Glucose and lactose as cryoprotectants for fungal strains immobilised in sodium alginate: an emphasis on the conservation of the zygomycetes Rhizopus and Mucor (original) (raw)
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Viability of ectomycorrhizal fungi following cryopreservation
Fungal Biology, 2013
The use of ectomycorrhizal (ECM) fungi in biotechnological processes requires their maintenance over long periods under conditions that maintain their genetic, phenotypic, and physiological stability. Cryopreservation is considered as the most reliable method for long-term storage of most filamentous fungi. However, this technique is not widespread for ECM fungi since many do not survive or exhibit poor recovery after freezing. The aim of this study was to develop an efficient cryopreservation protocol for the long-term storage of ECM fungi. Two cryopreservation protocols were compared. The first protocol was the conventional straw protocol (SP). The mycelium of the ECM isolates was grown in Petri dishes on agar and subsequently collected by punching the mycelium into a sterile straw before cryopreservation. In the second protocol, the cryovial protocol (CP), the mycelium of the ECM isolates was grown directly in cryovials filled with agar and subsequently cryopreserved. The same cryoprotectant solution, freezing, and thawing process, and regrowth conditions were used in both protocols. The survival (positive when at least 60 % of the replicates showed re-growth) was evaluated before and immediately after freezing as well as after 1 week, 1 m, and 6 m of storage at À130 C. Greater survival rate (80 % for the CP as compared to 25 % for the SP) and faster re-growth (within 10 d for the CP compared to the 4 weeks for the SP) were observed for most isolates with the CP suggesting that the preparation of the cultures prior to freezing had a significant impact on the isolates survival. The suitability of the CP for cryopreservation of ECM fungi was further confirmed on a set of 98 ECM isolates and displayed a survival rate of 88 % of the isolates. Only some isolates belonging to Suillus luteus, Hebeloma crustuliniforme, Paxillus involutus and Thelephora terrestris failed to survive. This suggested that the CP is an adequate method for the ultra-low cryopreservation of a large set of ECM fungi and that further studies are necessary for the more recalcitrant ones.
Cryopreservation of filamentous micromycetes and yeasts using perlite
Folia Microbiologica, 2007
The viability, growth and morphology of 48 strains of Ascomycota (including 17 yeasts) and 20 strains of Zygomycota were determined after a 2-d and then after 1-year storage in liquid nitrogen using a new cryopreservation method with perlite as a particulate solid carrier. In case of Ascomycota, 45 strains (94 %) out of 48 survived both 2-d and 1-year storage in liquid nitrogen, respectively. In case of Zygomycota, all 20 strains survived both storage. In addition, 3 strains of Basidiomycota counted among yeasts were tested and all survived the 1 year storage. In all surviving cultures no negative effects of cryopreservation by this method have been observed after 1-year of storage in liquid nitrogen. The results indicate that the perlite protocol can be successfully used for cryopreservation of taxonomically different groups of fungi and also for fungi which failed to survive other routinely used preservation procedures. 154 L. HOMOLKA et al. Vol. 52
Actualidades Biologicas, 2014
Cuatro hongos basidiomycetes, Agaricus blazei Murrill (Agaricomycetideae), Ganoderma lucidum (W.Curt.: Fr.) P. Karst. y Grifola frondosa (Dicks.: Fr.) S.F. Gray (Higher Basidiomycetes), y Pleurotus pulmonarius (Fr.) Quél. (Agaricomycetideae) fueron evaluados bajo tres métodos de conservación durante 12 meses, observando su viabilidad con el fin de establecer el mejor método de conservación. La cinética de crecimiento, producción de biomasa y polisacáridos fueron estudiados. Los métodos de conservación implementados incluyeron: agua destilada a 24 ºC; aserrín y salvado de arroz con glicerol 10% a-20 ºC; aserrín y salvado de arroz con glicerol 10% a-80 ºC y; liofilización de biomasa con trehalosa o leche desnatada. Luego de realizar el análisis de los resultados de 12 meses de conservación, se determinó que el tratamiento de agua destilada a 24 ºC fue el mejor método de conservación con el porcentaje de recuperabilidad más alto un 83,3% en el mes 12, seguido por el tratamiento de crioconservación a-80 ºC donde se recuperó el 75%, sin afectar negativamente la producción de biomasa y polisacáridos. El tratamiento a-20 ºC y la liofilización no fueron efectivos; con la crioconservación a-20 ºC solo se recuperaron cepas en el primer mes y con la liofilización no fue posible recuperar cepas en el período de 12 meses evaluado.
Mycopathologia, 2016
Serious mycological work requires a reliable source of cultures that are maintained under safe long-term storage. In this study, 1186 clinical fungal isolates consisting of molds (20 species in 11 genera) and yeasts (21 species in seven genera) maintained in water, under mineral oil at room temperature and cryopreserved at −80°C for periods ranging from 1 to 12 years, were evaluated for their viabilities and stabilities. The strains were subcultured onto either Sabouraud dextrose agar or potato dextrose agar to determine the viabilities and purities. The stabilities of the dermatophytes were investigated using urease test medium, the Trichophyton agar test and morphological examination. The stabilities of yeasts were evaluated by microscopic morphology and by determining the antifungal susceptibilities of random samples of yeasts (n = 120). Additionally, 365 strains (dermatophytes, n = 115; yeasts, n = 250) were further characterized by "matrix-assisted laser desorption/ionization timeof-flight mass spectrometry." After 12 years of preservation, the survival rates with the three different preservation techniques, i.e., in water, under mineral oil and by freezing, were assessed as 94.7, 82.0 and 97.4 %, respectively. Viability was generally unrelated to the duration of storage. More stable and consistent growth was achieved after storage in water and freezing compared with mineral oil preservation. Our results demonstrate that the procedure for maintaining fungal cultures in water is a simple and inexpensive method, next to cryopreservation, and that both can be reliably used for the long-term preservation of most fungal isolates.
Viability of fungal cultures maintained at -70 degrees C
Journal of clinical microbiology, 1992
One thousand four hundred forty-seven clinical and environmental isolates of molds, yeasts, aerobic actinomycetes, and algae belonging to 164 genera (382 taxa) maintained on potato dextrose agar at -70 degrees C for periods ranging from 6 months to 13 years were subcultured and then incubated at 25 degrees C to determine their viabilities. Thirty-three isolates, Alternaria alternata (n = 1), Apophysomyces elegans (n = 1), Bipolaris spicifera (n = 1), Blastomyces dermatitidis (n = 4), Cokeromyces recurvatus (n = 1), Coremiella cubispora (n = 1), Cryptococcus ater (n = 1), Curvularia sp. (n = 1), Exserohilum monoceras (n = 1), Exserohilum pedicillatum (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), Sporothrix sp. (n = 1), and Mycelia Sterilia (n = 8), did not grow after repeated attempts at subculturing. Neithe...
Mycoscience, 2012
The present investigation was designed to observe the survival of the anaerobic fungus Caecomyces sp. in various routine preservation methods. Among all the treatments, cryopreservation of fungi at-70°C with glycerol was found to be most effective for long-term maintenance (more than 90 days) of rumen fungi, followed by dimethyl sulfoxide (DMSO) and ethylene glycol (up to 60 days). In contrast, at-196°C, DMSO showed maximum survival (more than 90 days), followed by glycerol (up to 90 days) and ethylene glycol (up to 30 days). At 39°C, maximum survival (up to 30 days) was observed with soft agar and wheat straw; at refrigeration temperature, preservation with Orpin's media containing straw showed maximum survival (up to 30 days).
Vitality and genetic fidelity of white-rot fungi mycelia following different methods of preservation
Mycological Research, 2009
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Freeze-drying fungi using a shelf freeze-drier
Methods in molecular biology (Clifton, N.J.), 2007
Lyophilization, the removal of water by freezing and then volatilization at low pressure and temperature, has been employed as a standard long-term preservation method for many filamentous fungi. The method outlined involves the use of standard shelf freeze-drying and skimmed milk as a suspending solution/lyoprotectant. This approach has been employed to freeze-dry the majority of the 50,000 fungal strains that have been successfully lyophilized at the Centraal bureau voor Schimmelcultures (CBS) culture collection (http://www.cbs.knaw.nl/).
A new low-cost method for long-term preservation of filamentous fungi
Biocatalysis and Agricultural Biotechnology, 2019
Preservation of fungi for commercial and research purposes for a long time, is a very tedious task. Cryopreser-vation and lyophilization have been extensively used as long-term preservation techniques. Here, we introduced a novel method for long preservation and fast revival of filamentous fungi using sterile cotton balls (CB). A 16 fungal genera and 47 species represented by 135 strains have been preserved on cotton balls moistened with Potato Dextrose Agar (PDA) and incubated at 25 � 2 � C until giving sufficient growth, then preserved at 17-20 � C for 36 months. The revival of the fungal growth on PDA was monitored every 6 months. A 135 fungal strains have been revived by the CB method with an overall revival rate of 100% after two years of long-term preservation. While, 76 strains were revived after three years of preservation and the revival rate was 59%. The viability or the mycelial morphology was unaffected by the new method even after three years of preservation. It is an easy, cost-effective, and convenient method for preservation of filamentous fungi for long period without contamination risk. We believe that the CB method is a very convenient method for a cheap delivery of fila-mentous fungi between research institutes.
Experimental comparison: Methods for the preservation of fungal cultures
Current Research in Environmental & Applied Mycology, 2019
Fungal preservation is of utmost importance both with respect to commercial as well as research purpose; however its long term maintenance is troublesome. Frequent sub-culturing and restocking of the preserved culture often result in mutations. Most preservation techniques like cryopreservation, lyophilisation and silica gel are widely used but are researcher unfriendly for regular recovery and longstanding research. In this study different fungal species were preserved using the slant method, stab method and glycerol-slice method to elucidate the most effective technique feasible for both regular use and long term fugal preservation. Dataset revealed that stoage in glycerol-slice method lasts longer than the other methods and is recommended for laboratories where specialized conservation facilities are unavailable.