The development of a cryopreservation method suitable for a large cyanobacteria collection

Laboratories worldwide working with cyanobacteria have created culture collections to carry out related studies. However, the lack of manpower (especially after the studies were finished), maintenance costs and proper preservation methods often results in the loss of research materials and a waste of isolation effort. Several parameters are generally considered very important in cryopreservation, including the choice of the cryoprotectant, cryoprotectant concentration, freezing rate, physiological status of the culture and thawing procedure. This makes it very difficult to establish universal guidelines for cyanobacteria cryopreservation. Herein, we present a cryopreservation method suitable for a range of strains, using two cryoprotectants (methanol and dimethyl sulfoxide at a final concentration of 5 and 3 %, respectively) along with a combined vital staining and reproductive viability criteria for the post-thawing recovery. The obtained results are very encouraging as more than 83 % of tested cyanobacteria were amenable for cryopreservation, 80 % of strains (111 in total) showing more than 90 % recovery with at least one of the cryoprotectants used.     

Long-term preservation of some Rhodospirillaceae by freeze-drying

Lyophilization of phototropically grown cultures is difficult as it is essential to maintain strict anaerobic conditions or to avoid the exposure of the cultures to light. During a systematic investigation it was observed that several species of Rhodospirillaceae are able to grow heterotrophically in darkness on organic media and are not damaged by air oxygen under such conditions. Based on this character several oxygenic species of Rhodospirillaceae were grown under heterotrophic conditions and were lyophilized using raffinose (5% w/v) along with skim milk (20% w/v) as a protective agent. More than 30 strains from nine species of Rhodospirillaceae were successfully freeze-dried with this method. All tested cultures proved viable and showed 10–100% survival after lyophilization. During 2–3 years of storage at 9°C no further loss in viability was observed. In such lyophilized cultures no loss in photoautotrophy, diazotrophy or other desirable characters like hydrogen production, pigmentation, etc. was detected. The method is not suited to such anoxygenic Rhodospirillaceae which are not able to grown aerobically in darkness.     

Low-cost and low maintenance preservation of Agaricus brasiliensis cultures

Agaricus brasiliensis cultures quickly lose viability when stored at cool temperatures, even for a short period of time. We evaluated several low-cost preservation methods using varied substrates, preservation solutions, and storage temperatures. Agaricus brasiliensis was intolerant to freezing temperatures, making liquid nitrogen use and deep-freezing methods impossible for its preservation. The best preservation conditions for the A. brasiliensis CS1 strain tested in this study were obtained by using rice as substrate and water as preservation solution, with storage at room temperature or when using soil, mushroom cultivation compost, or rice and stored at 10 °C without preservation solution. Those cultures that were reactivated showed the same productivity attributes as the control. In addition, no effect on productivity or biological efficiency was observed through successive subculturing of the strain (CS1). Parboiled rice was successfully used for other A. brasiliensis strains (CS2, CS5, CS7, CS9, and CS10), and also for Pleurotus ostreatus, P. sajor-caju, and Lentinula edodes.     

Long-term viability of preserved eukaryotic algae

Levels of viability of Chlorella emersonii after storage of dried material for one year were 0.1% on rehydration, all other dried organisms examined in this study failed to recover after prolonged storage. In addition, no detectable recovery was observed in any of the algae tested after storage of freeze-dried cultures. Methods have also been developed to cryopreserve a range of microalgae, but no single protocol has been found to be universally satisfactory. Some strains are apparently not able to withstand cryopreservation using known methods, whilst others may be frozen successfully in the absence of cryoprotectant by plunging directly into liquid nitrogen. A two-step protocol (cooling to an intermediate subzero temperature prior to plunging into liquid nitrogen) has been used to cryopreserve the majority of strains. Where this has proven successful, post-thaw viability levels of over 95% have been attained for some algae. This paper demonstrates that, where applicable, cryopreservation allows the long-term preservation of frozen algae with no significant reduction in viability up to 22 years storage. (Previous location of Culture Collection of Algae and Protozoa) This revised version was published online in September 2006 with corrections to the Cover Date.     

Genetic control of hydrogen metabolism in cyanobacteria

The development of methods for the use of phototrophic cyanobacteria as producers of molecular hydrogen via bioconversion of solar energy is a promising filed of hydrogen energetics. Optimization of hydrogen formation and release is based on studying the genetic control of hydrogen metabolism and the use of genetic approaches for obtaining efficient producer strains. Data on genes coding for the hydrogenases that are responsible for hydrogen uptake and production in cyanobacteria are summarized. Bioinformatic methods have been used to construct the scheme of the hydrogen metabolism gene network of nitrogen-fixing heterocystous cyanobacteria. The possible approaches to constructing the cyanobacterium strains producing molecular hydrogen that would be promising for photobiotechnology by mutagenesis and genetic engineering methods are discussed in terms of this model and analysis of the data on hydrogen-producing mutants.     

Genetic control of hydrogen metabolism in cyanobacteria

The development of methods for the use of phototrophic cyanobacteria as producers of molecular hydrogen via bioconversion of solar energy is a promising filed of hydrogen energetics. Artificial optimization of hydrogen formation and release is based on studying the genetic control of hydrogen metabolism and the use of genetic approaches for obtaining efficient producer strains. Data on genes coding for the hydrogenases that are responsible for hydrogen uptake and production in cyanobacteria are summarized. Bioinformatic methods have been used to construct the scheme of the hydrogen metabolism gene network of nitrogen-fixing heterocyst cyanobacteria. The possible approaches to constructing the cyanobacterium strains producing molecular hydrogen that would be promising for photobiotechnology by mutagenesis and genetic engineering methods are discussed in terms of this model and analysis of the data on hydrogen-producing mutants.     

Hydrogen formation by cyanobacteria cultures selected for nitrogen fixation

Seventy-one cyanobacteria containing cultures were enriched from various soil and water locations either under aerobic and/or anaerobic conditions on agar medium selective for nitrogen fixation. Kept under argon containing 1% CO₂ for 24 and 48 h most of these cultures evolved hydrogen at very variable rates up to 116 l per mg chlorophyll and hour as a mean value over a time period of 24h. Several samples evolved hydrogen more efficiently compared with known hydrogen producing pure strains from culture collections. Thirty-one of the investigated cultures showed a hydrogen formation higher than 10 l per mg chlorophyll and hour measured over 24 or 48 h. Among these all the morphological forms of cyanobacteria i.e. unicellular and filamentous with or without heterocysts are found. Hence, selecting for nitrogen fixing cyanobacteria seems to be a practical method to find efficient hydrogen producers.     

Viability of fastidious Phytophthora following different cryopreservation treatments

Living stock cultures with constant phenotypes and genotypes are required for a wide range of research and industrial applications; however, long-term, stable preservation of fastidious Phytophthora strains has been challenging. In this study, we systematically evaluated different cryopreservation treatments to identify and clarify freezing, thawing, and other conditions appropriate for long-term maintenance. Optimal preservation conditions were largely strain-specific, with robust strains remaining fully viable and the fastidious yielding lower recovery under all test conditions. Nevertheless, several procedures were shown to be generally applicable for effective cryopreservation of most Phytophthora organisms. Fastidious strains retained higher viability following the −1 °C min⁻¹ freezing protocol (Mr Frosty's) than either of two widely used programmed freezing procedures. Revival was higher when frozen mycelium plugs were thawed at 37 °C for 2 min or 25 °C for 5 min, while lower viability was apparent for fastidious strains thawed at 55 °C for 1.5 min. Among 15 cryoprotective solutions assessed, 5 % dimethyl sulfoxide produced the highest viability for all fastidious strains. The effect of prefreeze and postfreeze treatments on revival was mild, if any, and strain-dependent. This study has generated reliable, practical, long-term preservation solutions applicable to a majority of Phytophthora species. It also has revealed a need for in-depth physiological and morphological investigations to further enhance the preservation methods for fastidious strains.     

Preservation of the hyperthermophile Pyrococcus furiosus

Methods are evaluated for the preservation of the hyperthermophile Pyrococcus furiosus . The use of glass capillary tubes stored over liquid nitrogen with dimethyl sulphoxide appears to be the preferred method of preservation. Lyophilization resulted in loss of viability and storage at room temperature and +4°C resulted in considerable loss of viability within 4 weeks.     

Preservation of live cultures of basidiomycetes – Recent methods

Basidiomycetes are used in industrial processes, in basic or applied research, teaching, systematic and biodiversity studies. Efficient work with basidiomycete cultures requires their reliable source, which is ensured by their safe long-term storage. Repeated subculturing, frequently used for the preservation, is time-consuming, prone to contamination, and does not prevent genetic and physiological changes during long-term maintenance. Various storage methods have been developed in order to eliminate these disadvantages. Besides lyophilization (unsuitable for the majority of basidiomycetes), cryopreservation at low temperatures seems to be a very efficient way to attain this goal. Besides survival, another requirement for successful maintenance of fungal strains is the ability to preserve their features unchanged. An ideal method has not been created so far. Therefore it is highly desirable to develop new or improve the current preservation methods, combining advantages and eliminate disadvantages of individual techniques. Many reviews on preservation of microorganisms including basidiomycetes have been published, but the progress in the field requires an update. Although herbaria specimens of fungi (and of basidiomycetes in particular) are very important for taxonomic and especially typological studies, this review is limited to live fungal cultures.     

Viability and sporulating capability of Coelomycetes preserved under a range of different storage regimes

The viability and sporulating capability of 45 Coelomycetes strains were evaluated. Strain subcultures were maintained under mineral oil, in soil and on agar slant for different periods of time lasting as long as 50 years, 39 years and 2 years, respectively. Of the 34 strains preserved under mineral oil, 20 maintained their viability but lost the sporulating capability with exception of one strain of Pestalotiopsis guepinii. Of the 16 strains also preserved in soil only one was viable and it was not able to sporulate. All 12 endophytic strains, 11 preserved on agar slant and one under mineral oil remained viable; however, the strain preserved under mineral oil lost its sporulating capability, while the strains on agar slant were only able to sporulate after culturing on sterilized alfalfa twigs. The results demonstrate that routine monitoring, and the use of different preservation methods, specially with the addition of sterilized plant tissue on the culture media for promoting conidiomata formation, is necessary for the success of the Coelomycetes long-term preservation.     

Effect of cultivation conditions on trehalose content and viability of brewing yeast following preservation via filter paper or lyophilization methods

The effects of variations in cultivation conditions on trehalose concentration and the viability of brewing yeasts following preservation by filter paper or lyophilization methods were evaluated. In case of filter paper preservation, the cultivation period had no affect on yeast viability, while agitation and aeration during cultivation had a positive effect regarding viability of the bottom-fermenting strains, Rh and Frank. For effective preservation, it was necessary to harvest yeast cells from the stationary phase during cultivation. For lyophilization preservation, the yeast strains tested showed a negative effect on viability, independent of strain or cultivation method. No significant correlation was found between trehalose concentration and yeast viability following either filter paper or lyophilization preservation. However, the filter paper preservation method was suitable for both bottom and top brewing yeast strains with regard to feasibility, viability, and maintenance of the yeast’s specific character.     

Preservation of Chytridiomycota in culture collections

Methods for the preservation of fungi in the Chytridiomycota in culture collections are reviewed in this paper. The Chytridiomycota can be preserved with varying degrees of success using a number of different protocols including cryopreservation. The survival of fungi in the Chytridiomycota is sensitive to environmental factors such as lack of moisture, high temperatures, high osmotic potential, and availability of oxygen, all of which must be considered in designing preservation methods. The age of the culture at the initiation of preservation appears to be a particularly important determinant of viability. Recently, commonly used methods for preservation of other groups of fungi have been modified to improve the survival of the Chytridiomycota in culture collections. High rates of survival have been reported after cryopreservation of aerobic and anaerobic chytrids in 10 % glycerol or dimethyl sulphoxide as cryoprotectants. The rates of freezing and thawing must be carefully controlled in the methods for cryopreservation considered in this review. Further research on increasing long-term survival rates and morphological, physiological and genetic stability of Chytridiomycota at low temperatures is necessary.     

Establishment and characterization of fibroblast cultures derived from a female common hippopotamus (Hippopotamus amphibius) skin biopsy

The population decline of the common hippopotamus (Hippopotamus amphibius) has necessitated the preservation of their genetic resources for species conservation and research. Of all actions, cryopreservation of fibroblast cell cultures derived from an animal biopsy is considered a simple but efficient means. Nevertheless, preserving viable cell cultures of the common hippopotamus has not been achieved to our knowledge. To this end, we established and characterized fibroblast cell cultures from the skin sample of a newborn common hippopotamus in this study. By combining the tissue explant direct culture and enzymatic digestion methods, we isolated a great number of cells with typical fibroblastic morphology and high viability. Neither bacteria/fungi nor mycoplasma was detectable in the cell cultures throughout the study. The population doubling time was 34 h according to the growth curve. Karyotyping based on Giemsa staining showed that the cultured cells were diploid with 36 chromosomes in all, one pair of which was sex chromosomes. The amplified mitochondrial cytochrome C oxidase subunit I gene sequence of the cultured cells was 99.26% identical with that of the registered H. amphibius complete mitochondrial DNA, confirming the species of origin of the cells. Flow cytometry and immunofluorescence staining results revealed that the detected cells were positive for fibroblast markers, S100A4, and vimentin. In conclusion, we generated the fibroblast cell cultures from a common hippopotamus and identified their characteristics using multiple techniques. We believe the cryopreserved cells could be useful genetic materials for future research.     

Effect of cryopreservation and lyophilization on viability and growth of strict anaerobic human gut microbes

Strict anaerobic gut microbes have been suggested as ‘next‐generation probiotics’ for treating several intestinal disorders. The development of preservation techniques is of major importance for therapeutic application. This study investigated cryopreservation (?80°C) and lyophilization survival and storage stability (4°C for 3 months) of the strict anaerobic gut microbes Bacteroides thetaiotaomicron, Faecalibacterium prausnitzii, Roseburia intestinalis, Anaerostipes caccae, Eubacterium hallii and Blautia obeum. To improve preservation survival, protectants sucrose and inulin (both 5% w/v) were added for lyophilization and were also combined with glycerol (15% v/v) for cryopreservation. Bacterial fitness, evaluated by maximum growth rate and lag phase, viability and membrane integrity were determined using a standardized growth assay and by flow cytometry as markers for preservation resistance. Lyophilization was more detrimental to viability and fitness than cryopreservation, but led to better storage stability. Adding sucrose and inulin enhanced viability and the proportion of intact cells during lyophilization of all strains. Viability of protectant‐free B. thetaiotaomicron, A. caccae and F. prausnitzii was above 50% after cryopreservation and storage and increased to above 80% if protectants were present. The addition of glycerol, sucrose and inulin strongly enhanced the viability of B. obeum, E. hallii and R. intestinalis from 0.03–2% in protectant‐free cultures to 11–37%. This is the first study that quantitatively compared the effect of cryopreservation and lyophilization and the addition of selected protectants on viability and fitness of six strict anaerobic gut microbes. Our results suggest that efficiency of protectants is process‐ and species‐specific.     

Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing

For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for genetic analysis.     

Yeast culture collections of the world: meeting the needs of industrial researchers

The importance of selecting optimal yeast strains for research or industrial applications is often underestimated. For example, utilizing a strain background that already provides the desired stress tolerance or nutrient utilization profile can eliminate costly strain optimization. Yeast culture collections can provide not only the yeast strains but also data and curator expertise to help narrow the search for the optimal strain. While some collections are known for a broad range of cultures and services, other "boutique" collections can provide a broader selection of strains of certain categories, a surprising amount of characterization data, and assistance in selecting strains. This article provides information on dozens of yeast collections of the world, profiles of selected yeast culture collections, and the services that they provide: e.g., strain preservation for patent or safe deposit purposes, species identification service, training workshops, and consulting on yeast identification and physiology. Utilization of these services can save industrial researchers valuable time and resources.     

Natural biopolymer for preservation of microorganisms during sampling and storage

Stability of microbial cultures during sampling and storage is a vital issue in various fields of medicine, biotechnology, food science, and forensics. We have developed a unique bacterial preservation process involving a non-toxic, water-soluble acacia gum polymer that eliminates the need for refrigerated storage of samples.The main goal of this study is to characterize the efficacy of acacia gum polymer for preservation of pathogenic bacteria (Bacillus anthracis and methicillin-resistant Staphylococcus aureus—MRSA) on different materials, used for swabbing and filtration: cotton, wool, polyester, rayon, charcoal cloth, and Whatman paper.Acacia gum polymer used for preservation of two pathogens has been shown to significantly protect bacteria during dehydration and storage in all tested samples at the range of temperatures (5-45 °C for MRSA and 40-90 °C for B. anthracis). Our results showed higher recovery as well as higher viability during the storage of both bacteria in all materials with acacia gum. Addition of acacia gum polymer to swabbing materials or filters will increase efficacy of sample collection and identification of pathogenic bacteria from locations such as hospitals or the environment. Proposed approach can also be used for long-term storage of culture collections, since acacia gum contributes to viability and stability of bacterial cultures.     

A simple and efficient method for cryopreservation of embryogenic triticale calli

Conventional breeding methods are now supplemented by modern in vitro techniques. However, long term maintenance of cultures has many disadvantages. It incurs risk of loss through microbial contamination, somaclonal variation or human error, but above all the regeneration capacity can decrease gradually during extended maintenance. Cryopreservation as a method of long term storage of biological material without genetic alteration was adapted for embryogenic triticale calli preservation. Callus of both winter and spring genotypes were successfully cryopreserved. The best viability rates (80–85%) were achieved with 6 weeks old winter genotypes treated with cryoprotective solution containing DMSO. This simple and efficient method of cryopreservation requires no special devices for controlled freezing and can be easily adapted for other cereals.     

Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains

Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts. Since 1930, successive subculture has been used to preserve C. neoformans isolates in our Fungus Collection. In the 1970s, some of these Fungus Collection samples were selected to be subjected to a different methods of maintenance--that of lyophilized. Our objective was to analyze C. neoformans isolates in order to make a comparative evaluation between these two methods of preservation. The overall aim of this study was to qualify the preservation technique used in our mycology laboratory since the technique used might affect the survival, stability and purity of the primary isolates in culture. The samples were analyzed using classical mycology methods and using the randomly amplified polymorphic DNA technique In the analysis of phenotypes and genotypes, the typical characteristics of C. neoformans were found to differ in relation to the different methods of preservation employed. The aim of this study was to demonstrate the importance of selecting the appropriate method of preservation for fungus collections. This selection can affect the survival and purity of the cultures, and preserve the stability of their physiological, biochemical, and genetic characteristics.