Keywords

Introduction

Preservation and long-term storage of type, authentic, and other kinds of fungal cultures in a living state is of high importance both for the fundamental and practical mycology.

Long-time storage of strains is performed in microbiological culture collections (biological resource centers). Various methods of preservation of fungal cultures have been reported [1–3].

Freeze-drying (lyophilization) and cryopreservation methods are utilized for thousands of fungal strains in microbial collections all over the world [4–6]. Nevertheless, it is clear that the fungal strains of different species vary in ability to survive after the long-time storage preservation under laboratory conditions. Some of them are very difficult to maintain ex situ, whereas others could be easily and successfully preserved alive by using almost any conservation technique. Available information on the maximal time periods in which the reliable storage of different fungal species are ensured does not cover those for the diversity of fungi maintained in culture collections. This chapter presents the methods of cryopreservation, freeze-drying, and preservation in sterile soil that are utilized in VKM fungal collection, accompanied by data on maximal storage time registered. The methods take into consideration the special features of cultures preserved as well as the equipment used.

VKM fungal collection (All-Russian Collection of Microorganisms, Russia) was established in 1955 and has a long-term experience for preservation and storage of fungal cultures. Collection of filamentous fungi is currently composed of approximately 5,000 strains (545 genera, 1,450 species) belonging to species of the kingdoms Chromista (Oomycetes) and Fungi (Zygomycetes, Ascomycetes, and Basidiomycetes). The current use of different preservation methods for more than 3,800 strains maintained in VKM for more than 40 years was analyzed using a specially designed database. The database keeps the protocols of conservation methods, storage conditions, the calculated time of reliable storage, special requirements of growth, and other information related to the issue. Data presented in this chapter are derived from this database. The information on preservation methods is also available in VKM catalogue (http://www.vkm.ru/Catalogue.htm), in data sheet for each strain.

Cryopreservation of Filamentous Fungi

According to published data, the fast cooling rates followed by storage in liquid nitrogen at −196°C allow secure and long-term preservation of some fungal cultures [7]. However, the ability to resist damage by freezing and warming differs considerably among genera/species and depends on their particular features (presence and type of sporulation, chemical composition of cytoplasmic membrane and cell wall, physiological state, etc.). Selection of optimal cryoprotectants, rates of cooling and warming has enabled increasing the number and diversity of taxa preserved by this method [8, 9].

More than 70% filamentous fungal of VKM (2,714 strains belonging to 1,148 species and 405 genera) are stored using various cryopreservation protocols. Cultures with abundant sexual and nonsexual sporulation usually were preserved by using fast cooling rates followed by storage either in liquid nitrogen or in ultra-low temperature freezers at −80°C.

It was noticed that some cultures of Zygomycetes belonging to the genera Mortierella, Basidiobolus, Coemansia, and Lobosporangium (syn. Echinosporangium) do not survive the ultrarapid freezing procedure even if they have abundant sporulation. Successful preservation of such strains was achieved by modification of the cryopreservation regime, for example using slow programmed freezing. The same method was used either for nonsporulating fungi or zoospore-forming ones (Basidiomycetes, Oomycetes).

According to our data, some part of strains of Oomycetes (20%), Basidiomycetes (4%), Zygomycetes (1%), and Ascomycetes (1%) did not survive cryopreservation at all freezing regimes and modification applied [10]. The strains most difficult to maintain belong to genera Brevilegnia, Dictyuchus, Phytophthora, and to some species of Achlya and Saprolegnia. Similar situations have also been seen with some species of Basidiomycetes (Suillus, Amanita, Dictyophora, Mutinus, etc.). They are usually maintained by subculturing and preservation under mineral oil.

It has been suggested that those microbial cultures that are able to survive the freezing and a short storage will permanently stay in the vital state after any length of storage [11]. According to our data this is not quite true: some strains of Achlya colorata, Antrodia serialis, Armillaria cepistipes, Athelia rolfsii, Ceratellopsis equiseticola, Choanephora conjuncta, Clitocybe nuda, Coemansia aciculifera, Collybia butyracea, Conidiobolus thromboides, Exobasidium karstenii, E. splendidum, Kickxella alabastrina, Lactarius deliciosus, Marasmius oreades, Mortierella gamsii, M. humilis, Mycena pura, Phallus impudicus, Rhizoctonia solani, Sclerotium tuliparum, Suillus variegatus, and Ustilago scabiosae have lost their ability to grow after 5–7 years of storage in liquid nitrogen, although they were in the vital state after 24 h of storage. The reason is not yet known. Nevertheless, the viability test showed that 350 strains of fungi remain alive after 19.5 years of storage (Table 3.1).

Table 3.1 Storage time of VKM fungal cultures
Full size table

The cooling equipment being used in VKM is storage tanks “Bioproducts-0.5” with capacity of 500 liters of liquid nitrogen and ultra-low temperature freezers (−80°C, Sanyo, Japan).

Freeze-Drying of Filamentous Fungi

Currently, freeze-drying is used to preserve approximately 80% of filamentous fungi maintained in VKM (2,991 strains, belonging to 1,010 species and 303 genera). Fungi from different taxonomical groups (Zygomycetes, Ascomycetes— both teleo- and anamorph) able to produce dormant structures (spores, sclerotia, etc.) usually survive freeze-drying [12]. According to our data, from 87 to 92% strains of these fungal groups remain alive in this method. We noticed that 57% of freeze-dried cultures stored at 5°C for more than 20 years were in a vital state, and cultures of more than 190 species have been sustained for even 30–40 years of storage. Some species did not survive freeze-drying even when the sporulation is abundant, those are: Conidiobolus coronatus, C. obscurus (syn. Entomophthora thaxteriana), C. thromboides (syn. Entomophthora virulenta), Erynia conica (syn. Entomophthora conica), Pandora dipterigena (syn. Entomophthora dipterigena), Cunninghamella homothallica, Cunninghamella vesiculosa. Species of genus Botrytis (B. cinerea, B. fabae, and B. gladiolorum), forming only sclerotia as a dormant structure, remain in vital state in freeze-drying only for rather a short time—less than 10 years [10].

Nonsporulating microorganisms from Oomycetes and Basidiomycetes are not stored in VKM by freeze-drying, since sterile mycelium generally do not remain viable. However, some ectomycorrhizal fungi (e.g., Laccaria laccata) could be successfully lyophilized. For the positive result preliminary slow freezing (to −32°C) of fungal material is required [13].

The equipment used in VKM for freeze-­drying is centrifugal freeze-dryer system Micro­modulyo (Edwards, UK).

Drying in Sterile Soil of Filamentous Fungi

This simple and popular method for preservation of fungi was applied in the beginning of the twentieth century [14]. Species of Aspergillus, Penicillium can be maintained by this way more effectively than other micromycetes. According to T.P. Suprun [15] who investigated preservation of 78 Penicillium species (more than 1,000 strains) in sterile soil for 7–10 years, the best preserved strains were representatives of Assymmetrica section. Less effectively preserved species were Biverticillata-Symmetrica and the lowest effectiveness was observed with strains of the section Monoverticillata.

Species of Zygomycetes could be stored in soil for periods ranging from 6 months (Cunninghamella elegans) to 5 years (Rhizopus stolonifer var. stolonifer [syn. R. nigricans]) [16].

This method is also efficient for preservation of some human, animal, and plant pathogens with retaining their virulence [7]. For example, Alternaria japonica (syn. A. raphani), Fusarium oxysporum, and the species of Septoria (S. avenae, S. nodorum, S. passerinii, S. tritici) have retained their ability to infect a plant host after 2–5 years of storage [17–19]. Some degraded strains of micromycetes partly recuperated their lost qualities after preservation in soil [15].

Species of Alternaria, Pseudocercosporella, Septoria are genetically more stabile compared with Fusarium, therefore they did not show these kinds of changes and can be effectively preserved in soil [3].

Protocols

Protocol of Cryopreservation

Preparation of Cryovials (2.0 mL Externally Threaded Polypropylene, Nunc, Denmark)

  • Labelled (6 for each culture) with an index, a collection number of a strain and a date of cryopreservation (month, year).

  • Sterilized by autoclaving, at 121°C for 20 min.

Preparation of Cryoprotectant: 10% (v/v) Glycerol

  • Pour 5 mL of glycerol into 12 mL glass tubes.

  • Sterilized by autoclaving at 121°C for 20 min.

  • Stored at +5°C for no longer than a month.

Preparation of Cultures

  • Grow sporulating fungal cultures on slant agar under optimal growth condition and on suitable medium (www.vkm.ru).

  • Wash off spores from agar surface with 5 mL of cryoprotectant.

  • Titer of spores’ suspension should be not less than 106 spores/mL.

  • Grow nonsporulating fungal cultures on Petri dishes under optimal growth condition and on suitable medium with agar concentration 5% (w/v). Incubate culture to get a mature colony.

  • Cut mycelial plugs (5 mm diameter) from vigorously growing colony part.

Filling of Vials

  • For sporulating cultures add 0.2-mL aliquots of suspension to each cryovial using a Pasteur pipet. This procedure is carried out under sterile aerobic conditions.

  • For nonsporulating cultures place 4 mycelial plugs into each cryovial using transfer needle, and then add 0.2 mL of cryoprotectant.

Fast Cooling Rates Regime of Freezing (∼400 grad/min)

  • Place the cryovials with cultures (spore suspension) in special containers; thoroughly fixate a position of every vial.

  • Immediately place cryovials with cultures in the vapour phase of liquid nitrogen or in ultra-low temperature freezer.

Programmed Regime of Freezing

The First Protocol

  • Place 18 cryovials with cultures (mycelial plugs) in a special container “NALGENE™” (Cryo 1°C Freezing Container, Cat. No. 5100-0001).

  • Fill the container with 250 mL of isopropanol.

  • Place the container in a mechanical freezer (−80°C). Temperature in this container decreased by 1°C/min.

  • When temperature achieves −70°C, transfer cryovials in special container (thoroughly fixate a position of every vial), and place in the vapour phase of liquid nitrogen or in the ultra-low temperature freezer.

The Second Protocol

  • Place 18 cryovials with cultures (mycelial plugs) in a special metal container, thermostatic inside by expanded polystyrene (container made in VKM).

  • Place the container in the ultra-low temperature freezer (−70°C). It was empirically shown in VKM that temperature in this container decreased by 0.4°C/min.

  • When temperature achieves −70°C, transfer cryovials in a special container (thoroughly fixate a position of every vial), and place in the vapour phase of liquid nitrogen or in the ultra-low temperature freezer.

Thawing

  • Pull out the cryovial of container, in which it was stored—either in cryogenic tank or in an ultra-low temperature freezer.

  • Warm the cryovial rapidly by immersion in a shaking (for increase in heat exchange) water bath (37°C) for 1–2 min.

Control of Viability

  • To estimate the viability of fungal culture before cryopreservation, place either one volume of the suspension (0.2 mL) or 4 plugs under optimal growth conditions on a suitable medium. This procedure is carried out under sterile aerobic conditions.

  • To estimate the viability of fungal culture after cryopreservation, sterilize the thawing cryovials surface by wiping with 70% (v/v) ethanol. Asepticaly transfer the contents (spore suspension or mycelial plugs) using a Pasteur pipet or a transfer needle onto a suitable growth medium. This procedure is carried out under sterile aerobic conditions.

Protocol of Freeze-Drying

Preparation of Ampoules

Glass tubes (gray glass, diameter 7 mm, length 110 mm):

  • Wash successively with detergent, tap water, and distilled water.

  • Dry.

  • Plug loosely with cotton wool to a depth of 1 cm.

  • Label with an index, a collection number of a strain, and a date of freeze-drying (month, year).

  • Sterilize in dry oven at 160°C for 2 h.

Preparation of Lyoprotectant Agent

  • Pour 5 mL 10% (v/v) skimmed milk into each 12-mL glass tube.

  • Sterilized by autoclaving at 105°C for 30 min.

  • Stored at +5°C for no longer than a month.

Preparation of Cultures

  • Grow sporulating fungal cultures on slant agar under optimal growth condition and on suitable medium (www.vkm.ru).

  • Wash off spores from agar surface with 5 mL of skimmed milk.

  • Titer of spores’ suspension should be more, than 106 spores/mL.

Filling of Ampoules

  • Add 0.2-mL aliquots of suspension to each ampoule using a Pasteur pipet. This procedure is carried out under sterile aerobic condition.

The First Stage (Primary Drying)

  • Transfer the ampoules to the spin freeze-drier.

  • Freeze (temperature in a refrigerator −45°C) under the reduced pressure of the ambient gas during centrifugation (30 min).

  • Dry via water sublimation (temperature of a freeze dryer is −45°C) in vacuum (from 4  ×  10−2 to 6  ×  10−2 mbar) till the moisture level achieves 5–10%.

  • Duration of the first stage is 3 h.

  • Switch off the vacuum pump after the first stage. The system is filled with gas; the ampoules are removed from the centrifuge.

Preparation of Ampoules for the Second Stage of Freeze-Drying

  • Constrict ampoules to diameter of 2–3 mm using an air/gas torch with horizontal flame preventing overheating of cultures just below the cotton wool plug (approximately 50 mm from the ampoule bottom)

The Second Stage (Secondary Drying)

  • Attach the constricted ampoules via rubber tubes to the manifold connecting with the vacuum pump.

  • Drying (vacuum 100 mm) till the moisture level reach 2%.

  • Duration of the second stage is 2.5 h.

  • Seal the ampoules across the constriction using an air/gas torch.

Vacuum Control

  • Immediately after sealing, test vacuum in ampoules using a high-voltage spark tester.

Control of Culture Viability

  • To estimate the viability of fungal strains prior to freeze-drying one volume of the spore suspension (0.2 mL) place under optimal growth condition on suitable medium. This procedure is carried out under sterile aerobic conditions.

  • To estimate the viability of fungal culture after freeze-drying, test ampoules after 24 h storage.

  • This procedure is carried out under sterile aerobic conditions.

  • Sterilize a control ampoule’s surface with 70% ethanol and open ampoules using a cutter.

  • Reconstitute the dried suspension with sterile tap water (0.2–0.3 mL) using a Pasteur pipet.

  • After 30 min (when rehydration is complete) transfer suspension under optimal growth condition on suitable medium.

Storage

  • Store the ampoules d at +5–8°C in the dark.

Protocol of Drying in Sterile Soil

Preparation of Sterile Soil

  • Place 5 g of finely cultivated (garden) soil into 12-mL glass tube.

  • Sterilized by autoclaving, at 121°C for 30 min for three consecutive days.

Preparation of Cultures

  • Grow sporulating fungal cultures on slant agar under optimal growth condition and on suitable medium (www.vkm.ru).

  • Wash off spores from agar surface with 5 mL of sterile tap water.

  • Titer of spores’ suspension should be not less than 106 spores/mL.

Soil Inoculation

  • Add 1 mL spore suspension to glass tubes with sterile air dry soil (moisture is under 20%).

  • Incubate at room temperature till soil dry up (near 1 month).

  • Store in the refrigerator at 4–7°C.

Control of Viability

  • Transfer a few grains of soil onto fresh agar medium, add a little water and incubate under optimal conditions.

Result

The real storage time estimates obtained in VKM are given in Table 3.1. They are not final data: the cultures are still being stored, and we expect to get longer storage times later on. Some cells of the table are empty; this is the case if the culture is not stored this method.

Conclusion

The conservation techniques used in VKM presents effective preservation of the stock of filamentous fungi from different taxonomic groups. The possibility and practical time estimates of secure long-term storage of fungal cultures belonging to 1,221 species and 424 genera was shown. The represented information could be used as a reference for researchers intending to maintain pure cultures of microorganisms for a long time. The data produced are also accessible online on the VKM Web site.