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C H A P T E R V-5

Fungi: Preservation of
cultures
RICHARD A. H U M B E R
USDA-ARS Plant Protection Research Unit, US Plant, Soil & Nutrition Laboratory, Tower Road,
Ithaca, New York 14853-2901, USA

needs to store cultures go beyond the most casual


1 INTRODUCTION level, it is very important to choose the preservation
technology that best fits one's needs with a con-
All research or applied studies using live organisms venient, affordable level of technological sophistica-
requires a constant supply of them in a suitable con- tion. Much time, anguish and money can be saved by
dition. Work with fungi usually requires keeping cul- carefully weighing the real purposes and needs to
tures, a task that is both easier and facilitates more preserve cultures before committing one's effort and
possible research approaches than dealing, for exam- financial and physical resources to any specific stor-
ple, with migratory birds, marine mammals, moun- age technique. The demands for space, materials,
tain gorillas, mature redwood trees, or even many record keeping and labour are much lower for
insects. researchers maintaining a few cultures being used in
The isolation and growth of microbial cultures are current research or teaching than for laboratories
dealt with elsewhere in this book. Although this keeping small archival collections with dozens to
chapter focuses on fungi, the techniques described several hundred cultures, or for general service cul-
here apply equally for nearly all other types of ento- ture collections that are actively acquiring, storing
mopathogens. and distributing large numbers of cultures.
No matter why or how one may store cultures, all Although it is not usually recognized as such, for-
preservation techniques increase the time between mulations of microbial biocontrol agents usually
transfers to periods ranging from several weeks or serve to preserve a living infective virus, bacterium
months to many years with a minimal loss of viabil- (except, possibly, for B. thuringiensis), microsporid-
ity or other key properties of the organism during ium or fungus. The 'active' ingredient of a formula-
storage. Each of these preservation techniques has tion remains in a quiescent but viable state during
strengths and weaknesses (Table 1). Once one's shelf storage to be activated upon application.
MANUALOF TECHNIQUESIN INSECTPATHOLOGY
ISBN 0-12-432555--6
~.70 Richard A. Humber

Table 1 Advantages and disadvantages of preservation techniques.


Preservation method Advantages Disadvantages
Storage temperature >_O~
Serial transfer (stored at 4 ~C) 9 Technologically simple 9 Basic (phenotypic) characters may
change
9 Allows continuous monitoring of 9 Continuing need for materials and
phenotype labour
9 Some fungi do not tolerate cold
Mineral oil 9 Inexpensive and technologically 9 Space intensive; tubes must be stored
simple upright
9 Must inspect periodically for
contaminants
Distilled water stasis 9 Inexpensive and technologically 9 Must check for water levels and
simple contaminants
9 Needs little space if using small vials
Lyophil (Freeze-dried) 9 Standard methods can be used for 9 Equipment is relatively expensive
many fungi 9 Ampoules should be refrigerated
9 Dried cultures can be mailed 9 Not suitable for some fungi

Storage temperature <0 ~C


Silica gel 9 Inexpensive and technologically 9 Long-term success depends on security
simple of screw-top seal
9 Uses standard appliance-type freezers
9 Can store only one tube per culture
9 These may be kept at room temp. or
-20~

Electric (mechanical) freezers


Standard (-20 ~C) 9 Inexpensive and readily available 9 Not recommended (except for silica gel
storage)
9 Subject to losses in event of power
failures
Ultracold (-80 or -120 ~C) 9 Convenient and available in many labs 9 Freezer temperatures favour ice crystal
9 Long-term cryostorage without liquid formation
NE 9 Subject to losses during power failures
9 Nitrogen back-up is recommended

Liquid nitrogen (LN2)freezers 9 Unexcelled for duration of storage 9 High initial and continuing costs
9 Long-term viability is maximized 9 Continuous access to LN2 is vital
9 All types of culturable fungi can be
stored
Vapour phase (-120 to 9 LNa supply cannot contaminate vials 9 Temperature is relatively unstable
-196~ 9 No risk of vials exploding when 9 LNE levels must be monitored
thawed continuously
9 Automatic LN2 level control depends on
uninterrupted electrical service
Liquid phase (-196 ~C) 9 Temperature is stable during immersion 9 Exclusion of nitrogen from entering
9 Ice crystal formation is unlikely plastic cryovials cannot be guaranteed
9 Monitoring of nitrogen levels is 9 Vial contents may be contaminated by
minimal nitrogen leakage during storage
9 Unaffected by loss of electrical service 9 Vials containing LN2 may explode when
thawed
,,

Examples of this approach include techniques to pre- spores and/or hyphae in alginate and starch (Pereira
pare dried mycelium that resumes sporulation when & Roberts, 1991; Salgado & Campos, 1993). The
rehydrated (McCabe & Soper; 1985; Rombach et al., production and formulation of entomopathogens is
1989; Verkleij et al., 1992) or the encapsulation of not covered further in this book.
F u n g i : P r e s e r v a t i o n of C u l t u r e s 271

There is a rich literature on the preservation of for such morphological changes and bioassayed peri-
fungal cultures. This chapter is not a comprehensive odically for changes of essential pathological charac-
review of all possible techniques but it does treat a teristics. Fennell (1960) and Onions (1971) discuss
range of techniques used for fungal pathogens of what fungal structures (hyphae, spores, etc.) consti-
invertebrates. More detailed discussions of these tute appropriate inoculum for serial transfers but the
techniques can be found in (uncited) journal articles staff of the USDA-ARS Collection of Entomo-
and in compendia on fungal preservation (Onions, pathogenic Fungal Cultures (ARSEF; Ithaca, NY)
1983; Simione & Brown, 1991; Smith & Onions, uses both hyphae and, if available, spores as the pre-
1994). ferred inoculum.
The protocols discussed here are not fixed in
stone; they can be adapted to meet individual needs
and limitations. The exact reproduction of published B Mineral oil
preservation protocols should not the ultimate goal;
that goal must be to preserve viable cultures by the Storing culture slants under a layer of sterile mineral
most successful, practical and reasonable means oil is one of the oldest, simplest and least expensive
suited to one's operating conditions. methods for long-term culture preservation. This
approach is still widely used, especially for fungi
that do not tolerate freeze drying or where cryogenic
storage is too costly.
2 PRESERVATION AT TEMPERATURES The oil both prevents desiccation and diminishes
ABOVE FREEZING gas exchange, thus reducing fungal metabolism to a
very low level. If space is available to store racks of
Storing fungi at ambient temperatures rather than in tubes, this is a common alternative for the storage of
refrigeration may require fewer resources than any a very diverse range of fungi. Onions (1971) warns
other preservation strategy, but it also seems to be the that McCartney bottles or other screwcap bottles
least dependable and shortest-term technique. with rubber gaskets should be avoided unless the
Several differing techniques - including serial trans- gaskets are removed since oil-soluble components in
fer, storage under mineral oil or distilled water, on the rubber may be toxic to cultures.
silica gel crystals, and even after lyophilization Cultures kept under mineral oil may remain viable
(freeze-drying) - may be used to prepare fungi for for decades (Cavalcanti, 1991; Silva et al., 1994).
storage at ambient temperatures. The phenotypic and Pathogenicity of entomopathogenic fungi may be
genotypic stability of the culture will be affected by undiminished after several months of storage
the choice of preparative method used and by both (Balardin & Loch, 1988), but whether pathogenicity
the overall range and stability of temperature during or virulence decline after many years of storage
the storage period. under mineral oil remains uncertain.

Set up for storage


A Serial transfer 9 Use vigorously growing culture slants in glass
culture tubes.
This most obvious way to store cultures is best suited 9 Autoclave a supply of heavy mineral oil and re-
for relatively short-term studies (over a few weeks or autoclave 24-48 h later to kill any bacterial
months), although it can serve adequately to main- spores activated by the first autoclaving.
tain small numbers of cultures for many years. 9 Aseptically cover the culture slant with sterile oil
There are several potentially serious drawbacks to to the depth of 1 cm.
relying on serial transfer of fungi. Repeated subcul- 9 Cover tubes with tight caps or plugs and apply a
turing may lead to such deleterious changes as losses couple of layers of paraffin film as a further
of pathogenicity, virulence, or sporulation, but there vapour barrier.
is no way to predict if or when repeated subculturing 9 Store tubes upright in racks. Viability may be
might result in losses of vital characters. Cultures of retained longer if refrigerated than if kept at room
invertebrate pathogens must be inspected carefully temperature.
272 R i c h a r d A. H u m b e r

Culture recovery 9 Dispense sterile water into sterile storage tubes.


9 With a sterile scalpel, loop, or needle, recover an 9 Inoculate tubes with small (ca. 1 mm 3) blocks of a
explant from the submerged culture. culture and/or an aqueous suspension of spores.
9 Drain excess oil from the explant and place on The volume of water must be >40 times the total
fresh medium. volume of the culture inoculum preserved in it.
9 Reseal and return the tube to long-term storage. 9 Cover tubes with tight fitting caps. For more
9 Monitor the culture for viability and/or contam- security, also seal with paraffin film or by dipping
ination. tube tops in melted paraffin.
9 Store tubes uptight at room temperature or in
refrigerator.
C Distilled water stasis
Culture recovery
Storage of metabolically inactive fungi under sterile 9 Recover a block from the tube with a sterile
distilled water may be least technologically demand- scalpel, loop, or needle and place (fungus side
ing of any preservation techniques discussed here. down) onto fresh medium.
This method is useful for many diverse fungi, but may 9 Monitor the culture for viability and/or contam-
be especially welcomed by those needing to preserve ination.
fungi that cannot be freeze-dried (e.g. Oomycetes; 9 Reseal the tube and return to long-term storage.
Clark & Dick, 1974). This method has been used suc-
cessfully with a wide range of fungi including human
or plant pathogens (Castellani, 1967; Figueiredo &
Pimentel, 1975). Viability of some fungi for up to 20 3 PRESERVATION BY FREEZE-DRYING
years has been documented with this method (Hartung (LYOPHILIZATION)
de Capriles et al., 1989) although many fungi lose via-
bility much sooner. No literature specifically describes Lyophilization may be the most widely used of the
using this technique for entomopathogens but it can 'technologically sophisticated' approaches to pre-
be used with the Entomophthorales (Humber, unpub- serving fungal germplasm and is the primary
lished). Water stasis is probably a suitable storage tech- technique used at most general service culture col-
nique for nearly all fungal pathogens of invertebrates. lections. Cultures can be processed for storage in a
This technique requires having only sterile screw- relatively short time after which the lyophilized
cap tubes or vials and sterilized water (tap water can ampoules can be filed and then sent out for revival by
be used if distilled or deionized water is unavail- the recipient.
able). Material in water stasis can be kept at room Freeze-drying is effective for nearly all conidial
temperature or refrigerated. fungi (Hyphomycetes and Coelomycetes), asco-
The most serious problems of this technique are mycetes, and basidiomycetes. It is not usually useful
easily avoided. Too much inoculum for the volume for fungi with very 'watery' cells (with large vacuo-
of water may jeopardize the ability of the fungus to lar volumes) such as many Oomycetes and the
withstand long-term storage; this can be avoided by Entomophthorales.
using a volume of water at least 40 times greater than Many different sorts of ampoules or other con-
that of the inoculum blocks. Evaporative loss of miners may be used to hold cultures during
water from poorly sealed storage tubes can be over- lyophilization (Simione & Brown, 1991; Smith &
come by periodically adding sterile water to those Onions, 1994). Most small- to medium-scale users
storage units that might need it. rely on manifold or centrifugal freeze-dryers and use
commercial lyophil ampoules or glass tubing sealed
Set up for storage at one end to make small tubes. Large-scale opera-
9 Use vigorously growing, relatively young cul- tions may use serum bottles dried on shelves in a
tures for inoculum. large vacuum chamber and sealed under vacuum by
9 Autoclave a supply of distilled water and screw- lowering a pressure plate onto the unseated tops of
cap vials or tubes (or use presterilized screwcap the bottles.
tubes, tissue culture flasks, or centrifuge tubes). The following discussion on methods for
F u n g i : P r e s e r v a t i o n of C u l t u r e s 273

lyophilizing fungi is a condensed summary of the quantity of liquid will usually be indicated by the
process; detailed protocols are needed only in labor- culture's sender) to reconstitute the culture.
atories with access to a lyophilizer and are readily 9 Rest the material in a sterile hood for 1-30 min to
available in general reference works on culture soften and to rehydrate the dried pellet.
preservation (e.g. Simione & Brown, 1991; Smith & Resuspend and pipette the reconstituted mixture
Onions, 1994). However, because nearly any labo- onto fresh culture medium.
ratory studying fungi may receive and need to revive 9 Monitor the culture for viability and/or contam-
lyophilized cultures, detailed instructions for doing ination.
so are included below.

Set up for storage


9 Use sporulating cultures for inoculum. (Note: 4 PRESERVATION IN A FROZEN STATE
Spores may retain viability better than hyphae but
non-sporulating cultures can also be lyophilized.) Standard domestic freezers (operating a t - 2 0 ~
9 Sterilize and dry cotton-plugged ampoules. might seem to be an ideal and economical tool for
9 Cover sporulating cultures with sterilized skim keeping frozen cultures. However, such freezers are
milk solution or another nutritionally complex not recommended for preserving cultures although
carder, and suspend spores and hyphae. Transfer Carmichael (1962) successfully kept peptone-yeast
small quantities of this suspension to sterile extract cultures of a wide range of fungi a t - 2 0 oC.
ampoules and 'cure' the contents for several The most reliable long-term cryopreservation tech-
hours in a refrigerator. (Note: If left overnight in niques require much colder temperatures; it is
the milk solution, some fungi such as critical to understand that the colder the storage tem-
Metarhizium anisopliae may clear this carrier but perature, the greater the likelihood will be for long-
do so without affecting the viability of the term viability.
lyophilized culture.)
9 Rapidly freeze the preparations in a mixture of
dry ice and either ethanol or propylene glycol or A Silica gel
by placing ampoules in an ultracold freezer or in
the vapour phase in a liquid nitrogen dewar). Storage of spores on sterile, anhydrous silica gel
9 Attach frozen ampoules to a strong vacuum on crystals is the only storage technique for micro-
the lyophilizer. Preparations must remain frozen organisms that recommends use o f - 2 0 ~ freezers.
during the initial stages of vacuum desiccation. It is possible to store fungus-inoculated silica gel at
After desiccation, ampoules are flame-sealed room temperature if freezer space is unavailable but
while still under vacuum. fungal viability will usually decrease sooner. Fungal
9 Store ampoules at ca. 4 oC. They may also be kept spores stored on silica gel may remain viable for
at ambient temperature if necessary but viability more than ten years (Windels et al., 1993), and this
will probably decline sooner than if refrigerated. method is inexpensive, simple and reliable for many
fungi (Smith, 1993) including entomopathogens
Culture recovery (Bell & Hamalle, 1974).
9 Most culture collections that send out lyophilized The use of anhydrous silica gel crystals as a carrier
cultures provide detailed directions on how to for culture propagules is limited to aerobic bacteria
open and reconstitute such preparations. and fungi that grow on solid culture media. Fungi
9 If an ampoule is not pre-scored, score the neck with high ratios of vacuolar to cytoplasmic volumes
with a file or diamond pencil. (e.g. Oomycetes and Entomophthorales), viruses,
9 Surface sterilize in 70% ethanol or sodium microsporidia and anaerobes are unsuited for storage
hypochlorite solution (e.g. a 1 : 1 dilution of com- on silica gel.
mercial bleach), wrap the scored ampoule in a Although several grades of silica gel are available
sterile paper wipe moistened (but not soaking!) commercially, only some of them are acceptable for
with ethanol and break at the scoring. storing fungi. The crystals should be relatively large
9 Add sterile water or liquid medium (the type and and uncoloured (with no indicator dye); the blue
274 R i c h a r d A. H u m b e r

indicator dye that turns pink when hydrated can be longer-term than storage in ultracold freezers, is both
toxic to fungi (Perkins, 1962). more expensive and wholly dependent on a continu-
Because the uptake of water by anhydrous silica ous supply of liquid nitrogen, a commodity that is
gel is a strongly exothermic reaction, silica gel tubes either unavailable or prohibitively expensive for
must be put in an ice bath or to chilled in a freezer to most laboratories.
-20 ~ to dissipate the heat of hydration during their Virtually every fungus that can be preserved by
inoculation. any of the techniques noted above or that can be
cultured in vitro can be preserved cryogenically.
Set up for storage Further, many fungi that cannot be preserved by
9 Use sporulating cultures for inoculum. such techniques as lyophilization can be stored
9 Fill 25 x 200 mm screw-cap glass tubes one-third cryogenically.
full with 6-12 mesh, grade 40, uncoloured anhy- Despite the versatility of cryogenic techniques,
drous silica gel crystals. they are no panacea; all techniques to preserve
9 Sterilize the tubes and silica gel in an oven at germplasm have flaws and weaknesses (see Table 1).
160-180 ~ for 1-6 h to assure that the silica gel The viability of frozen cultures may be strongly
is both sterile and fully anhydrous. affected by such factors as the choice of cryoprotec-
9 Dispense 1-5 ml sterile w a t e r - or an autoclaved tant, the rate of freezing, and temperature stability
solution of 5-7% (v/v) skim m i l k - into a sporu- during storage; these same factors may also affect
lating culture. Cap and agitate the tube or rub the the viability of freeze-dried cultures (Tan et al.,
surface of the plate with a sterile glass rod. 1995). The physical factors affecting the freezing,
9 Dispense 1 ml of the suspension by drops onto storage and recovery of living cells demand an
cold silica gel crystals. Tilt tubes during inocula- awareness of some basic principles of chemistry and
tion to expose the greatest possible surface area physics; these points and other general considera-
and rotate or agitate tubes while adding the inocu- tions about cryopreservation are synthesized and dis-
lum suspension. cussed by Calcott (1978).
9 Hold inoculated tubes at room temperature for a What happens to water in and around cells during
few days, rotating or agitating them periodically freezing, storage and thawing may be the most criti-
until all water has been absorbed and the crystals cal such factor: Water freezes in one of two states,
are separated. either the amorphous (glassy or vitreous) state or the
9 Store a t - 2 0 ~ although tubes may also be kept crystalline (icy) state. Cryoprotectants serve to
at room temperature. favour the freezing of intracellular water as a glass
9 Check viability and sterility of the stored prepara- rather than as ice crystals whose growth may disrupt
tion after ca. 1-2 weeks. or destroy a cell's membrane systems. Frozen water
can convert from the glassy into the crystalline state
Culture recovery (leading to the initiation and growth of ice crystals
9 Sprinkle a few granules of inoculated silica gel while remaining frozen!). This devitrification, the
from a tube onto fresh culture medium. shift out of the glassy state by ice nucleation, is
9 Tightly reseal tube and return to long-term storage. increased by fluctuation of the storage temperature
(discussed below) and is favoured at a few tempera-
tures that are troublingly close to those in ultracold
mechanical freezers. One of these glass transition
B Cryogenic preservation in mechanical or
temperatures for frozen protein solutions is - 8 0 ~
liquid nitrogen freezers (Chang & Randall, 1992). In water-glycerol mix-
tures, ice nucleation (without much crystal growth)
1. General considerations and protocols
is favoured throughout the temperature range of -93
The widespread proliferation of ultracold freezers in t o - 1 2 3 ~ (Vigier & Vassoille, 1987).
biological laboratories has enabled growing numbers
of biologists to store cultures of their research organ- a. Cryoprotectant
isms cryogenically. Storing cultures in liquid nitro- Many different cryoprotectants can be used to help
gen dewars, while theoretically still more secure and prevent the formation of ice crystals during freezing,
Fungi: Preservation of Cultures 275

storage and thawing of cultures. Some of the cryo- chosen since they are more difficult to use during the
protectants that are widely used in various laborato- freezing process and may be especially hazardous if
ries include glycerol, dimethylsulphoxide (DMSO), they shatter when recovering material from storage.
polyethylene glycol and propylene glycol. In prac- The exclusion of liquid nitrogen from the interior
tice, however, most laboratories preserving fungi use of storage units is the most critical issue for choos-
only 10% glycerol (v/v); this dependence on a single ing a unit to be stored by immersion in liquid nitro-
cryoprotectant is based on the convenience and suit- gen. The leakage of nitrogen into storage units may
ability of this cryoprotectant rather than from any result in contamination of the preserved material as
experimental demonstration of its optimal effective- well as explosions of these units during thawing.
ness (see Sanskar & Magalh~es, 1994). Glycerol is Sections of polypropylene drinking straws whose
used for virtually every fungus in the ARSEF collec- ends are heat-sealed are an inexpensive alternative
tion (more than 300 species from several fungal to screwcap cryovials (Challen & Elliott, 1986;
classes); the few fungi that have been frozen in 6% Stalpers et al., 1987) and, if their end seals are
(v/v) DMSO are entomophthoraleans. DMSO is not secure, can be immersed safely in liquid nitrogen.
usually used as the cryoprotectant for fungi, how- Cryogenic vials can be protected from nitrogen infil-
ever, until after repeated failures to freeze an isolate tration during immersed storage by sheathing entire
with glycerol as a cryoprotectant but while varying sets of vials or individual vials in plastic tubing that
the inoculum by using very young or somewhat can be heat-shrunk onto the vials and sealed by heat-
senescent cultures or cultures grown on a different crimping either end of the tubing section before
medium from that routinely used for the fungus. In freezing the material. However, cultures delicate
many instances, isolates that resist repeated attempts enough to require such sheathing may be unable to
to freeze them are those that grow poorly in culture, tolerate the heating received during the sealing
and which tend to die after several serial transfers. process. It has been the experience of the ARSEF
The choice of cryoprotectant determines the tem- collection staff that those cultures grown in tissue
perature, the heat of fusion, at which the cryoprotec- culture media- the isolates that are most vulnerable
tant freezes with a strongly exothermic reaction. to contamination during storage immersed in liquid
Electronically controlled cell freezers can dissipate nitrogen- are the cultures least able to tolerate this
this heat while allowing material to continue freez- heat-sealing.
ing at a highly uniform overall rate, usually ca. -1 ~ C
per minute. c. Auxiliary equipment for culture freezing
Regardless of the cryoprotectant used, cryoprotec- An inexpensive, commercially produced polycar-
tant solutions should be made and filter sterilized bonate freezing container (Nalgene TM Cryo 1 ~
(not autoclaved!) in relatively small batches to Freezing Container) can be a very suitable low-tech-
restrict contamination hazards since a contaminated nology tool for producing a semi-controlled rate of
cryoprotectant stock solution will contaminate every freezing approximating-1 ~ This device has
culture treated with it. been used successfully to freeze entomophthoralean
protoplasts (A.E. Hajek, unpublished), a growth
b. Cryogenic storage units form that is among the most difficult to freeze among
Plastic cryogenic vials, plastic straws, or glass all of the entomopathogenic fungi. This commercial
ampoules are used to contain material to be frozen. freezing device accommodates 18 cryovials. After
The choice among these storage units is driven by conditioning the loaded cryovials at refrigerator tem-
the racking system chosen for the physical contain- perature for several hours, the rack containing them
ment of individual storage units, by convenience in is placed on a pool of isopropanol in this freezing
handling, by cost and by safety considerations. container and the whole assembly put in an ultracold
Although they are not the least expensive alternative, freezer for 24 h. The temperature in the vials in this
commercially available plastic cryogenic vials are container drops at about 1 ~C/min.
probably the most commonly used storage units; Styrofoam boxes with sides 2.5 cm thick and tops
polypropylene drinking straws (which may be the and bottoms 1.25 cm thick can be used as freezing
least expensive storage containers) are less com- containers, but these may have much faster freezing
monly used. Glass ampoules or tubes are rarely rates than an isopropanol-mediated freeze or those in
276 R i c h a r d A. H u m b e r

electronically controlled (and vastly more expen- after a new lot is frozen with acceptable post-
sive) cell freezing units. recovery characteristics.
Electronically controlled, programmable freezing
units are most useful in large-scale operations Culture recovery
because of their high capacity and precise controls of 9 Thaw all cryogenically stored material directly in
freezing curves. These expensive freezing units are 37 ~ water. Leave units in the warm water only
driven with liquid nitrogen vapours and are best until the ice is completely melted. (IMPORTANT
suited for use in laboratories that depend heavily on SAFETY WARNING: For material stored
cryogenic storage of germplasm or on highly immersed in liquid nitrogen, visually check trans-
controlled freezes to ensure viability of valuable but parent or translucent storage units upon removal
relatively delicate germplasm. from storage for moving liquid (nitrogen) inside. If
McLaughlin et al. (1990) suggested that there was motion is seen, allow nitrogen to vaporize by
little difference in viability of human spermatozoa warming at room temperature for a minute or two
frozen in an uncontrolled manner in nitrogen vapour under a metal container Placing a glass or plastic
or in an electronically controlled freezing unit. It has unit enclosing liquid nitrogen directly into warm
been the experience of the ARSEF collection staff water can result in a small but violent explosion,
that most entomopathogenic fungi, especially flying shards of the storage unit (and risks of seri-
hyphomycetes, have excellent recoverability after ous bodily injury), and the widespread dispersion
having been frozen by direct plunging into liquid around a laboratory of a microbial culture. When
nitrogen. thawing frozen cultures, always use proper pro-
tective g e a r - lab coat, cryogenic gloves and a
d. Freezing and thawing protocols full face m a s k - and keep unprotected workers
2 - 3 m away from thawing vials.)
Set up for storage
9 For cultures grown on solid media: Dispense e. Checking viability
sterile cryoprotectant into storage units (vials, No matter what preservation method is chosen,
straws, etc.) and then add 2 - 4 cubes (1-3 mm on always check the viability of a preserved sample rel-
a side); make sure that there is >40:1 ratio of atively few days after its preparation. If the test
cryoprotectant to inoculum (as in Section 2C for sample is viable and uncontaminated upon recovery,
water stasis). the remaining samples should be reliable for long-
9 For cultures grown in liquid media: Add an term storage. If the sample is not viable or is con-
appropriate volume of undiluted, sterile cryopro- taminated, it is likely that rest of the lot is similarly
tectant directly to cultures and disperse quickly unacceptable. Once a new, successful freeze is com-
by gentle agitation to minimize osmotic damage. pleted and confirmed, the older, inviable material
Aliquot directly into sterile cryovials. should be discarded. If a viability check indicates
9 'Cure' fungus in cryoprotectant at 4~ for contamination or any loss of a culture's essential
2-48 h to allow uptake of the cryoprotectant. properties, it is best to check the original culture and
Most ARSEF cultures are cured overnight (ca. to pull a second confirmatory unit from storage. If
12 h) before freezing. the second unit also fails to meet the required stand-
9 Freeze at an uncontrolled rate by direct placement ards, try a new freeze procedure and, when the
into the storage freezer or in a controlled freezing isolate has been successfully frozen, discard all
device (see Section 4A.lc). With controlled and unsuccessfully preserved material.
semi-controlled freezes, cultures are usually
frozen to between --40 a n d - 5 0 ~ and held there f. Stability of temperature during storage
for 30-120min before being moved to the The stability of temperature during frozen storage
storage facility. may be more important than the actual temperature
9 1-7 days after freezing, thaw a unit of each cul- at which storage occurs. The fewer and smaller the
ture to check for viability, growth rate, morphol- temperature fluctuations during frozen storage, the
ogy, etc. If this viability check does not meet all less likely it will be that ice crystals can form and
expectations, the entire lot should be discarded grow to damaging sizes in the preserved cells.
F u n g i : P r e s e r v a t i o n of C u l t u r e s 277

MacFarlane et al. (1992) suggest that optimal stor- 200 ~ hotter than the long-term storage temperature
age conditions require both the coldest possible tem- each time an individual stack of boxes is retrieved
perature and greatest possible temperature stability. from storage, and may be submitted to lesser warm-
The optimal conditions for long-term storage of liv- ing each time the stack is partially raised to gain
ing material should be, therefore, immersion in li- access to higher-placed boxes in it. Even though
quid nitrogen a t - 1 9 6 ~ However, the exclusion each of these warming periods may only last a few
from storage units of nitrogen and nitrogen-borne seconds, it may still be enough time and temperature
contaminants is a challenging problem for material change to damage the least robust fungi. For an
immersed in liquid nitrogen. Storage in nitrogen instructive comparison about the magnitude of such
vapour over a pool of liquid nitrogen avoids this con- temperature fluctuations, consider that plunging
tamination hazard but is neither so cold nor so stable your hand into boiling water for only a few seconds
as immersion in the liquid phase. In a nitrogen might entail a relatively smaller temperature change
dewar, the temperature in the vapour phase ranges for a comparable time span to that experienced by
from-196 ~ at the vapour/liquid interface up to ca. frozen material every time it is pulled from a nitro-
-120~ at the top of the dewar. gen dewar or ultracold freezer. Despite the theoreti-
In addition to the absolute temperature during cal possibility for damage due to large and rapid
storage, the physical racking system in which stor- temperature fluctuations, practical experience with
age units are placed within a freezer is also critical. cryogenic storage in many laboratories suggests that
This racking dramatically affects the overall stability this sort of handling-induced temperature fluctuation
of temperature for individual units during storage. does not routinely cause obvious damage to most
Vials or straws clipped to vertical aluminium canes cultures.
stored immersed in liquid nitrogen need never be
removed from liquid nitrogen except to remove the
2. Comments about specific cryopreservation
topmost unit; such storage conditions are the theo-
facilities
retical optimum. To facilitate the location of specific
vials, canes are usually grouped in tall boxes that a. Mechanical freezers
are, in turn, arranged in a single palisade layer. Standard freezers (-20 ~C). Except for cultures pre-
Stacked racking systems like those usually illus- served on silica gel (see Section 4A), stand-alone
trated in sales literature for large cryogenic storage standard (upright or chest) freezers or the freezing
systems - sets of vertical stainless steel racks hold- units of two-door refrigerator-freezer combination
ing vertical stacks of covered boxes that are intern- units are not recommended for preserving fungal
ally divided to hold individual cryogenic u n i t s - cultures. The freezing units inside single-door refrig-
should be avoided because the manner in which they erator-freezer combination appliances may only be
must be handled assures enormous temperature fluc- able to achieve temperatures of ca. -5 ~C. The tem-
tuations for stored material every time any material perature of all of these freezers is too high (too close
is added or removed from the stack. In such a sys- to freezing) and too unstable to be truly reliable for
tem, the most temperature-stable positions are those germplasm storage. Frost-free freezer units undergo
at the top where a stack may be lifted a minimal dis- regular, periodic heating cycles to maintain a frost-
tance in order to withdraw a box, place it on top of free interior; this fluctuating temperature, as noted
the other stacks, remove its top and the desired stor- above for cryogenic storage, is potentially harmful to
age units from it, and then to close and return the cultures and should be avoided.
box to its permanent storage position. To recover Ultracoldfreezers (-80 or-120~ Many laborato-
material from the lowest boxes in a stack (in the ries store cultures in ultracold mechanical freezers.
coldest storage positions), all the boxes in the stack Chest-type freezers are inherently more temperature
must be lifted out of nitrogen (or its vapours) until stable and preferable to uptight designs. Although
the desired box is accessible and removed; after ultracold freezers may be very adequate for storing
material is recovered, the stack must be lifted out many fungi, liquid nitrogen back-up facilities are
again to return the box to its position. In this worst- recommended to be attached to them to maintain the
case scenario, material in the stack will be exposed temperature during power failures. Further, as was
to temperatures from 70~ to potentially more than noted above, the temperatures in these freezers are
278 Richard A. Humber

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b. Liquid nitrogen freezers entomopathogenic fungal insect control agent. US
Storage in or over liquid nitrogen is the most expen- Patent No. 4,530,834 (23 July 1985).
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tion methodologies. Dependence on such an A comparison of the freezing of human semen in
uncirculated vapor above liquid nitrogen and in a
approach to germplasm preservation requires a seri-
commercial semi-programmable freezer. Hum.
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