Journal of Microbiological Methods 47 Ž2001. 307–313 www.elsevier.


Cryopreservation of basidiomycete strains using perlite
Ladislav Homolka) , Ludmila Lisa, Ivana Eichlerova, Frantisek Nerud ´ ´ ˇ
Institute of Microbiology AS CR, Vıdenska 1083, 142 20 Prague 4, Czech Republic ´ ˇ ´ Received 6 July 2001; received in revised form 23 July 2001; accepted 20 August 2001

Abstract A new alternative method using perlite as a particulate solid carrier in the growth medium with a cryoprotectant was successfully tested for cryopreservation of several basidiomycete species from different genera Ž Armillaria, Pleurotus, Pluteus, Polyporus . which failed to survive or retain their properties in cryopreservation procedures routinely used in our laboratory. Frozen basidiomycete strains were kept in cryovials submerged in liquid nitrogen and were either immediately after the freezing process or after a 6-month storage thawed and checked for viability, purity and changes in growth, morphology and biochemical characteristics. All cultures survived the cryopreservation procedure and no negative effects of cryopreservation by this method have been observed after 6 months of storage in liquid nitrogen. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: Basidiomycetes; Cryopreservation; Liquid nitrogen; Perlite

1. Introduction Efficient microbiological Žand consequently also mycological. work requires a reliable source of cultures, i.e. well-defined and taxonomically determined starting material, which is ensured by its safe storage. Routine subculturing is not a very practical method of storing large numbers of fungal cultures. It is time-consuming, prone to contamination and does not prevent genetic and physiological changes

) Corresponding author. Tel.: q 420-2-475-2397; fax: q 420-2475-2384. E-mail address: ŽL. Homolka..

during long-term and frequent subculturing. Various storage methods have been developed in order to eliminate these disadvantages. Among them, the storage in liquid nitrogen has been considered the best and most widely applicable preservation technique available for filamentous fungi ŽSmith, 1998.. It is a safe and perspective method of a long-term maintenance of most fungal species, especially those not amenable to freeze-drying. This storage technique seems to surpass all others in the ability to preserve genomic and phenotypic features and is, therefore, effective in most microbial biodiversity maintenance programmes ŽSmith, 1982; Heckley, 1978; Prescott and Kernkamp, 1971.. This method, useful for both sporulating and nonsporulating fungal cultures, has several other important advantages: protection of cultures from contamination Žreduced

0167-7012r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 7 0 1 2 Ž 0 1 . 0 0 3 3 8 - 4

The ´ cryogenic technique for long-term storage of large numbers of fungal species was first tested and then introduced to ATCC in the 1960s. feed concentrates. 1986. 1983.. we found no negative effect of cryopreservation or the cryoprotective used on the production of ligninolytic enzymes ŽStoychev et al. The technique was subsequently introduced to many other prominent collections. IFO ŽInstitute for Fermentation Osaka. This papers deals with four such strains and the culture of Pleurotus ostreatus CCBAS 472 serving as a reference strain which can be successfully preserved by all tested techniques. 1976. the most widely used method of culture maintenance in lyophilized state is rather limited in basidiomycetes. particularly in the construction.. nonsporulating cultures of basidiomycetes are stored by cryopreservation at y80 8C in electric freezers ŽIto. and also mycorrhizal relationships play often a certain role. Homolka. Agar blocks immersed in an appropriate cryoprotectant were originally used as carriers of fungal mycelium for cryopreservation process ŽHwang.. Therefore. enzymes and other metabolites. which can be released as needed.. Hwang et al.. Later. Homolka et al. 1960. We also tested if . do not survive the process ŽAntheunisse.308 L. etc.r Journal of Microbiological Methods 47 (2001) 307–313 number of transfers. ´ Perlite is a unique aluminosilicate volcanic mineral holding and retaining substantial amounts of water. 1968. e. Many of these fungi do not form asexual spores.. The effect of cryopreservation on the production of the antibiotic mucidin by the basidiomycete Oudemansiella mucida was studied by Homolka Ž1976. 1971. the results have been highly satisfactory ŽHwang. herbicides and other similar applications. glass ampoules were replaced with more safe polypropylene cryovials andror straws. We have not found any report on using solid carriers for cryopreservation of nonsporulating cultures of filamentous fungi and any report concerning the use of such carriers for preservation of any fungal cultures in liquid nitrogen. their dominant form is mycelium which is rather sensitive to environmental conditions. A comprehensive and detailed overview of the methods and results of cryopreservation of microorganisms.. in particular with nonsporulating cultures ŽChallen and Elliott.. Another technique using straws in cryotubes without a cryoprotectant solution was described by Hoffmann Ž1991. In some collections. 1973. and improved by Stalpers et al. CAB International Mycological Institute ŽOnions. work and room. including basidiomycetes. ´ Mycelium andror spore suspensions with or without a cryoprotectant in sealed glass ampoules were originally used for cryopreservation of filamentous fungi. 1998. A useful straw technique with agar miniblocks for the preservation of fungi in liquid nitrogen was developed by Elliott Ž1976.. production of antibiotics.g. Polystyrene beads as carriers were used for cryopreservation of sporulating Aspergillus fumigatus cultures at y80 8C by Belkacemi et al. Ž1997. who found that the production ability was practically unaffected... 1976.. and the saving of time. Data on the production of metabolites or other features are relatively rare ŽHubalek. and porous ceramic beads were employed for cryopreservation of several sporulating fungal cultures and for a Saccharomyces cereÕisiae culture at y70 8C by Palagyi et al.. Chvostova et al. 1993. It is also used as a solid support in solid-state fermentations ŽKerem and Hadar. It is recommended as an efficient purifying agent and as a carrier for pesticides. On the other hand. was published by Hubalek Ž1996. In our previous successful experiments with some white-rot basidiomycetes. e. A need for an alternative method of cryopreservation arose after several partially or completely unsuccessful attempts to preserve some of our basidiomycete strains or their specific properties by our current routinely used procedure using straws in cryovials and by a standard cryotechnique using agar discs in polypropylene vials with a cryoprotectant solution. Routine tests carried out in large collections of microorganisms to estimate the success of surviving the process of cryopreservation usually involve growth and sporulation tests together with morphology assessment of fungal colonies. 1996. horticulture and other various industrial fields. because most of them Žespecially nonsporulating ones. Expanded perlite is being used in many applications. Ž1997. the maintenance of basidiomycetes is rather difficult. 1966. ´ 1996. Smith..... A principal requirement for successful maintenance of production strains is the full preservation of their important properties Že. the method of cryogenic culture maintenance seems to be mostly successful. According to the literature and our personal experience..g.g. Ž1987. 1995.

in Bres. purchased from Keramik. supplemented with glycerol at a final concentration of 5% Žvrv. prepared as above and then incubated for 10 days at 24 8C. These cultures have not been previously stored in LN.. Armillaria socialis ŽDC. which is a slightly modified Hoffmann’s protocol ŽHoffmann.5%. All experiments were carried out twice in 10 replicates. as a cryoprotectant. After thawing. moistened with 1 ml of wort Ž48 Balling. at 24 8C.r Journal of Microbiological Methods 47 (2001) 307–313 309 the cryopreservation of different basidiomycete species on perlite had any negative effect on their essential characteristics.. — P. the perlite particles overgrown with mycelium were at least partially separated by shaking. CCBAS 472. cut from the actively growing part of a colony on a Petri dish containing wort medium Žwort 48 Balling. enriched with glycerol ŽSigma. agricultural grade. Materials and methods 2. with 200 mg of perlite ŽAgroperlit. CCBAS 850—were maintained by serial transfers and kept on wort agar slants at 4 8C. CCBAS 483 and Polyporus squamosus ŽHuds. Herink CCBAS 331. ostreatus var. 1.4.:Fr. Difco agar 1. three individual straws or agar discs were used instead of perlite particles.5 % agar Difco. Organisms and cultiÕation Fungi obtained from the CCBAS collection ŽInstitute of Microbiology.8 ml.:Fr.. The cryovials with perlite overgrown by the mycelium were frozen in a programmable freezer IceCube 1800 SyLab to y70 8C at a freezing rate of 1 8C per minute. These filled straws are then transferred to the sterile cryovials. Another tested freezing protocol Ždisc protocol— DP. columbinus ŽQuel.. Enzyme and hydrogen peroxide assays Enzyme activities were measured in the filtrates from four replicate flasks after removing the . ostreatus ŽJacq. Preparation of organisms and freezingr thawing protocols Fungal cultures for cryopreservation experiments with perlite were grown directly in sterile plastic Nunc CryoTube Vials 363401 Ž1. Prague. The flasks were inoculated with two agar plugs Ž6 mm diameter. 2. are used to punch the mycelium with agar from the colony. using agar discs with mycelium submerged in 10% glycerol solution in polypropylene cryovials was performed according to Butterfield et al. P. Prior to opening the surface of the cryovials was disinfected with alcohol. Enzyme activities and overall extracellular H 2 O 2 production were measured on the 10th and 14th days of cultivation in the filtrates from three parallel flasks after separation of the mycelia. This procedure we named Aperlite protocolB —PP. Homolka et al. from cultures before or after cryopreservation. The mycelia were harvested from the cultivation flasks which were incubated as above.5 mm.1. They were then placed in liquid nitrogen in a HARSCO TW-5K container. Growth estimation Submerged growth in liquid medium was evaluated by determining the dry mass of mycelia. Thawing—reactivation of cultures—was common for all tested protocols and was carried out by transferring the cryovials to warm water Ž38 8C. 0. 1988.:Fr.. Gill. Czech Republic. Static submerged cultivation for growth and enzyme estimation was carried out in 100-ml Erlenmeyer flasks containing 10 ml of N-limited Kirk medium ŽTien and Kirk.. Prague. open at both ends.3.2%. the content of the cryovials was divided into three approximately equal aliquots and these were plated onto wort solid medium in Petri dishes and incubated at 24 8C. All measurements were done in quadruplicate. final concentration 5%. which are tightly sealed with screw stoppers provided with silicone gaskets and frozen as above. 2. yeast extract 0. Quel. is the following: strains are grown on Petri dishes on MEYA medium Žmalt extract 2%. plastic straws. Sterile thin Ždiameter 1 mm. 2.. Kumm. In case of CP or DP.L. inoculated with an agar plug Ž6 mm diameter. Pluteus petasatus ŽFr. Ž1978. Our current routine freezing protocol Žcontrol protocol—CP... 2.. This is repeated until the straws are filled with agar plugs containing the mycelium. 1991.2. washed with distilled water. dried at 105 8C for 24 h and weighed. ´ ´ CCBAS 462.

in medium filtrates. The cultures were checked for their viability and other characteristics immediately after the cryopreservation process and then after 6 months of storing under liquid nitrogen. socialis CCBAS 331 to survive the process of freezing by our routine protocol CP could be partially ascribed to the fact that the fungus forms thick rhizomorphs which make difficult to cut out the miniblocks using a thin straw as a plunger. were partially or completely unsuccessful. using open straws in cryovials and by a cryotechnique according to Butterfield et al. ostreatus CCBAS 472 served as a reference strain which can be successfully preserved by all tested techniques.r Journal of Microbiological Methods 47 (2001) 307–313 mycelium by filtration. ostreatus CCBAS 472 100 100 100 P. Laccase activity was determined according to Bourbonnais and Paice Ž1990. squamosus CCBAS 850—were tested for the ability to grow on perlite with nutrients. ŽABTS. a The recovered cultures lost their ability to produce laccase. in case of P. by monitoring the oxidation of 2. petasatus CCBAS 483 30 65 100 Pol. socialis CCBAS 331 55 0b 100 Plu. Freezing protocol DP CP PP Fungal strain P.. and 3-dimethylaminobenzoic acid ŽDMAB.2X-azinobisŽ3-ethylbenzothiazoline-6-sulfonic acid.. ostreatus CCBAS 472. Homolka et al. socialis CCBAS 331. their growth and biochemical characteristics are shown in Table 2. or 590 nm ŽMnP.and micromorphological characteristics. columbinus CCBAS 462. and the ability to retain the selected biochemical characteristics Žproduction of lac- Table 1 Recovery Žin percent. Moreover. Activities of extracellular laccase and manganese peroxidase ŽMnP. the culture lost its ability to produce laccase after freezing.310 L. columbinus CCBAS 462.. to survive the cryopreservation in liquid nitrogen with glycerol as a cryoprotective and to maintain their important characteristics—growth rate. using agar discs in polypropylene vials with a cryoprotectant solution ŽDP.. 3. squamosus CCBAS 850 50 70 100 CP s control protocol. Results and discussion Five basidiomycete strains—P. The failure of A. DP s disc protocol. The overall production of extracellular hydrogen peroxide was measured using the phenol red method according to Pick and Keisari Ž1980. The principal criteria for a successful recovery of fungal strains were the ability to survive the cryopreservation process Žexpressed as an ability to retain the original growth and macro. modified according to Daniel et al. A. . were oxidatively coupled to give a purple indamine dye product by the action of the enzyme in the presence of added H 2 O 2 and Mn2q ions. Ž1994. The cultures were tested before and immediately after the freezing process and then after a 6-month storage in liquid nitrogen. petasatus CCBAS 483 and Pol. were determined spectrophotometrically by monitoring the absorbance increase at 425 nm Žlaccase. ostreatus v. The recovery of the tested strains subjected to the three different cryopreservation protocols is summarized in Table 1. morphology. 3-methyl-2-benzothiazolinone hydrazone ŽMBTH. was defined as catalyzing the production of 1 mmol of green or purple dye per milliliter per minute. columbinus CCBAS 462 0 60a 100 A. The culture of P. The above-listed strains were chosen because the attempts to preserve them by our routinely used procedure ŽCP. Plu. PP s perlite protocol Žsee Section 2. Determination of MnP activity was based on the method of Ngo and Lenhoff Ž1980. of different fungal strains frozen using three alternative cryoprotocols after 6-month storage in cryovials Ž20 replicates of each strain were tested in two experiments.. production of hydrogen peroxide and enzymes in- volved in lignin modification Žlaccase and manganese peroxidase. One unit of enzyme activity ŽU. b Mycelia with rhizomorphs were recalcitrant to punching and entering the straws. ostreatus var. Ž1978. P. ostreatus var.

89 102. Unlike the other two protocols tested.10 12. MnP Ž%.39 " 5.and micromorphological characteristics.81 " 0.21 82.26 98. squamosus 1.65 " 0. The improved cryopreservation procedure has several additional advantages.26 101. One cultivation step is saved by using the cryovials directly for the cultivation of cultures.11 95.44 111.32 " 0. petasatus 3. Twenty replicates of each fungal strain were tested with each protocol. or control protocol ŽCP. In our experience.19 " 0.82 101.29 118.45 P.01 55. the perlite protocol was fully successful in cryopreservation of all tested strains.14 100.02 1. Laccase Ž%. the recovery was counted as percentage of surviving replicates.05 104.06 0.69 " 6.15 92.50 97.02 19. the process of recovery using the disc protocol can be complicated in some fungal strains due to the separation of the mycelial mat from the discs in the cryopreservation solution. Even after another two transfers on fresh solid media the results were almost the same Ždata not shown.25 110.28 " 4.84 Pol. MnP Ž%. Freezing curves of P. ostreatus 4.45 104.06 93.56 31. No contamination was detected during the whole study. 3.88 " 0.60 8. unchanged. H 2 O 2 ŽnM.44 " 1.12 7.19 2.84 " 1. the growth and biochemical characteristics as percentage of original values.45 88.58 98. MnP ŽmUrml. Dry mass Ž%.22 96.05 " 0.45 10.00 95. The same held for the macro. The mycelium on perlite grows continuously and its damage caused by punching it from the agar plate and by subsequent handling is prevented and specific mycelial structures Žrhizomorphs etc.09 95. Special features of perlite Žretention and release of water and air.26 109.59 98.86 1. The growth rates of all strains tested were virtually unaffected by the process of freezing and thawing..42 111.. ostreatus CCBAS 472 cultures frozen using perlite protocol ŽPP.29 " 0. The activities of all enzymes before cryopreservation and after the revival did not differ substantially and the differences between the obtained values did not exceed the usual fluctuation.00 Plu.37 " 1.95 98.25 Immediately after freezing After 6-month storage in LN case.48 " 0.51 102. Laccase Ž%.94 0. socialis 4.52 136.64 " 3.95 112.16 99. columbinus Nonfrozen culture Dry mass Žmgrml.08 108.16 5.11 A.r Journal of Microbiological Methods 47 (2001) 307–313 311 Table 2 Growth and biochemical characteristics of fungal cultures before freezing. affect the ice formation and thaw- Fig.48 15.15 5. Homolka et al.26 96.18 115. thin line. Dry mass Ž%.46 89. Laccase ŽmUrml. the transparency of the cryovials makes it possible to check the growth of the culture inside and to prevent possible problems with insufficient inoculation and contamination.70 " 0.. .65 108.50 110. ostreatus v. H 2 O 2 Ž%.44 105. Experimental conditions Strains P.96 " 0.L.12 95. can be preserved more easily.65 " 1.28 96.00 105.95 " 0. bold line. immediately after freezing and after a 6-month storage in liquid nitrogen ŽLN. H 2 O 2 Ž%.87 117.91 " 2. Mn peroxidase and hydrogen peroxide.16 110. 1.

Hwang. Microbiol. Soc.. 1973. Trans.. Elliott. W. This expectation is supported by the promising results of preliminary testing of the method on another 60 fungal strains from 19 different genera and 36 species Ž14 strains of whiterot basidiomycetes from eight species of two genera —Inonotus and Pholiota—were also successfully cryopreserved on perlite without visible changes and further 46 strains from 28 species of 17 genera were at least able to grow on perlite and survive the process of freezing and thawing. On the problem of maintenance and cultivation of higher fungi. Cryoprotectants and the cryopreservation of fungi... L. Environ. Stalpers. Soc.. 59. 193–197. S.. E. AV0Z5020903.312 L.. 161–170. M. Haynes.. Z. Mycologia 52. Nerud. S. 1986. R. Barton. Prescott. 267.C. 92–94. Methods in Microbiology. The described method is evidently suitable for different fungal strains requiring special treatment and the authors believe that it is generally applicable to most fungal cultures. Antonie van Leeuwenhoek 39.. Hwang.. Vastag. 80. World J.. V. 1978. A.J. R. L. Oxidation of non-phenolic substrates. Mycologia 68. 1968. 99–102. Homolka.. Microbiol. D. 1976.. Academic Press. T. Kerem.. they are at present under further study. van der Mei.. S. Onions. 4. 527–529. 67. 103–104. Hubalek. Mycoses 40. Polypropylene vials for preserving fungi in liquid nitrogen... Trametes Õersicolor. Microbiol. Kernkamp. Br. Paice. Another potential advantage of the procedure is the partial prevention of a large increase in the temperature of the frozen sample around the crystallization point. 14. E. Biotechnol. 389–397. R. 243–248.. 40.-W. 1996.. L. 1–53.A. Smith. 2524–2532. Alexander. FEBS Lett. 1997.M. Lenhoff. Evans. Long-term preservation of fungus cultures with liquid nitrogen refrigeration. Homolka et al. 113–115. Elliott. Acknowledgements This work has been supported by Institutional Research Concept no. C. ŽEd.. Cryopreservation of fungi. Ito. Heckley. Culture Collections to Improve the Quality of Life. 360–363. Trans. H. Kubatova. 1971.. A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture. Polypropylene straw ampoules for the storage of microorganisms in liquid nitrogen. 613– 621. 1982. J. ŽEds. Kwolek. 4115–4120. Daniel. 249–250.. Mycologia 60. 1990. Volc. Anal. C. Trans. Br.. 1122–1124.T. Microbiol. 60.. The presence of perlite seems to partially counteract the generation of heat in the sample and the corresponding curve is flattened. 1991.. M. Appl. pp. and Oudemansiella mucida. where the temperature course curves of the samples frozen on perlite and in a straw with agar miniblocks are compared. Appl. pp. 1994. Adv.F. 1996..C. Microbiol. 1971.. Keisari. Ngo. Y.. An expanded role for laccase in lignin biodegradation.. Bourbonnais. The Netherlands. Microbiol. ´ Palagyi.. M. 1995. A. Soc. 24. ´ Academia Publishing House. 784– 788. Preservation of microorganisms.. Investigation of ultra-low temperature for fungal cultures: I. This can be seen in Fig. M. Pick. Nagy. 1966.G. Mycol.. J. Viability and growth rate of mycelial cultures following cryogenic storage. 21. Effect of manganese on lignin degradation by Pleurotus ostreatus during solid-state fermentation. Hwang.-W. 545–546.H. Z. E.. 1980. vol. Biochem. Appl.M.H. Centraalbureau voor Schimmelcultures. S. Viability of wood´ inhabiting basidiomycetes following cryogenic preservation.-W. J.r Journal of Microbiological Methods 47 (2001) 307–313 Chvostova. Hadar. Belkacemi. Stouthamer. T. a ´ ´ major source of H 2 O 2 during wood degradation by Phanerochaete chrysosporium. 377–387. 55. Cryopreservation of Microorganisms. 1976. Challen. Effects of ultra-low temperatures on the viability of selected fungus strains. 11–23. Alternative ampoule for storing fungal cultures in liquid nitrogen.G. D. Rep. In: Booth. W. Folia Microbiol. Smith. W.. Baarn. J. Maintenance of fungal strains on cryopreservative-immersed porous ceramic beads. 1983. 1. Mycol.. R. Cryopreservation of Aspergillus fumigatus stock cultures with a commercial bead system..-W. 189..P.. 1976. 11.V. 415–421. Jong... 1978. Hwang.. New York. Immunol. T. 695–696. A. References Antheunisse. Vagvolgyi. Genetic stability of Puccinia graminis tritici in cryogenic storage.T. 1980. 105. Preservation of fungi.. 7. J. Mycol. A sensitive and versatile chromogenic assay for peroxidase and peroxidase-coupled reactions. 79. L. Ferency.. Pyranose oxidase. D. P. Preservation of fungal cultures at the Institute of Fermentation. Tech. Methods 5. An evaluation of liquid nitrogen storage for preservation of selected fungal cultures. M. Osaka ŽIFO. T. 210–211.. Viability of lyophilized microorganisms after storage.J. Y.. Butterfield.S. Homolka.. Methods 38. Liquid nitrogen storage of fungi. Prague. In: Samson. . G.F. 1993. S. Plant Dis.. Folia Microbiol..C. Environ... Br. Appl.A. Mycologia 70. ´ ´ ¨ 1997. Z.. Biotechnol. F. 1960. ing and this can be useful for culture protection and preservation. Hoffmann. Investigation of ultra-low temperature for fungal cultures: III.. J...

1998. L. Kirk. Methods Enzymol. Improvement of the straw technique for the preservation of fungi in liquid nitrogen.. Stoychev. T. Lignin peroxidase of Phanerochaete chrysosporium. 1998. Activities of ´ 313 ligninolytic enzymes in some white-rot basidiomycete strains after recovering from cryopreservation in liquid nitrogen. 161B... Stalpers. Cryo-Lett.L.J. Vlug. D.K. Nerud. 79–90. A. Mycologia 79.r Journal of Microbiological Methods 47 (2001) 307–313 Smith... 19. Homolka.. Homolka et al. J. 238–248. I.. 1987. F. Antonie van Leeuwenhoek 73.A. The use of cryopreservation in the ex-situ conservation of fungi.. Lisa. M. 1988. Tien. 82–89.. I. L. .. de Hoog. 211–214.