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Securing the UK National Microbial Genetic Resource: cryopreservation protocols for recalcitrant organisms - GC0126

This study will establish vital preservation protocols needed to cryo-store key groups of recalcitrant organisms that currently cannot be successfully retained for the long-term. It will follow-up the results of a MAFF sponsored pilot study and provide the tools to secure the UK microbial genetic resource held by the United Kingdom National Culture Collection (UKNCC). The holdings of the UKNCC are part of the national heritage for future research and exploitation and must be secured to enable the collections to discharge their public service functions. The holdings are of relevance to MAFF activities in many ways. Of the 73 000 strains held over 10 000 are from food related sources, a similar number are plant pathogens and there are several thousand organisms involved in the natural processes of soil fertility, plant health, biocontrol and agrochemical production. Examples of these organisms are supplied to MAFF laboratories. The UKNCC is a repository for replicate samples of strains with MAFF investment ensuring their availability for future use. The UKNCC living biological resources form part of the UK science base and, as such, contribute to the economy and are essential for the education and training of scientists. To facilitate protocol improvement a number of techniques will be employed to determine when damage to the cell is incurred and to assess physical stability of the preserved material. Utilising the procedure that induces least damage and exploiting such storage methods as the alginate gel bead technique and vitrification should allow long-term preservation of recalcitrant organisms and give an insight on how to develop cryopreservation protocols for organisms yet to be isolated and grown. A preservation protocol development project has been submitted to the Quality of Life Programme of the Fifth Framework. Should this be successful this study will enable enhanced UK participation.
1. Investigation of cryopreservation techniques used for the preservation of micro-organisms and cell lines by a series of techniques to elucidate critical stages where cell damage is incurred to allow development of improved techniques. At least 5 representatives of recalcitrant algae, bacteria, fungi, insect cells, nematodes, plant cells, protozoa and yeasts will be examined to give an indication how the different organism groups respond. As a result recommendations can then be made on how cryopreservation protocols can be manipulated. The effect of vitrification, protection in alginate bead and variation of protectant formulation will be investigated. This work will be undertaken in months 1-12

2. Production of a set of preservation protocols for validation by members of the UKNCC and extend its quality management procedures to ensure standardisation and quality for UK Collections. Protocol development: months 6-18. Protocol validation: months 24-36.

3. Selection of a series of phenotypic and genotypic tests to use pre and post preservation to provide data on stability of stored organisms and successful implementation of the test procedures at UK Biological Resource Centres. Months 1-12

4. Application of alternative preservation techniques including vitrification and encapsulation by the alginate bead cryopreservation technique to improve stability of strains in storage. Objective 1 will provide an indication which new methods will be applicable to the different organism groups and appropriate new methodologies will be applied to further examples of recalcitrant organisms. The alginate bead technique offers a procedure with enormous potential to protect sensitive cells, preserve not yet culturable organisms and as a collection and delivery system. A hundred strains will be preserved over the short-term representing the organism types listed in objective 1, longer-term assessment will be under-taken under objective 6 utilising appropriate stability tests identified in objective 3. Months 6-12

5. Preservation and storage of representative strains of all organism types listed in objective 1, laying down a minimum of 500 strains in the first year and a total of 2000 over the course of the three-year project. The strains will be tested pre and post preservation by the phenotypic and genotypic stability tests selected under objective 3. Months 25-36

6. Test a series of collection management standards that are included in the UKNCC Quality Management System that cover characterisation, viability testing and supply of cell lines and micro-organisms to be adopted by UK Biological Resource Centres. This assessment will be undertaken in the final year of the project. Months 25-36

7. The improved preservation technology and collection standards will be desseminated through the publication of at least two papers in peer reviewed scientific journals, inclusion in the UKNCC quality management system and publicised on the UKNCC web site, and oral and poster presentation of results to major collection symposia including the International Congress for culture Collections and the European Culture Collection Organisation. This will be done at appropriate times during the study, particularly in the final year and the year following completion of the project.

Project Documents
• Final Report : Securing the UK National Microbial Genetic Resource: cryopreservation protocols for recalcitrant organisms   (9863k)
Time-Scale and Cost
From: 2000

To: 2003

Cost: £206,182
Contractor / Funded Organisations
CABI Bioscience
Plant Genetic Resources              
Plants and Animals