Keywords

21.1 Introduction

Fungi, integral component of the Earth, are associated with various ecosystem functions and services. Over the years, our understanding about fungi has developed for their utilization as biological ‘tools’ in biotechnology. Fungal-based fertilizers, growth stimulators and bioinsecticides have the potential to enhance production and to ameliorate barren and degraded lands. In medical sciences, novel pharmaceutical products, blood proteins, hormones, interferon, cell growth stimulators, insulin, therapeutic products, etc. are being produced by employing fungi and other microbes (Blanch et al. 1985). Gases, power-alcohols, petroleum substitutes and other renewable energy sources can be produced using fungi. Recycling processes relying on fungal based biotechnology are cost-effective by means of disposing effluent (Robinson and Howell 1985). Besides, authentic and viable fungal cultures are needed to use as reference for identification of human, plant and animal pathogens and also for carrying out taxonomic and ecological studies. Scientific development in the field of mycology is incumbent upon the availability of authentic cultures defined in various publications and patent applications for independent study. Fungal studies are based on the availability of quality cultures that are well defined and this can be taken care of by their long-term storage. That is why culture collections are important for the preservation of diverse fungi and also serve as a source from which material can be had for teaching, research and other purposes (Onions 1971). The upkeep of fungal cultures in collections is a tardy task. It is advantageous to have a procedure that ensures the viability of valuable strains. One of the methods which is very simple and widely used for extending the ordinary stock cultures is mineral oil method (Buell and Weston 1947; Hartsell 1953). It is, therefore, of paramount importance to preserve cultures of high quality for use in agriculture, medicine, industry, etc. and also in the area of taxonomy (Malik and Claus 1987). This chapter will discuss about fungal culture collection or resource centres as well as methods for preservation of fungi.

21.2 Fungal Culture Collections

Fungal culture collections have an important place in microbiology and biotechnology. Their primary role is to preserve cultures and competent to provide information on the availability, identification, maintenance, nomenclature of cultures and regulations pertaining to transportation and patent regulations. Specialized culture collections give advice and provide services to industry. To characterize and identify fungal cultures is a time-consuming task. These cultures are invaluable and should be preserved and deposited at Culture Collection Centres at national and international level (Tables 21.1 and 21.2). At these centres, the experts properly maintain the cultures that are subjected to rigorous rules and regulations to safeguard the intellectual property rights of the depositors. The culture can be procured from these centres on payment. Therefore, these cultures are very valuable from commercial point of view (Anonymous 2008). As per World Data Centre for Microorganisms, total 3,119, 654 microorganisms comprising1,341,588 bacteria, 824,696 fungi, 38,622 viruses and 32,220 cell line are preserved in 781 culture collections in 76 countries and regions are registered in CCINFO (http://www.wfcc.info/ccinfo/. Accessed as of 13th June, 2019).

Table 21.1 National status of fungal culture collections: 15 out of 32 culture collections in India are fungal collection centers
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Table 21.2 Global status of fungal culture collections: 4 out of 5 regions in world have fungal culture collections as follows
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21.3 Preservation Techniques

Various techniques are available to preserve microorganisms (Table 21.3). These can be classified into three categories:

  1. 1.

    Continuous growth.

  2. 2.

    Dehydration.

  3. 3.

    Frozen storage.

The preservation methods aim to keep the viability and genetic stability of the culture through reduction in metabolic rate of microbes. This helps to prolong the period between subcultures. Continuous growth is achieved through various techniques that permit the fungi to grow and metabolize during period of storage. Various elements, namely, controlling growth conditions by restricting carbon, nitrogen and energy sources, decreasing the temperature, preventing dehydration, etc. step up the time period between subcultures (Anonymous 2008). Fungi can also be preserved by dehydration or drying techniques that comprise air-drying, drying in a vacuum either from the liquid or frozen phase, desiccation in or above a desiccant. In frozen storage, the fungal culture is stored at a temperature which freezes the culture, its metabolic rate is reduced and there is no physical change in it. The success of the preservation depends on medium and cultivation method used and the age of the culture at the time of preservation. There are two preservation methods: Short term and long term. Short-term methods of preservation are serial transfer of fungi to fresh medium followed by low temperature storage and keeping spores in dry sterile soil. Long-term methods include freeze-drying or ultra-freezing in vapour phase liquid nitrogen (−156 °C). There is no one method that can be used to preserve all types of fungi (Nakasone et al. 2004). Different fungal taxonomic groups react differently to different methods of preservation. Biological properties of fungi and their reaction to changes in their environment determine the type of methods for success of their preservation (Fennell 1960; Lloyd 1994; Simione and Brown 1991).

Table 21.3 Methods of preservation of fungal cultures
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Different preservation methods withhold availability of nutrients, water and oxygen to fungi leading to reduction in their metabolic rate. This is achieved by reduction in storage temperature or by a combination of these. The method of preservation to be used is determined by a number of factors, viz. nature of fungi, objective of preservation, availability of equipment and skilled manpower, probable preservation time frame, culture number and their use in future, ease of carrying them, frequency of use of cultures and keeping costs (Collee et al. 1996). All preservation methods have more or less similar protocol with distinct stages, viz. checking culture purity, preparing ampoules, growing the culture, cells suspension in preservation medium, putting cell suspension into ampoules, preservation, storage of ampoule stocks, updation of ampule stock records and testing viability, purity and genetic stability of preserved culture stocks.

21.3.1 Serial Transfer

Fungal cultures can be maintained by periodic transfer on fresh and sterile medium. Alternate cycles of active growth and storage periods are achieved by a series of subcultures. Periodicity of transfer differs with the kind of fungi.

21.3.2 Storage in Soil

Dry, sterile soil or sand is a very good medium for storage of some fungi for many years. This method is cheap and suitable for fungi such as Rhizoctonia (Sneh et al. 1991), Septoria (Shearer et al. 1974) and Pseudocercosporella (Reinecke and Fokkema 1979). Dryness induces dormancy over a period of time. Changes in morphology of some fungi due to it have been reported. The available moisture is used by the fungi during their growth and then they reach dormant state. The bottles are kept in refrigerator. The culture can be regrown by putting few soil particles on a suitable medium.

21.3.3 Storage in Silica Gel

Perkins (1962) developed this method for preservation of Neurospora species. He observed that viability of sporulating fungi can be maintained by using skimmed milk to protect them and then storing on silica gel. Silica gel powder can be used to store bacteria and yeast for a period of 1–2 years at low temperature. In this method, fine powder of silica is made followed by heat sterilization, cooling and then mixing with a thick suspension of cells and storing at a low temperature. This technique relies on quick desiccation at low temperature, and this helps to keep the cells viable for a long period.

21.3.4 Liquid Nitrogen

Liquid nitrogen can be used to preserve many fungi. The fungi which are not amenable to lyophilization can be preserved in liquid nitrogen. It is a costly method of preservation because continuous supply of liquid nitrogen has to be maintained. Dictyostelids (Raper 1984), amoebae (Davis 1956; Evans et al. 1982), zygomycetes including Entomophthorales (Humber 1994), oomycetes (Nishi and Nakagiri 1991), phytopathogenic fungi (Dahmen et al. 1983) and yeasts (Kirsop 1991) can be preserved using liquid nitrogen. Cell division and metabolic rate determines the mutation rate of cultured fungi. Therefore, any storage method that prevents division of cells and slows down metabolism while retaining viability is considered as the best method. Freezing fungal cultures at or below −139 °C prevents growth of ice crystals and slows down biophysical processes considerably, thereby ensuring cell survival.

21.3.5 Storage by Freeze-Drying

Spore-forming fungal cultures can be preserved by freeze-drying or lyophilization. In this method, cultures are rapidly frozen and a cryoprotectant is added to dissolve ice crystals and minimize their growth. Skimmed milk powder (sterile 5% or 10% solution) and filter-sterilized bovine serum are the commonly used cryoprotectants besides the proteinaceous materials.

21.4 Mineral Oil Overlay

A mineral oil is a petroleum distillate and contains a light mixture of higher alkanes. It is colourless and odourless. Mineral oil is also obtained as a liquid by-product while refining crude oil to make gasoline and other petroleum products. This liquid by-product is transparent, colourless and contains alkanes and cycloalkanes, related to petroleum jelly. It has a density of around 0.8 g/cm3. It is also known as liquid paraffin and white oil.

This method is simple, cost-effective and preserves fungal cultures for long duration time at ambient temperature (Fig. 21.1). Slant culture of microbes is immersed in mineral oil and the tubes are stored in upright position at room temperature.

Fig. 21.1
figure 1

Mineral oil storage of fungal cultures at NAIMCC, ICAR-NBAIM, Maunath Bhanjan, U.P. (a) Tubes containing fungal culture in mineral oil; (b) Rack to place the box in store room; (c) Box containing tubes

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Points to be taken into account while preserving the microbial cultures in oil:

  1. 1.

    The medium dries out and separates from the tube wall and floats to the wall surface if the medium is not completely immersed in oil leading to death of fungal culture.

  2. 2.

    The oil must be of good quality. The fungi are harmed if it is rancid or contains any toxic substance.

  3. 3.

    During autoclaving, the oil becomes milky due to mixing of moisture with it. Therefore, it is advisable that the oil be sterilized in the hot air oven at 150–170 °C for 1 h to remove milkiness.

21.4.1 History of Mineral Oil Preservation

Professor Frantisek Kral (1846–1911) of Prague was the first person to understand the importance of Culture Collections. He collected cultures and made these available to other workers by charging a fee. Professor Ernst Pribram later shifted this Collection to the University of Vienna in 1915. The other very old Collection, Centraalbureau voor Schimmelcultures (CBS), was founded in 1906 and is still in existence at Baarn, the Netherlands (Malik and Claus 1987). Mineral oil was used to preserved bacterial cultures about 100 years ago by Ungermann in 1918. He conserved bacterial cultures in dilute sera by overlaying them with oil. M. Michelle in (1921) used this method to preserve gonococci, meningococci and pneumococci in broth (Krasilnikov 1967). He slightly modified the method by using solid media. Morton and Pulaski (1938) used this method to maintain 45 cultures of bacteria. He compared this method with other storage methods and found mineral oil method to be useful to maintain viability of bacterial cultures for longer duration (Uzunova-Doneva and Donev 2005; Hartsell 1953).

Sherf (1943) used this method to conserve filamentous fungi, viz. Fusarium and Alternaria, and found cultures to be viable even after 6 months. In Australia, Norris (1944) preserved fungi by the said method and published a note on reporting cultures of seven genera of plant pathogens still viable after 18 months under oil (Hartsell 1956).

There are reports mentioning that fungi depending on their properties can be conserved without cultivation for 1–12 years under Vaseline oil. Optimal time limits have been worked out for cultivation of different taxonomical groups (Uzunova-Doneva and Donev 2005).

21.4.2 Process of Preservation of Fungi in Mineral Oil

  1. 1.

    The culture slants in glass tubes should be fresh and vigorously grown.

  2. 2.

    Take good quality mineral oil of low specific gravity, i.e. 0.830–0.890 g/cm3.

  3. 3.

    Heavy mineral oil should be autoclaved twice. First autoclaving may cause activation of bacterial spores followed by keeping the vials at room temperature for 24–48 h for their germination. Second round of autoclaving would kill germinated spores presented in oil.

  4. 4.

    Mineral oil is dried in oven at 170 °C for 1–2 h to remove entrapped moisture. It is an important step to remove water molecules from oil water.

  5. 5.

    The culture slant should be covered with sterile oil up to the depth of 1 cm.

  6. 6.

    Tightly cap tubes and put paraffin film to act as a vapour barrier.

  7. 7.

    Tubes should be stored in upright position. The cultures remain viable for a longer time if stored at low temperature as compared to ambient temperature.

  8. 8.

    Periodically check oil level in the tubes and add oil if need be.

21.4.3 Process of Culture Revival

  1. 1.

    Remove a part of culture immersed in mineral oil with a sterile needle/loop.

  2. 2.

    Excess oil should be drained from the explants by keeping on sterile filter paper and then placing it on fresh medium.

  3. 3.

    Afterwards, the tube should be resealed and returned for long-term storage.

  4. 4.

    Cultures should be monitored for viability and contamination.

  5. 5.

    The culture may be required to be subcultured several times to remove oil from it.

    Mode of action of mineral oil preservation.

The mode of action of mineral oil is yet to be fully unravelled. The evidence points to its action via checking dehydration of the cultures, retarding metabolic activity and growth of the fungi and by slowing down the gases exchange within fungi and surrounding (Table 21.4).

Table 21.4 Maximum survival time of Aspergillus species under mineral oil (Christina 1989)
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21.4.4 Advantage and Disadvantage of Mineral Oil Preservation

The mineral oil method has merits such as:

  1. 1.

    The procedure is simple.

  2. 2.

    Preserved cultures can be easily transferred.

  3. 3.

    The medium does not dry due to presence of paraffin oil.

  4. 4.

    All fungi including non-sporulating ones can be preserved.

  5. 5.

    The cultures do not get contaminated with mites.

  6. 6.

    Cultures remain in dormant state due to aerobic conditions.

  7. 7.

    Is useful for laboratories with limited amenities.

The mineral oil method has some disadvantages also

  1. 1.

    Changes in characterization of fungal cultures still occur due to growth of fungi and there can be selection of mutants capable of growing under adverse conditions.

  2. 2.

    Slow growing on retrieval.

  3. 3.

    Contamination of microbial spores from air.

21.5 Conclusion

Utmost care has to be exercised while preserving and maintaining fungal cultures in fungal culture collections. A number of fungal collections are available in India and abroad. Quality control is also important so that the revived cultures are true to type when compared with the original cultures. One has to be conversant with various methods of preservation so that suitable preservation method may be chosen keeping in view the characteristics and requirements of the microorganisms. Various factors, namely, the strain, culture medium, storage temperature, the sub-culturing medium, and the periodicity and technique of transfer determine viability duration and the maintenance of cultural characteristics of the strain. Of these methods, the mineral oil method appears to be an easy, convenient, economical and effective tool for the preservation of fungal strains for their long-term preservation under low temperature.