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Contenido archivado el 2024-04-15

DEVELOPMENT OF IMPROVED TECHNIQUES FOR THE PRESERVATION AND MAINTENANCE OF CULTURES OF FUNGAL STRAINS OF BIOTECHNOLOGICAL IMPORTANCE

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Our research programme has resulted in the development of improved long-term preservation procedures to retain purity, viability and stability of fungi. Cryomicroscopy has been used for the first time on fungi and the observed responses to cooling used to predict their survival following freezing techniques.
The techniques of freeze drying and cryopreservation have been used for many years but were not specifically developed for fungi. No fundametnal research had been carried out to determine the effect of cooling on fungal cells; this was needed for a better understanding of preservation in order to develop improved methods. To optimize the techniques of freezing and freeze drying for the perservation of viability and stability of properties of fungi of biotechnological importance. The primary approach was to determine the effects of cooling and subsequent rewarming on selected strains of fungi, and employing the findings to devise optimal methods of preservation. The viability, morphology, physiology, biochemistry and other aspects of genetic stability were monitored before and after perservation and during storage under a variety of conditions. The results of preservation by the newly developed techniques were compared with those by traditional methods of freeze drying and cryopreservation. This enabled recommendations to be made on optimum techniques to adopt for different fungi. 25 of 26 fungi were shown to have optimum cooling rates for recovery. Death was related to shrinkage at slow cooling rates and, in Lentinus edodes, membrane integrity was lost during fast cooling. Many fungi gradually lost viability during storage at temperatures of -40 C or above. Most strains of fungi belonging to the Mastigomycotina failed to recover after 1 year of storage at -20 and -40 C but remained viable in liquid nitrogen vapour (approximately -175 C). 45 (60%) fungi failed to survive or died in storage when freeze dried using methods involving accelerated drying. The properties of the survivors did not deteriorate or change with loss of viability. Change seemed to relate more to storage time. Recovery time preserved strains became longer with storage time. In general, the properties of the fungi and yeasts tested were retained (3 y) but must be reassessed in the long term.

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