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Preservation and recovery

of filamentous fungi
Introduction and pool the suspensions into one tube. A

Preservation methods for filamentous fungi vary depending on

concentration of at least 106 spores per mL
of milk solution is needed.
Tech
the type and degree of sporulation. Spore-forming strains (with Bulletin
the exception of zoosporic fungi) can usually be freeze-dried 5. Dispense 0.2 mL of the suspension into
successfully. Similar success with nonsporulating strains is far less each vial for freeze-drying. Many spores No. 2
likely. Both types can be frozen and stored for long periods in liquid will begin to germinate when suspended
nitrogen or liquid nitrogen vapor. All plasmid-containing or mutant in liquid, so timing is critical when filling
strains should be frozen directly from the original material if vials. Spores should not be in the skim milk solution for more than
possible to prevent any alteration or loss of desired characteristics. two hours before being processed. Refrigerate filled vials while
waiting for further processing.
The following overview discusses preservation methods used
at ATCC and is presented for descriptive purposes only; it is not Freeze-drying methods
intended as a laboratory protocol. Anyone planning to preserve Although the process is somewhat labor-intensive, freeze-drying
cultures by these methods is strongly advised to study detailed, spore-forming fungi greatly facilitates their distribution and
published protocols before proceeding. storage. The following four methods are described in detail in
Simione and Brown (1991).

Freeze-drying sporeformers 1. Component freeze-dryer—Samples are freeze-dried in cotton

plugged glass inner vials which are then sealed inside glass outer
Preparing the culture ampules under vacuum. The components of the system (vacuum
1. Grow fungi under conditions that will induce maximum pump and condenser) are assembled on a benchtop.
sporulation so that sufficient spores will survive the freezing and
drying process. Optimum media and growth conditions are listed 2. Commercial freeze-dryer—Samples are prepared in glass
in the strain descriptions in ATCC’s online catalog at www.atcc. vials in ampules (as in no. 1 above), and then freeze-dried in a
org. Literature cited there may also give further guidance on commercial freeze-dryer.
appropriate cultivation procedures.
3. Serum vial—Samples are processed in glass serum vials sealed
2. Prepare a 20% solution of skim milk and autoclave at 116°C for with rubbers stoppers and metal caps. A commercial freeze-dryer
20 minutes in 10 mL tubes. One tube is usually more than enough is used.
for 10 freeze-dried vials unless the culture is very mycelial. Store
the milk solution at 2-8°C until needed so that it will be cold 4. Manifold—Samples are processed in bulbshaped or tubular
when used. glass ampules attached with latex tubing to a manifold. (This
method is not used at ATCC, but is relatively inexpensive and uses
3. Prepare the spore suspension by slowly introducing about 2 mL of equipment that a lab may already own.)
milk solution into the culture tube or plate while gently scraping
the surface of the culture with a pipette. Take care to avoid Recovery
raising a cloud of spores, especially with Aspergillus, Penicillium, 1. If the culture to be recovered was obtained directly from
and other fungi that produce large amounts of dry spores. For ATCC, check it thoroughly upon receipt. If it is found to be
example, Neurospora spores are very difficult to contain. It is unsatisfactory in any respect, notify ATCC so that the strain in
therefore recommended that cultures for freeze-drying are question can be investigated.
grown on agar in 250 mL Erlenmeyer flasks.
2. Open the ampule as directed and, using sterile distilled water
4. Transfer the suspension back into the tube containing the and a sterile pipette, transfer the contents of the preparation to
remainder of the skim milk solution and mix thoroughly. If more approximately 5 mL of sterile distilled water in a test tube.
than one plate or tube is used, repeat the procedure for each
 3. Allow the contents to rehydrate for at least one hour (two hours into one sample containing only sterile medium. Prior to starting
is better, and overnight is not too long) before transfer of a few the cooling program, allow the material to cool to within 2°C of
drops to broth or agar. Use the media and growth conditions the chamber temperature.
specified in the strain descriptions when first subculturing to
ensure optimal recovery. 6. Cool the material at a rate of 1°C per minute to –40°C, then cool
10°C per minute to –90°C.
4. Incubate at the appropriate temperature. (The remainder of the
suspension may be stored for a few days if refrigerated, allowing 7. When the program is complete, transfer the canes to boxes
for another recovery attempt if the first should fail.) Given proper in the vapor phase of a liquid nitrogen unit for storage. If the
treatment and conditions, most cultures will grow in a few days. unit is more than a few feet from the programmable freezer,
However, some may exhibit a prolonged lag period and should be transport the canes in an insulated container with liquid nitrogen.
given twice the normal incubation time before being discarded as Be careful not to store the vials directly in liquid nitrogen. See
nonviable. notes under “Safety” below. Liquid nitrogen has a temperature of
–196°C. The vapor will have a temperature gradient which is near
5. For special media, growth conditions, and tips on maintenance, –196°C at the level of the liquid and which gradually becomes
carefully read the literature cited in the strain descriptions. This is warmer near the top of the freezer. For best long-term storage,
especially important for producers of secondary metabolites and keep frozen materials below –130°C. To ensure that materials
quality control strains. are stored at proper temperatures, liquid nitrogen freezers must
first be validated by placing a thermometer at the top of the
unit and adding liquid nitrogen until a working temperature of
Freezing Filamentous FungI at least –130°C is maintained at the top of the freezer. This level
is then continuously monitored and an alarm system activated
Preparing the culture if the levels fluctuate above or below predetermined limits.
Grow sporulating strains on solid media as for freeze-drying; grow Occasionally, strains may require special handling. If a strain does
nonsporulating strains on either solid or liquid medium. If the not survive freezing in glycerol, try 5% DMSO. Some cultures, such
mycelium is easily broken, grow the culture on agar in test tubes, as some Agaricus strains, may be grown on sterile seeds, grains or
scrape with a pipette, and suspend the fragments in sterile 10% pollen, and may be frozen without a cryoprotectant.
glycerol. Dispense 0.5 mL into each plastic vial. If the mycelium is
sticky, will not break up, or grows embedded in the agar, grow the Recovery
culture on agar in plates, cut out plugs containing new growth Thaw frozen cultures quickly in a 37°C water bath, transfer
(hyphal tips) with a sterile cork borer, and place three or four plugs immediately to appropriate growth media, and incubate at an
into each plastic freezing vial with approximately 0.4 mL of 10% appropriate temperature. Given proper treatment and conditions,
glycerol. most cultures will grow in a few days. However, some may exhibit
a prolonged lag period and should be given twice the normal
Freezing methods incubation time before being discarded as nonviable. For special
To ensure long-term viability of fungal cultures, ATCC recommends media, growth conditions, and tips on maintenance, carefully read
freezing and storage at liquid nitrogen temperatures. Storage in the literature cited in the strain descriptions. This is especially
the liquid itself is not always convenient or safe. Storage in liquid important for transformation hosts, genetic mutants, producers of
nitrogen vapor is a more practical alternative. It is critical, however, secondary metabolites, and quality control strains.
to constantly monitor the liquid level of the liquid nitrogen freezer
to ensure that material is maintained below –130°C. Storage at
warmer temperatures can compromise the stability of many strains. Safety

1. Select a container appropriate for the material to be preserved. Safety precautions must be considered when preserving living
Plastic screw-capped vials with internal tube threads (1.0 to cells and microorganisms by freeze-drying, freezing, and storing at
2.0 mL) are appropriate for most fungi. They are sterilized by cryogenic temperatures.
the manufacturer and, when properly handled, remain sterile
throughout labeling and dispensing. Cryogenic storage
Because of its extremely cold temperature, liquid nitrogen can
2. Label each vial clearly and accurately. Whatever labeling method be hazardous if improperly used. When handling liquid nitrogen,
is chosen, labels must be able to withstand subsequent freezing take precautions to protect your face and exposed skin from
and thawing procedures. exposure to the liquid. Wear protective clothing, including a
laboratory coat, gloves designed for handling material at cryogenic
3. Prepare the cultures for freezing as described above. Seal the temperatures, and a face shield. To reduce your exposure to
plastic vials as tightly as possible with the screw cap. cryogenic temperatures, design inventory systems for storing
frozen specimens that allow for easy retrieval and that minimize
4. Load the vials onto aluminum canes and record the location of the time required to look for specimens. Prolonged exposure to
each culture. cryogenic temperatures can lead to a loss of sensation in the hands
that can only be recovered after warming. This loss of sensation
5. Cool the chamber of a controlled-rate cooling apparatus to 4°C can lead to a false sense of security regarding damage to tissues by
and place the canes into the unit. Insert the thermocouple probe the low temperatures. When the temperature in a liquid nitrogen
unit becomes tolerable and working in the unit is no longer

2 www.atcc.org.
uncomfortable, the operator has reached a point where damage 3. Brown RW, Gilbert P. Microbiological Quality Assurance: A Guide
from the cryogenic temperatures is likely. When liquid nitrogen is Towards Relevance and Reproducibility of Inocula. Boca Raton,
used in confined and inadequately ventilated areas, the nitrogen Fla.: CRC Press; 1995.
can quickly displace the room air. Liquid nitrogen freezers should be 4. Booth C (ed.). Methods in Microbiology. Volume 4. London:
located in well-ventilated areas, and special precautions should be Academic Press; 1971.
taken during fill operations. In facilities with several liquid nitrogen
freezers, an oxygen monitor should be installed to warn occupants 5. Chang ST, Hayes WA. The Biology and Cultivation of Edible
of any deterioration in the air quality due to the nitrogen gas. Mushrooms. New York: Academic Press; 1978.
Plastic screw-capped vials can present a hazard if stored directly 6. Chang ST, Miles PG. Edible Mushrooms and Their Cultivation.
in liquid nitrogen. Vials with an inadequate seal between the cap Boca Raton, Fla.: CRC Press; 1989.
and the vial can fill with liquid nitrogen. Upon retrieval to warmer 7. Chang ST, Quimio TH. Tropical Mushrooms: Biological Nature
temperatures the vials may explode violently or may spray liquid, and Cultivation Methods. Hong Kong: The Chinese University
potentially disseminating the contents of the vial. Likewise when Press; 1982.
opening plastic vials after thawing some dissemination of the
8. Dhingra OD, Sinclair JB. Basic Plant Pathology Methods. 2nd ed.
contents may occur. Therefore material in plastic ampules should be
Boca Raton, Fla.: CRC Press; 1995.
stored in the vapor above the liquid nitrogen.
9. Elliott TJ (ed). Science and Cultivation of Edible Fungi. Volumes
Freeze-drying 1 and 2. (Mushroom Science XIV) Rotterdam: A.A. Balkema;
When freeze-drying microorganisms in vials or ampules without 1995.
cotton plugs or other bacteriological filters, the microorganisms 10. Fassatiova O. Moulds and Filamentous Fungi in Technical
can be carried from the container and contaminate the freeze- Microbiology. Amsterdam: Elsevier; 1986.
drying system. Microbial contamination can be found on the
11. Flegg PB, Spencer DB, Wood DA (eds.). The Biology and
outside of the vial or ampule, and on parts of the freeze-drying
Technology of the Cultivated Mushroom. New York: John Wiley;
system such as the condenser. A system should be designed to
1985.
monitor the contamination level, and decontamination procedures
should be implemented if necessary. Take care to properly treat 12. Fletcher JT, White PF, Gaze RH. Mushrooms: Pest and Disease
freeze-dried cultures prior to disposal. To autoclave freeze-dried Control. 2nd ed. Andover, Hants, England: Intercept; 1989.
cultures, open the vial or ampule to allow penetration of the steam. 13. Fuller MS, Jaworski A (eds). Zoosporic Fungi in Teaching and
An alternative to autoclaving is to heat the preparations in a hot air Research. Athens, GA: Southeastern Publishing Corp; 1987.
oven at 180°C for four hours.
14. Hunter-Cevera JC, Belt A. Maintaining Cultures for
Biotechnology and Industry. New York: Academic Press; 1996.
Culture handling
When opening frozen or freeze-dried cultures, take care to prevent 15. Johnston A, Booth C. Plant Pathologist’s Pocketbook. 2nd ed.
dispersion of the ampule contents. Open these preparations in Farnham Royal, Slough, England: CAB International; 1983.
a biological safety cabinet if possible, and perform all work with 16. King AD Jr., Pitt JL, Beuchat LR, Corry JEL (eds). Methods for
hazardous cultures in a biological safety cabinet. There are varying the Mycological Examination of Food. New York: Plenum Press;
degrees of pathogenicity among microorganisms. All laboratory 1986.
personnel should be aware of the hazards posed by the cultures 17. Nakasone KK, Peterson SW, Jong, SC. Biodiversity of Fungi:
they are handling. Detailed discussions of laboratory safety Inventory and Monitoring Methods. Amsterdam: Elsevier
procedures are provided in the latest U.S. Dept. of Health and Academic Press; 2004.
Human Services / CDC publication Biosafety in Microbiological and
Biomedical Laboratories. This publication is available in its entirety on 18. Simione F, Brown EM. ATCC Preservation Methods: Freezing and
the CDC Office of Health and Safety website at: Freeze-drying. 2nd ed. Rockville, Md.: ATCC; 1991.
www.cdc.gov/od/ohs. 19. Smith D, Onions AHS. The Preservation and Maintenance
of Living Fungi. 2nd ed. Wallingford, Oxon, England: CAB
International; 1994.
Cultivation and Preservation Literature 20. Stamets P. 1993. Growing Gourmet & Medicinal Mushrooms.
Berkeley, Calif.: Ten Speed Press.
For any strain listed on ATCC’s website, please note the
recommended media and incubation conditions. The literature 21. Tuite J. Plant Pathological Methods: Fungi and Bacteria.
cited below is useful for general knowledge on the cultivation and Minneapolis, Minn.: Burgess Publishing Co; 1969.
preservation of a wide variety of fungi. 22. van Griensven LJLD. The Cultivation of Mushrooms. Rustington,
Sussex, England: Darlington Mushroom Laboratories; 1988.
23. Wuest PJ, Royse DJ, Beelman RB (eds.). Cultivating Edible Fungi.
reFerences Amsterdam: Elsevier; 1987.

1. Atlas RM. Handbook of Microbiological Media. 2nd ed. Boca

Raton, Fla.: CRC Press; 1996.
2. Atlas RM. Handbook of Media for Environmental Microbiology.
Boca Raton, Fla.: CRC Press; 1995.

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