ABSTRACT
BACKGROUND: The use of a Stirling cycle freezer for cryopreservation is considered to have significant advantages over traditional methodologies including N2 free operation, application of low cooling rates, reduction of sample contamination risks and control of ice nucleation. OBJECTIVE: The study assesses the suitability of an 'N2-free' Stirling Cycle controlled rate freezer for fungi cryopreservation. METHODS: In total, 77 fungi representing a broad taxonomic coverage were cooled using the N2 free cooler following a cooling rate of -1 degrees C min(-1). Of these, 15 strains were also cryopreserved using a traditional 'N2 gas chamber' controlled rate cooler and a comparison of culture morphology and genomic stability against non-cryopreserved starter cultures was undertaken. RESULTS: In total of 75 fungi survived cryopreservation, only a recalcitrant Basidiomycete and filamentous Chromist failed to survive. No changes were detected in genomic profile after preservation, suggesting that genomic function is not adversely compromised as a result of using 'N2 free' cooling. CONCLUSION: The results demonstrate the potential of 'N2-free' cooling for the routine cryopreservation of fungi in Biological Resource Centres.
Subject(s)
Cryopreservation/instrumentation , Fungi/physiology , Genome, Fungal , Biological Specimen Banks , Cryopreservation/methods , DNA, Intergenic , Genetic Markers , Genomic Instability , Microbial Viability , Nitrogen , Species Specificity , Tandem Repeat SequencesABSTRACT
Ecological diversification of Bacillus thuringiensis soil isolates was examined to determine whether bacteria adapted to grow at low temperature and/or potentially pathogenic correspond to genetically distinct lineages. Altogether, nine phylogenetic lineages were found among bacilli originating from North-Eastern Poland (n = 24) and Lithuania (n = 25) using multi-locus sequence typing. This clustering was chiefly confirmed by pulsed-field gel electrophoresis. One third of the bacilli were found to be psychrotolerant, which strongly supports the hypothesis of the existence of thermal ecotypes among B. thuringiensis. PCR screening was also performed to detect potential enterotoxin genes and Bacillus anthracis pXO1- and pXO2-like replicons. The cytK-positive isolates (22%) were significantly associated with two phylogenetic lineages (potential CytK pathotypes), whereas there was no correlation between phylogenetic grouping and the presence of the potential tripartite enterotoxin pathotypes (86% of strains). A statistically significant association between phylogenetic lineages and ecologic properties was found with regard to the cry1-positive Lithuanian isolates, while the cry genes in Polish isolates and the pXO1- and pXO2 replicon-like elements showed scattered distribution across phylogenetic lineages. Our results support the hypothesis that B. thuringiensis comprises strains belonging to different phylogenetic lineages, which exhibit specific ecological properties.