.—In this study,
3199 individual cryogenic vials of fungal tissue were
preserved across 427 unique freezing batches (FIG. 6).
These samples represent 372 individual fungal strains
from 107 species across 61 genera (TABLE 1).
Post–cryogenic storage viability.—Of the 427 batches
that were frozen, only a single batch failed initial viabi-
lity testing (0.23%). Further investigation of this failed
batch revealed that one of two tested Ganoderma appla-
natum cryogenic vials that were removed for viability
testing on 29 October 2021 was contaminated by bac-
teria prior to testing, resulting in three of the six viability
plates becoming contaminated. This strain batch was
removed entirely from cryogenic storage and was suc-
cessfully preserved shortly thereafter, with two viability
test vials successfully colonizing all six inoculated test
plates. Additionally, of the 514 sets of vials that have
been removed from the CSS (typically in duplicate), 502
(97.7%) sets resulted in 100% of tested inoculum passing
viability standards, as outlined previously, by suffi-
ciently colonizing the test medium (FIG. 7).
Although the reported storage viability data primarily
feature short-term testing (typically within a few days)
during initial establishment of our cryogenic biobank,
many samples cryogenically stored for longer durations
have also been removed and tested, based on standard
organizational need, including 17 unique batches (15
species across 10 genera) that have been confirmed viable
after more than 2 years in storage (FIG. 7;
SUPPLEMENTARY TABLE 2). Every batch frozen was
tested between 1 and 3 days after freezing, and every
subsequent sample thawed was also tested, regardless of
initial viability score or duration frozen. Although the
majority of the frozen strains have only been tested once,
some commonly used batches have been tested up to five
times, with no evidence of decreased viability over time.
The longest frozen sample yet tested is an Agaricus bra-
siliensis strain inoculated on 28 March 2019 and frozen
for 1262 days prior to thawing; this single sample vial
successfully inoculated six MEA plates at a 100% success
rate (SUPPLEMENTARY TABLE 2).
Practical cost of maintaining cryostorage.—LN
2
was
delivered as needed by Airgas (Radnor, Pennsylvania) in
standard 180-L 22-psi cylinders. Our observed static eva-
poration rate from our cryogenic storage tank is 2.2 kg/
day, a calculated static use rate of about 2.5 L/day (FIG.
8). Further, internal data have shown significant LN
2
loss
associated with tank openings. Whether adding or
removing samples, a typical tank opening of between 3
and 5 min resulted in an immediate loss of approximately
0.5 to 1 kg of LN
2
. Additional LN
2
is lost through normal
off-gassing of N
2
from portable LN
2
cylinders. Although
precise LN
2
consumption rates will vary, we were able to
obtain LN
2
economically, at an approximate average cost
of $175 per week, including delivery.
Additional costs associated with maintaining a CSS
include personnel hours required for batch inocula-
tions and processing. A typical batch of 10 cryogenic
vials can be prepared, and all processing and viability
testing procedures that require active “hands on”
labor can be completed in roughly 2 h, spaced out
over the course of several weeks. After freezing,
Figure 6. Total sample vials preserved. The total number of
preserved cryogenic vials in gas-phase cryogenic liquid nitrogen
(LN
2
) storage over time.
Figure 7. Viability rate. The viability test plate success rate
shown as a percentage of the total number of inoculated plates
(minimum of three plates per cryogenic vial) over the frozen
duration of each sample. The dashed horizontal line represents
the threshold above which preserved batches are deemed
viable. Many data points are obscured by overlapping data at
or around day 1.
8ZALESKY ET AL.: FUNGAL CRYOPRESERVATION
ongoing labor costs are minimal, primarily consisting
of daily systems checks and periodic installation of
new LN
2
cylinders.
Comparison with commercially available species
from ATCC.—Conservatively, our biobank currently
contains 26 species not commercially available from
ATCC (TABLE 1). Additionally, our biobank currently
holds the world’s largest known collection of cryogeni-
cally preserved Laricifomes officinalis (syn. = Fomitopsis
officinalis; Han et al. 2016) strains (87 isolate strains as
of initial manuscript preparation).
DISCUSSION
Our cryopreservation initiative has succeeded in hous-
ing a diverse collection of fungal cultures, focused pri-
marily on Basidiomycota (TABLE 1). Moreover, as a
privately funded laboratory, we provide a unique per-
spective on the establishment of a fungal cryogenic
biobank in the absence of institutional resources asso-
ciated with traditional academic settings. We have
demonstrated that it is possible for small- to medium-
sized laboratories to economically implement a cryo-
genic fungal biobank for both commercial and biodi-
versity preservation interests.
The methods presented were found to be economical
and reasonable to implement without prior experience in
long-term cryogenic biological sample storage, although
considerable time and effort was needed to fully
implement and optimize procedures. Following the initial
establishment of the biobank, the CSS has proven easy to
maintain and this cryopreservation method has been
shown to be extremely reliable, as fungal cultures are
easily revived following long-term cryogenic storage.
Implementation costs.—Although cryogenic storage
methods can be more expensive to establish compared
with alternative methods of fungal culture preservation
(TABLE 2), this approach can be cost effective, especially
considering reduced labor costs over long time frames,
reduced risks of contamination, and mitigation of strain
senescence. Initial equipment costs include a cryogenic
storage tank and −80 C freezer. Equipment prices can
vary widely depending on factors such as manufacturer,
model, capacity, supplier, and energy efficiency.
Cryogenic storage tanks come in a variety of sizes, ran-
ging from a 3-L LN
2
Dewar costing as little as $150, up to
units exceeding 1500 L internal volume. Models may be
purchased new or used and are often highly customizable
with ergonomic designs and optional safety and security
features. Similarly, various models of −80 C freezers are
available with a wide array of optional features, with some
used models presently available for approximately $1500.
An additional piece of required equipment is a con-
trolled-rate cell freezing container, or an ultra-low free-
zer that allows for precision controlled freezing rates. In
the current study, we used the Mr. Frosty and the
CoolCell, both of which are economical, starting at
around $150. We did not observe any difference in
efficacy or freezing rate between the two devices.
Consumables include cryogenic vials and components
of culturing media. Although we prefer to use the 2-mL
externally threaded self-standing type, other styles of cryo-
genic vial may be appropriate depending on user prefer-
ence. Packs of 50 cryogenic vials can typically be obtained
for about $50, and cost per unit typically decreases by
purchasing in bulk. The culturing medium is a glycerol-
supplemented MEA, the components of which are com-
monly used in mycological laboratories. Lastly, although
size-sorted “cryogenic” perlite can be found for sale online,
sieving bulk organic perlite is far more economical.
Liquid nitrogen supply is also a major cost considera-
tion. Pricing and cost structures will vary by location
and vendor, as well as quantity and regularity of pur-
chases. Industrial gas suppliers are common in most
large cities and typically deliver. Depending on the
CSS location and size, an infrastructural LN
2
microbulk
system may be more cost effective compared with
exchangeable dewars or tanks.
Time and labor requirements are directly proportional
to a biobank’s size. The workload is heavily frontloaded
Figure 8. Static evaporative loss. The mass of a Forma Scientific
Cryo 100 model 740 90 L capacity (serial no. 14503-28) cryogenic
storage tank with >3100 inoculated 2-mL cryogenic vials of
preserved fungal tissue tracked over a 4-week period with the
lid sealed throughout testing. The mass lost represents the static
evaporative loss of liquid nitrogen (LN
2
) from the system,
whereas rapid increases in mass represent LN
2
fills.
MYCOLOGIA 9
with documentation, sample preparation, freezing proce-
dures, and viability testing followed by years or decades of
infrequent or intermittent use. Notably, implementation of
a cryogenic storage program often comprises two phases:
(i) initial establishment, requiring culture transfers from an
existing culture library, and (ii) periodic addition of newly
isolated cultures or removal of cultures as needed. After the
more intensive initial period required to transfer existing
cultures into cryogenic storage, newly isolated cultures will
require continued time investment proportional to the rate
of acquisition. Although full implementation timelines
from initial culture inoculation to viability confirmation
will typically take several weeks, most of this time is dedi-
cated to passive culture incubation. Steps requiring active
attention from trained personnel can be conducted quickly,
and a single technician using assembly-line practices can
easily process hundreds of samples per week.
Safety, security, and associated risks.—LN
2
use poses
potential hazards and can cause bodily harm from con-
tact with any liquid phase, vapor phase, or supercooled
surfaces. Thus, industry safety standards should be
reviewed and personal protective equipment (PPE)
acquired prior to implementing any cryogenic storage
methods (Jong and Birmingham 2001; Mückley et al.
2007; Schiewe et al. 2019). Required and recommended
PPE, along with price estimates, are listed in TABLE 2.
Besides sample loss due to negligent or malicious activ-
ity, the most likely situation resulting in critical or total
sample loss is uncontrolled thawing (i.e., tank failure) from
insufficient LN
2
supply (Alikani 2018). Although several
factors govern the evaporation rate of LN
2
from cryogenic
storage tanks, the integrity of the insulating vacuum jacket
seal is the most critical. For a full review of the risk of
sample loss due to tank failure and best practices for
mitigating risk, see Pomeroy et al. (2019).
Additional considerations.—For related batches that
are deemed viable, a 1:3 cryogenic vial:plate ratio should
be considered the minimum culture expansion value. For
viable cultures, we have successfully expanded to up to
eight plates per culture sample vial, but specific expansion
rates will depend on case-specific laboratory and project
needs.
For the safety of cultures, samples should preferentially
be stored in the gas phase of cryogenic CSSs, as opposed to
being submerged in liquid phase, as LN
2
can seep into
cryogenic vials, potentially vectoring contaminants
(Alikani 2018; Bielanski and Vajta 2009; Schiewe et al.
2019).
Consistent labeling conventions are highly recom-
mended from the outset and should include informa-
tion such as the genus, species, tissue transfer number,
and inoculation date, and any other relevant species or
project information, which can be encoded or abbre-
viated as desired. Additional cryogenic sample labels
and color-coded sample caps can, optionally, be pur-
chased from scientific equipment suppliers.
Potential considerations that may improve this method
include additional screening procedures for assessing cul-
ture viability. Qualitative assessment is largely based on
macroscopic culture characteristics, with microscopic fun-
gal anatomy investigated as needed. These quantification
methods could be improved by requiring detailed micro-
scopy and growth rate measurements.
Importance of diversity preservation.—As human
activities continue to impact species extinction rates
across the globe, protection and conservation of biodi-
versity becomes increasingly critical (Barnosky et al.
2011), especially given that the proportion of unidentified
species in Fungi is far greater than other monophyletic
eukaryotic groups such as vascular plants (Cheek et al.
2020). With so many undescribed fungal species globally
and considering their cryptic nature, the full extent of
fungal diversity loss due to climate change and other
factors remains difficult to estimate (Lughadha et al.
2020). Although public attitudes toward mushrooms
and fungi are changing, fungi have historically been an
understudied topic in biology (Troudet et al. 2017).
While research with plant, animal, and bacterial cultures
has benefited significantly from the application of cryo-
genic preservation, the availability of living, cryogenically
preserved fungal mycelium remains limited. In the inter-
est of fungal biodiversity preservation, we have provided
evidence of the suitability of cryopreservation for over
100 fungal species using this simple method, including 26
fungal species that are not known to be commercially
available through ATCC, as well as multiple genera that
are rarely (or have not yet been) cited in peer-reviewed
literature (e.g., Leratiomyces, Polyporus, Wolfiporia;
SUPPLEMENTARY TABLE 2; Linde et al. 2018).
In addition to preserving species diversity, strain (or
isolate) diversity is also an important consideration. As
has been shown by the history of Penicillium spp. strain
isolation for the scalable production of penicillin in the
early 20th century, there can be considerable variation
between strains within a particular species, especially
when considering optimization for a single secondary
metabolite or small molecule of potential pharmaceutical
or industrial interest (Fleming 1929, 1941; Gaynes 2017;
Lobanovska and Pilla 2017; Raper et al. 1944). With this
10 ZALESKY ET AL.: FUNGAL CRYOPRESERVATION
consideration in mind, certain species of interest have
been isolated repeatedly from widely disparate geo-
graphic locations, such as Laricifomes officinalis
(syn. = Fomitopsis officinalis). Throughout our biobank-
ing efforts, we have successfully cryogenically preserved
87 isolates of L. officinalis, which, to our knowledge, is the
largest strain collection for this species in the world.
ACKNOWLEDGMENTS
We would like to thank Allison Woodall for assistance in
fungal culturing and Jacqueline D. Morgado for photography
and practice of relevant cryopreservation methods. We also
extend our gratitude to numerous collectors, including David
Sumerlin and Jim Gouin, who have made significant contri-
butions to this fungal culture library.
DISCLOSURE STATEMENT
Zolton Bair, and Kyle Meyer report that they are employees of
Fungi Perfecti LLC, a company founded and owned by Paul
Stamets, which produces fungal-based products for commer-
cial sale. Although this work is funded by Fungi Perfecti LLC,
this study is not a product or service related to company
activity; rather, this study is presented as a practical metho-
dology deemed to be of interest outside of Fungi Perfecti LLC
commercial activities.
DATA AND CODE AVAILABILITY STATEMENT
Calculations, data management, and visualizations were per-
formed in R 4.1.0 (R Core Team 2021) using packages READXL
1.4.1 (Wickham and Bryan 2022), TIDYVERSE 1.3.2 (Wickham et
al. 2019), and LUBRIDATE 1.9.0 (Grolemund and Wickham
2011). Raw data and general use code can be found in the
GitHub repository at https://github.com/FungiPerfecti/
Cryopreservation.
ORCID
Travis Zalesky http://orcid.org/0009-0006-0181-3099
Alexander J. Bradshaw http://orcid.org/0000-0002-6261-
621X
Zolton J. Bair http://orcid.org/0009-0006-3215-8971
Kyle W. Meyer http://orcid.org/0000-0002-1933-2908
Paul Stamets http://orcid.org/0000-0003-1319-6914
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