0095-1137/92/041000-05$02.00/0
Copyright ©1992, American Society for Microbiology
Viability of Fungal Cultures Maintained
at
-70°C
LESTERPASARELLtAND MICHAELR. McGINNIS* Department ofPathology, UniversityofTexasMedical Branch,
Galveston, Texas 77550
Received 12November1991/Accepted 14 December 1991
One thousand four hundred forty-seven clinical and environmental isolates of molds, yeasts, aerobic
actinomycetes, and algaebelongingto164genera(382 taxa) maintainedonpotatodextroseagarat -70'C for
periods ranging from 6 monthsto13years were subcultured and then incubatedat25°Ctodeterminetheir
viabilities. Thirty-threeisolates, Alternaria alternata(n = 1),Apophysomyceselegans(n = 1),Bipolaris
spicifera
(n = 1), Blastomyces dermatitidis (n = 4), Cokeromyces recurvatus (n = 1), Coremiella cubispora (n = 1),
Cryptococcusater(n = 1), Curvulariasp. (n = 1),Exserohilummonoceras(n = 1), Exserohilumpedicillatum(n
= 1), Exserohilum rostratum (n = 1), Filobasidium floriforme (n = 1), Madurella mycetomatis (n = 1),
Oedocephalum spp. (n = 2), Penicillium marneffei (n = 1), Pseudomicrodochium spp. (n = 4), Saksenaea
vasiformis (n = 1),Sporothrixsp. (n = 1), and MyceliaSterilia(n = 8), didnotgrowafter repeatedattempts atsubculturing. Neither time in storage nortaxonomic classificationwasassociated with a lackof viability.
Storageatlowtemperaturefor either shortorlong periodsoftime isanexcellent method for maintainingmost medically importantfungi.
Culture collections containing fungi and related
microor-ganisms are of importance to biologists, microbiologists, epidemiologists, andothers involved in the healthcareand natural sciences. Several methods of preservation of fungi
have been proposed and studied since Castellani (4) first
suggested thepreservation offungiin steriledistilledwater. Since then, fungi have been preserved for long periods of time insoil,onoil-coveredslants,at -20to -70'C,inliquid nitrogen, andby lyophilization (1-8). All of these methods
haveproven very successfulin preserving cultures for 1 to
many yearswithout requiringcontinuous subculture.
Prob-lemsarisewith thebiological orinnatevariabilitiesofsome of the organisms tested after storage by all of the ap-proaches.
Onethousand four hundredforty-seven isolatesof
medi-callyandagriculturally important species ofmolds, yeasts, aerobic Actinomycetes, and algae that were maintained in our culture collection for periods ranging from 6 months to 13 years were selected for study. Only pure
cultures that were identified by contemporary taxono-mic criteria and methods were included in the study. All cultures were checked for purity and were then subcul-tured into two test tubes containing potato dextrose agar (PDA). The medium was prepared by mixing 500 ml of
potato infusion (100 g of potatoes boiled in 500 ml of distilled water and then filtered), 5 g of glucose (Difco, Detroit, Mich.), and7.5 gofagar(Difco); the mediumwas sterilized and dispensed in 5-ml aliquots into screw-cap glass test tubes (16 by 125 mm). After approximately
10 days of incubation at 25'C, the test tubes were placed directlyinto anultra-low-temperature freezermaintained at
-70°C.
In order to determine culture viability, one tube of each isolatewasremoved fromtheultra-low-temperaturefreezer,
* Correspondingauthor. t Reprint requests.
after which a small portion of the colony was chipped from the frozen PDA slant and then placed onto
the surface of PDA in a test tube (16 by 125 mm). The
frozenslantwasnot allowedtothaw before itwasreturned
to -70°C. The subculture was incubated at 25°C for at least 4weeks. If the fungus grew and thecolony character-istics agreed with the previously known identification,
the culturewasrecorded asviable. Ifcultures didnotgrow initiallyuponsubculturingorifthecolony characteristicsdid
not agree with the isolate's identification, the fungus was thensubcultured from the second tube maintainedat -70'C
by using the procedure described above. The isolate was considered nonviable ifnogrowthoccurred.
The 1,447 cultures tested included representative species
ofanamorphsandteleomorphsofyeastsand molds classified as Ascomycetes, Basidiomycetes, Fungi Imperfecti, and Zygomycetes (Table 1). Surprisingly, only33(2.29%) of the total number of cultures did not survive storage at -70°C (Table 2). There was nocorrelation with specificgenera or species,nor wasthereacorrelation with thelengthof time in storage. Amongthe isolates thatsurvived, thereweremany
cultures oforganisms thatwere nonsporulating fungi, such asthezygomycetesApophysomyces elegansandSaksenaea
vasiformisand thedermatophytes Trichophytonschoenleinii andMicrosporum audouinii. The ability of these kinds of
fungitosurvive isimportant.Ourstudyalso showedsuccess in preserving the oomycetePythium insidiosum, the
zygo-mycete Basidiobolus ranarum, and the dimorphic fungus Paracoccidioides brasiliensis, organisms which are very
fastidious and difficult tomaintain forlong periodsof time. Inourexperience,storage at -70°Cisan excellent, conve-nient, and easy method of storing fungi and some other microorganisms for many years. The results ofour study agreewiththefindingsofCarmichael(3),who storedfungiat
-20°C, aswell as those of Meyer (7), who kept dermato-phytes at -52 to -22°C. Storage at -70°C is effective because isolates are maintained in a metabolically inactive
state.
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TABLE 1. Medically important fungi, aerobic actinomycetes, and algae maintained at -70°Cfor 1 to 13 years
Strain (no. of viable isolates/no. ofisolatesat-70°C) Absidia corymbifera (2/2)
Acladium castellani (1/1) Acremoniumspp. (8/8) Acremonium alabamensis (1/1) Acremoniumstrictum (1/1) Acrodontium sp. (1/1) Acrodontium salmoneum (1/1) Acrodontium simplex (1/1) Acuadendron ovatum (1/1) Acuadendrontriangularis (1/1) Alternariaspp. (3/3)
Alternariaalternata (2/3) Ambrosiozymacicatricosa (1/1) Aphanocladium album (1/1) Apophysomyces elegans (2/3) Arniumleporinum (1/1) Arthrinium sp. (1/1)
Arthriniumphaeospermum (1/1) Arthrographis spp.(6/6) Arthrographiscuboidea (2/2) Athrographiskalrai (2/2) Arthropsis deltoidea (1/1) Arthropsistruncata (1/1) Ascorynesarcoides (1/1) Aspergillus sp. (1/1) Aspergillusflavus (1/1) Aspergillusfumigatus (7/7) Aspergillus glaucusgroup (1/1) Aspergillusniger(1/1) Aspergilluspenicillioides (1/1) Aspergillusterreus(2/2) Aureobasidium spp. (4/4) Aureobasidiumnpullulans (9/9) Auxarthron sp. (1/1)
Bahusakala olivaceonigra (1/1) Basidiobolus ranarum (2/2) Basipetosporarubra (1/1) Beauveriaalba (2/2) Beauveriabassiana (1/1) Bipolarisaustraliensis (4/4) Bipolariscaptivora(1/1) Bipolaris cynodontis (2/2) Bipolarishawaiiensis (23/23) Bipolaris maydis(1/1) Bipolarismelinidis (1/1) Bipolarissorokiniana (3/3) Bipolaris spicifera (57/58) Blastomyces dermatitidis (25/29) Blastoschizomyces capitatus (11/11) Botryoascussynnaedendrus(1/1) Botryomyces caespitosus (1/1) Botryosporium sp. (1/1) Brettanomyces claussenii(1/1) Bullera alba (1/1)
Candidaahagu (1/1) Candidaalbicans (41/41) Candida
ciferii
(2/2) Candidadiddensiae (1/1) Candidaguilliermondii (2/2) Candida humicola (3/3) Candida intermedia (1/1) Candida krusei (3/3) Candida lusitaniae (6/6) Candida macedoniensis (1/1) Candidamembranaefaciens (1/1) Candida norvegensis (2/2) Candidaparapsilosis (3/3)TABLE 1-Continued
Strain(no.of viableisolates/no.of isolatesat-70°C)
Candidapseudotropicalis (2/2) Candida ravautii (1/1) Candidastellatoidea (3/3) Candida utilis (2/2) Chaetomium sp. (1/1) Chaetomium globosum (3/3) Chrysosporium spp.(2/2) Chrysosporium asperatum (1/1) Cladosporiumspp. (9/9) Cladosporiumcamionii(4/4) Cladosporium cladosporioides(7/7) Cladosporiumelatum (1/1) Cladosporiumherbarum (1/1)
Cladosporium sphaerospernum (13/13) Coccidioides immitis (1/1)
Cokeromycesrecurvatus (0/1) Coniothyriumfuckelii(1/1) Coremiella cubispora (0/1) Cryptococcus albidus (1/1) Cryptococcus ater (3/4)
Cryptococcusneofornans(56/56) Cunninghamella spp. (3/3) Cunninghamellabertholletiae (2/2) Curvularia spp. (3/4)
Curvularia affinis(2/2) Curvulariaclavata (1/1) Curvularia inaequalis(2/2) Curvularialunata (8/8) Curvularia pallescens (2/2) Curvularia senegalensis (9/9) Curvulariaverruculosa(12/12) Debaryomyceshansenii(1/1) Debaryomyces marama (1/1) Dissitimurusexedrus (1/1) Drechslera avenae (1/1) Drechslera brizae(1/1) Drechslera teres (4/4) Drechsleravagans (1/1) Embellisia abundans (1/1) Embellisia allii (1/1) Epicoccum sp. (1/1) Epicoccum nigrum (1/1) Epicoccumpurpurascens (1/1)
Epidermophytonfloccosum (3/3) Epidermophytonstockdaleae (1/1)
Evxophiala spp. (10/10) Exophialaalcalophila (2/2)
Esxophialacastellanii (7/7)
Evxophialadopicola (1/1) EJxophiala jeanselmei(71/71)
Exophialamoniliae (2/2)
Esxophialapisciphila (3/3)
Exophialasalmonis (1/1) Esxophiala spinifera (9/9)
Esxserohilumsp. (1/1)
EJxserohilumholmii(2/2) Exserohilum longirostratum (1/1) Exserohilum mcginnisii(2/2)
Evxserohilummonoceras(0/1) Exserohilumpedicillatum (0/1)
Esxserohilumrostratum (21/22) Exserohilumturcicum(1/1) Filobasidiellaarachnophila (1/1) Filobasidium capsuligenum (2/2) Filobasidium
floniforme
(1/2) Fonsecaeacompacta(4/4)Fonsecaeapedrosoi (46/46)
Fusarium spp. (9/9)
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TABLE 1-Continued
Strain (no.ofviableisolates/no. of isolatesat-70°C)
Fusarium anthophilum (1/1) Fusarium chlamydosporum(1/1) Fusariumdimerum (1/1) Fusarium graminearum (1/1) Fusariummoniliforme (3/3) Fusariumoxysporum (3/3) Fusariumproliferatum (1/1) Fusarium semitectum (1/1) Fusarium solani(2/2) Geomycesasperulatus (1/1) Geotrichumsp. (1/1)
Geotrichum
fici (1/1)Geotrichum penicillatum (1/1) Glenospora graphii (1/1) Gliomastixspp. (3/3) Hanseniospora uvarum (1/1) Hansenula anomala (2/2) Hansenula califomica (1/1) Hansenula fabianii (3/3) Hansenulajadinii(1/1) Histoplasma capsulatum (4/4) Homiactis sp. (1/1)
Hormonemadematioides (1/1) Horteae sp. (1/1)
Humicola spp. (2/2) Humicola fuscoatra (1/1) Humicola grisea (1/1)
Humicolopsis cephalosporoides (1/1) Hyalodendrumspp. (4/4)
Hyalodendrum lignicola (2/2) Hyphozyma variabilis (1/1) Issatchenkia orientalis (1/1) Kluveromycesbulgaricus (1/1) Kluveromycesfragilis (1/1) Kluveromyces marxianus (1/1) Kluveromycesthermotolerans(1/1) Lecythophorahoffinandi (5/5) Lecythophora lignicola (3/3) Lecythophora mutabilis (6/6) Lodderomyces elongisporus (1/1) Madurellamycetomatis (0/1) Malassezia furfur (3/3) Malasseziapachydermatis (1/1) Malbranchea aurantiaca (1/1) Malbranchea chrysosporoidea (1/1) Malbranchea circinata (1/1) Malbrancheadendritica (1/1) Malbranchea filamentosa (1/1) Malbrancheaflavorosea (1/1) Malbrancheaflocciformis (1/1) Malbrancheagramnicola (1/1) Malbrancheagypsea(1/1) Metarrhizium sp. (1/1) Metarrhiziumanisopliae (1/1) Metschnikowiapulcherrima (1/1) Microascustrigonosporus(2/2) Microsporum audouinii(2/2) Microsporumcanis(7/7) Microsporumcookei(1/1) Microsporumdistortum (3/3) Microsporumfulvum (2/2) Microsporumgallinae(3/3)
Microsporumgypseum (5/5)
Microsporumnanum(1/1) Microsporum persicolor (2/2) Microsporumvanbreuseghemii (1/1) Monascus sp. (1/1)
Monascusruber(1/1)
TABLE 1-Continued
Strain(no.of viableisolates/no. of isolatesat-70°C)
Moniliasp.(1/1)
Moniliella acetoabutens (1/1) Moniliella suaveolens(5/5) Monocillium sp. (1/1) Mortierella sp. (1/1) Mucorspp.(4/4)
Mycelia
Sterilia(30/38) Myrothecium sp. (1/1) Myxotrichumspp.(2/2) Nattrassiamangiferae (3/3) Neocosmosporavasinfecta (1/1) Nigrospora sp.(1/1)Nocardiaasteroides (11/11) Nocardiabrasiliensis (2/2) Nocardia otitidiscaviarum (1/1) Nodulosporiumspp.(4/4) Oedocephalumspp. (0/2) Oidiodendron Spp.(4/4) Ostracodenna sp.(1/1)
Ovadendronsulphureo-ochraceum (1/1) Pachysolen tannophilus (1/1)
Paecilomyces sp.(5/5) Paecilomyces lilacinus (1/1) Paecilomycesvariotii(7/7) Paracoccidioides brasiliensis(1/1) Penicillium Spp. (5/5)
Penicilliummameffeii (1/2) Penicilliumnotatum(6/6) Periconiella Spp.(4/4) Pestalotia Spp. (3/3)
Phaeoannellomyces Spp. (2/2) Phaeoannellomyces elegans (8/8) Phaeoannellomyceswerneckii (15/15) Phaeococcomyces sp. (5/5)
Phaeococcomycesalcalophilus (3/3) Phaeococcomycescatenatus (1/1) Phaeococcomycesexophialae (10/10) Phaeococcomycesnigricans (1/1) Phaeoscleradermatioides(2/2) Phaeothecafissurella (1/1) Phialemoniumcurvatum(6/6) Phialemoniumdimorphosporum (3/3) Phialemoniumobovatum(6/6) Phialophora Spp. (3/3) Phialophorabubakii(4/4) Phialophoracyanescens(1/1) Phialophora cyclaminis (4/4) Phialophora fastigiata(5/5) Phialophora heteromorpha(2/2) Phialophora lagerbergii (1/1) Phialophora luteo-viridis (1/1) Phialophora malorum (4/4) Phialophora parasitica (5/5) Phialophora repens(4/4)
Phialophoranichardsiae (7/7)
Phialophoraverrucosa (20/20) Phoma Spp. (2/2)
Phoma eupyrena (1/1) Pichiafarinosa (1/1) Pichiaguilliermondii (1/1) Pichiamembranaefaciens (1/1) Pichia ohmeri(2/2)
Piedraia hortae(1/1) Pithomyces sp. (1/1)
Pithomycesaltro-olivaceus (1/1) Pithomyceschartarum (1/1) Pleurophoma pleurospora (1/1) Protothecastagnora (1/1)
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TABLE 1-Continued
Strain(no. of viableisolates/no. of isolates at -700C)
Prototheca zopfii (1/1)
Pseudallescheria angustum (1/1) Pseudallescheriaboydii (31/31) Pseudallescheria ellipsoideum (1/1) Pseudallescheriafimetii (1/1) Pseudallescheriafusoideum (1/1) Pseudomicrodochium spp. (2/6) Pyrenochaeta sp. (1/1) Pythium insidiosum (1/1) Ramichloridium spp. (4/4) Ramichloridiumapiculatum (1/1) Rhamichloridiumobovoideum (1/1) Rhamichloridium schulzeri (1/1) Rhamichloridium subulatum (1/1) Rhinocladiella spp. (5/5) Rhinocladiellaatrovirens (19/19) Rhizomucorpusillus(1/1) Rhizopus spp. (3/3) Rhizopus arrhizus (1/1) Rhizopusmicrosporus (1/1) Rhodotorulaglutinis(1/1) Rhodotorulagraminis(1/1) Rhodotorula minuta (2/2) Rhodotorula rubra (2/2) Saccharomycescerevisiae(5/5) Sagrahamelaspp. (3/3) Sagrahamela bacillispora (1/1) Saksenaea vasiformis(1/2) Sarcinomycesmuriformis (1/1) Sarcinomycesphaeomuriformis (1/1) Sarcinosporon inkin (1/1)
Scedosporium apiospermum (18/18) Scedosporium prolificans (17/17) Schizophyllumcommune (1/1) Scolecobasidium spp. (2/2) Scolecobasidium constrictum (4/4) Scolecobasidium humicola (2/2) Scopulariopsisspp. (3/3) Scopulariopsisbrevicaulis(3/3) Scopulariopsis brumptii (7/7) Scopulariopsiscroci(2/2)
Scopulariopsis maduramycosis (1/1) Scytalidium spp.(2/2)
Scytalidiumdimidiatum (2/2) Scytalidiumfiavo-brunneum (1/1) Scytalidiumhyalinum (1/1) Sepedonium spp. (2/2) Spegazzinia sp. (1/1) Sporobolomyces sp. (1/1)
Sporobolomycessalmonicolor(1/1) Sporothrixspp. (2/3)
Sporothrixcyanescens (1/1) Sporothrixschenckii(2/2) Sporotrichumpruinosum (2/2) Stachybotryssp. (1/1) Stachybotryschartarum (1/1) Stephanosporiumcereale (2/2) Streptomycesgriseus(2/2) Tetraposporium sp. (1/1) Thermoascuscrustaceus (1/1) Thielavia sp. (1/1)
Thielaviaterrestris (2/2) Torulomycesspp. (2/2) Torulopsiscandida(6/6) Torulopsis glabrata (9/9) Torulopsisinconspicua(1/1) Torulopsis magnoliae (1/1) Torulopsismanis (1/1)
TABLE 1-Continued
Strain(no.ofviableisolates/no.ofisolates at-70°C) Torulopsisrosea (1/1)
Torulopsis stellata (1/1) Triadelphia pulvinata (1/1) Trichodermalongibrachiatrum (1/1) Trichophyton concentricum(1/1) Trichophyton equinum (1/1) Trichophyton fischerii(1/1) Trichophyton georgiae (1/1)
Trichophyton mentagrophytes (43/43) Trichophyton rotundum (1/1) Tnchophyton rubrum (15/15) Trichophyton schoenleinii(2/2) Trichophyton terrestre (3/3) Trichophyton tonsurans (19/19) Trichophyton verrucosum (4/4) Trichophyton violaceum (1/1) Trichosporon beigelii(6/6) Trichosporonoides sp. (1/1) Tnichosporonoides madida (2/2) Trichosporonoides nigrecens (3/3) Trichosporonoidesspathulata (1/1) Trimmatostromabetulinum (1/1) Uncinocarpusreesii(1/1) Ustilago sp. (1/1) Wangiella spp. (14/14) Wangielladermatitidis(29/29) Xylohypha spp. (3/3) Xylohypha bantiana (27/27) Xylohypha curta (1/1) Xylohypha emmonsii (6/6) Xylohyphalignicola (1/1) Xylohyphanigrescens(1/1) Zopfiarosatii(1/1)
Zygosaccharomyces bailii (1/1)
TABLE 2. Cultures that didnotsurvivestorage at-70°C
Strain(yr instorage)
AlterncaiaalternataUTMBa2587(2) Apophysomyceselegans UTMB 1999 (4) Bipolaris spicifera UTMB2472(3)
BlastomycesdenrmatitidisU1MB 174, UTMB 207, UTMB 216, UTMB 236(12, 13)
CokeromycesrecurvatusUTlMB2733(2) Coremiella cubispora UTMB354(11) CryptococcusaterUTMB2042(4) Curvularia sp. UTMB2123(4) Exserohilummonioceras UTIlMB2413(3) Excserohilum pedicillatum UTMB 2306(3) ExserohilumrostratumUTMB2282(4) Filobasidium
floriforme
UlTMB
2735 (2)Madurellamycetomatis Ul MB 1950(5)
Mycelia Sterilia UTMB 1886, UTFMB1890, U1MB1901, UTMB2088, UTMB2416,UTMB2424, UTlMB 2449, UTMB 2450 (4, 5, 6) Oedocephalum spp. UTMB2107,UTMB 2280(4)
PenicilliummarneffeiUTMB89(13)
Pseudomicrodochium spp.UTMB1350, UTMB1351, UTMB1352, UTMB 1353(7)
SakseenaeavasifornmisUl MB 2725 (2) Sporothrixsp.U1TMB1653(5)
i UTMB, Universityof TexasMedical Branch Culture Collection.
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We thank Shirley Wright for secretarial assistance, Zane Gray for
computerexpertise, and Nora Dunnell for technical help.
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