Securing the UK National Microbial Genetic Resource: Development of Cryopreservation Protocols for Recalcitrant Microorganisms

BASED AT

Bakeham Lane, Egham, Surrey, UK TW20 9TY.

TEL +44 (0) 1784 470111 : FAX +44 (0) 1491 829100

For further information contact: M.Ryan@cabi.org

AIMS OF RESEARCH

To develop and improve cryopreservation protocols for recalcitrant microorganisms and cell lines:

* Filamentous Fungi * Yeast's * Animal Cells * Insect Cells * Algae * Protozoa * Bacteria * Nematodes

To safely cryopreserve organisms in order to secure the long-term future of the UK National Microbial Resource

BACKGROUND

Many microorganisms and cell lines are relatively easy to maintain and preserve. However, a large number of microorganisms and cell lines are difficult to preserve whilst some cannot be preserved at all, these organisms are collectively known as 'Preservation Recalcitrant'. Through this DEFRA funded project, new techniques and existing protocols are being developed in an attempt to successfully preserve these organisms, many of which are of immense scientific and economic importance.

Importantly, biochemical and molecular methods are being utilised to allow in-depth assessment to be made of the stability of an organism's genetic and physiological characters post preservation. Protocols that provide stability of characteristics or at the very least minimise the prospects of damage will be developed for key target organisms.

 TARGET ORGANISMS

The UKNCC / DEFRA project focuses on the cryopreservation of organisms that are economically important:

Aquatic Pathogens of fish and amphibians (Aphanomyces spp., Saprolegnia spp.)

Plant Pathogens (Phytophthora spp., Moniliophthora spp., Diplocarpon spp.)

Biological Control Agents (Steinernema spp., Puccinia spp., Metarhizium spp. Beauveria spp.)

Human Pathogens (Helicobacter spp.)

 APPROACHES TO CRYOPRESERVATION

Traditional Approaches

The application of strictly defined controlled rate cooling programmes in association with different combinations of cryoprotectants. Optimal regimes often previously determined using light cryo-microscopy and/or the results from viability assessments.

This has been the ‘classical’ approach to cryopreservation for many years and remains the most adequate mechanism for the cryopreservation of many microorganisms. A cooling rate of -1^oC min^-1 is suitable for most organisms. Common cryoprotectants include: Glycerol, Trehalose, Dimethylsulphoxide (DMSO), Polyvinylpyrrolidone (PVP), Desferrisoxamine

Vitrification

The addition of highly concentrated cryoprotectant solutions to cell-systems, that when exposed to ultra-low temperatures, initiates the formation of an amorphous glass, which prevents the development of damaging ice crystals. Vitrification solutions consist of concentrated combinations of osmotically active compounds (e.g. glycerol, trehalose, ethylene glycol, DMSO & sucrose) and may have the potential to be extremely toxic. Vitrification has previously been applied to animal, plant and human cells and has proved successful for some basidiomycete fungi although post preservation physiological/genomic stability tests and toxicological assessments have not been undertaken. The use of vitrification/cryopreservation has potential for use for a wide range of organisms including oomycete fungi, algae and protozoa.

Immobilisation

The encapsulation of microorganisms in calcium alginate beads or other appropriate matrix. The use of immobilisation has been tested for fungi e.g. Serpula lacrymans, (Ryan, 2001), and will be extended for nematodes, protozoa & algae. Once immobilised, cells can be preserved by traditional preservation methodology. Specimens can be pre-treated before preservation; for example, dehydration before cryopreservation, can reduce the potential of damaging ice crystal formation. The properties of the alginate matrix can be altered by varying the chemical concentration of the constituents and by adding "fillers" such as bentonite

In situ techniques

The cryopreservation of an organism in association with its host (or host material) is used for organisms that can not be cultured using traditional in vitro techniques or that can not be cryopreserved using standard cryo-protocols. In situ cryopreservation has potential for a wide range of organisms. For example, obligate pathogens, unculturable organisms or organisms involved in symbiotic associations such as Mycorrhizal fungi. In situ cryopreservation has been used in an attempt to preserve isolates of a microcyclic rust species (Puccina spp.). Traditional approaches to its preservation failed whereas cryopreservation of the fungus in association with its host material proved to be successful.

Monitoring success

To assess the success of cryopreservation regime, a number of techniques are being utilised to provide an indication of the stability of an organisms characters after preservation. When characters are influenced by cryopreservation, alternative protocols are applied in an attempt to reduce strain damage.

Qualitative methods (observations, microscopy)

Quantitative methods (growth rate, sporulation capacity)

Biochemical methods (chromatography of metabolites, enzyme assays & protein analysis)

Molecular methods (PCR, sequencing, functional genomics)

FURTHER READING

Ryan, M.J. (2001). The use of immobilisation for the preservation of Serpula lacrymans. Mycologist 15: 66-69

Ryan, M.J., Jeffries, P., Bridge, P.D., & Smith, D. (2001). Developing cryopreservation protocols to secure fungal gene function Cryo-Letters 22: 115-124

Ryan, M.J., Smith, D. & Jeffries, P. (2000). A decision-based key to determine the most appropriate protocol for the preservation of fungi. World Journal of Microbiology and Biotechnology 16: 183-186.

Smith, D. & Ryan, M.J. (2001). Culture collections in the twenty first century. Biologist, 48: 125-128

Smith, D., Ryan, M.J. & Day, J.G. (2001) The UKNCC Biological Resource: Properties, Maintenance and Management. 382 pages. UKNCC Secretariat, Egham, Surrey, UK. ISBN 0954028503.

Collaborators

Our collaborators include individuals at the following institutions: