Culture collections — their services to biotechnology

Trends in B,otechnology, VoL 1, No. 1, 1983

Culture collections - their services to biotechnology Barbara Kirsop The need to recruit qualified and experienced employees is a major concern of many ort~'~zations engaged in ~ n n o l c ~ . Universities and industry are ~ to ftnd the t i ~ peof~, but where are we to find promising new candidates for the most r~merous, and arguably most talented section of the bio,technotc~ workforce - microorganisms and other types of cel~?The ~ t i o ~ l network c~ culture collections offers the best answer, and a proper ~ of their roles and capabilities can save biotechnologists an immense amount of time and money. Among the millions of microbial, plant and animal cells stored in the world's culture collections it is often possible to f~ld one that exactly suits a biotechr~logist's particular reqd~err'~ts or one that can be developed to perform a specifK:task. This article out~-~,~sthe se~-~---~soffered by culture coUections, generally orang the UK collections as examples of the main

activities throughout tY~ world.

The positive use of microorganisms for the production of industrially important substances cannot succeed without a supply of reliable, authenticated cultures and there is now a growing awareness that culture collections provide essential resources and services, The collection, maintenance and characterization of microorganisms form part of the infrastructure of microbiology and together with advisory, identification, preservation and patent services make an important contribution to the development of biotechnology. Organization of culture collections

The World Data Center on Microorganisms lists 566 culture collections in its directoryL While many of these collections provide cultures only by special arrangement and do not publish catalogues, others not only provide cultures, advice and ancillary services on demand to researchers in industry and elsewhere but also publish catalogues. The number of these 'service collections' is small, but many countries are now seeking to establish national microbiological resource centres. In addition, a global network of eleven Microbiological Resource Centres (MIRCENS) has been set up by the International Cell Research Barbara Kirsop is Curator of the National Collection of Yeast Cultures, Food Research Institute, Colney Lane, Norwich NR4 7UA, UK. She is also President of the UK Federation for Culture Collections.

Organization. Each is concerned with developing collections of microorganisms, providing training and advisory centres and promoting regional collaboration. Service collections have developed and are administered in several different ways. The US and the FRG, for example, have single collections which maintain all types of microorganism; other countries, such as the UK and Czechoslovakia, have a decentralized system whereby each type of microorganism is housed in a separate institute and the individual collections are coordinated to form a national network. The United Kingdom supports eleven service collections (Table 1) housed in separate institutes and maintaining bacteria, fungi (including yeasts), algae, protozoa and animal cells. Some have specialist interests in medical, veterinary, industrial or genetical strains; others maintain taxonomic collections of more general interest. The services of these collections are available to scientists throughout the world. Table 2 lists some of the major culture collections in other countries which are particularly useful to biotechnologists. The decision to establish a decentralized system in the UK was taken by a specialist committee set up in 1947 to consider the future requirements in the country. The committee felt that there was much scientific advantage in a system whereby each type of microorganism was maintained in an

© 1983, Elsevier Science Pubhshcrs B V , Amsterdam 0167 - 9430/831501 00

institute with related interests. There was concern that centralized collections could become isolated from new developments. Although the decision has proved to be scientifically wise, it has led to funding difficulties. These have arisen partly from a natural concern by the funding bodies for departmental rather than national considerations and also from the absence of an official coordinating body empowered to ensure permanent funding. The new biotechnological developments, however, have led to government recognition of the role to be played by culture collections and recently the Interdepartmental Committee for Biotechnology (ICBT) has made clear its concern for the protection of culture collections and their funding in the future. The diversity of culture collections and wide range of interest of their users has led to a need to coordinate their activities and a number of bodies and societies have been set up for the purpose. In the UK there is both a national committee of culture collections to establish liaison between the curators of the service collections and a federation for culture collections (UKFCC) to further the interests of both collections and their users. National federations have been established in several countries and the World Federation for Culture Collections (WFCC) provides international coordination. The federations hold scientific meetings, arrange training courses and prepare books, documents and newsletters. In addition they provide essential links with government and funding bodies. T h e g e n e bank

Clearly, the prime function of culture collections must be to provide a comprehensive range of pure, authenticated microorganisms that are of past, present or potential interest. Not only must microbiologists be encouraged to deposit cultures, but curators must actively collect strains that will add to the value of a collection. Although most cultures will be deposited by scientists working in university or institute laboratories, industry is often prepared to make strains no longer in use generally available. This enlightened approach enhances the value of collections to the general benefit of industry. Culture collections must, nevertheless, exercise a degree of selectivity in accepting strains since the accession, characterization, documentation and

Trends in Biotechnology, Vol. 1, No. 1, 1983

Table 1. United Kingdom national culture collections Culture collection NC of Type Cultures (NCTC)

Parent instuute Central Public Health Laboratory, Colindale Avenue, London NW9 5HT, UK

Type of culture Bacteria (medical, veterinary)

Holding 4 000

NCs of Industrial and Marine Bacteria (NCIMB)

Torry Research Institute, 135 Abbey Road, PO Box 31, Aberdeen AB9 8DG, UK

Bacteria (general, industrial, marine)

5 000

Commonwealth Mycological Collection of the Commonwealth Mycological Institute, Ferry Lane, Kew TW9 3AF, Surrey, UK Institute (CMI)

Fungi

10 000

Culture Centre Algae and Protozoa (CCAP)

Institute of Terrestrial Ecology, Storey's Way, Cambridge CB3 0DT, UK

Algae, protozoa

2 000

NC of Yeast Cultures (NCYC)

Food Research Institute, Colney Lane, Norwich NR4 7UA, Norfolk, UK

Yeasts, other than known pathogens

2 000

NC of Dmry Organisms (NCDO)

National Institute for Research in Dairying, Shinfield, Reading, UK

Bacteria(general, dairying)

2 000

NC of Pathogenic Fungi (NCPD

Mycological Reference Laboratory, London School of Hygiene & Tropical Medicine, Keppel St, London WC1E 7HT, UK

Fungi pathogenic to man and animals

830

NC of Plant Pathogemc Bacteria (NCPPB)

Plant Pathology Laboratory, Ministry of Agriculture, Fisheries & Food, Hatching Green, Harpenden, Herts, UK

Bacteria(plant pathogens) and associatedphages

3 000

NC of Wood Rotting Fungi (NCWRF)

Wood rotting Princes Risborough basidiomycetes Laboratory, Building Research Establishment, Forest Products Research Laboratory, Princes Risborough, Aylesbury, Bucks, UK

National Animal Cells Culture Collection (NACCC)

PHLS Centre for Microbiology and Applied Research, Porton Down, Salisbury SP4 0JG, UK

Animal cells

620

Preventing loss and change Most industrial microbiologists have experienced the loss of experimental strains or the failure to reproduce results in carefully controlled fermentations using inocula derived from the same stock cultures. The need to preserve important strains for prolonged periods in a viable and enhanced condition is a major concern of biotechnology. Microorganisms vary greatly in their ability to resist the stresses of preservation and the methods appropriate for one group of microorganisms may be unsuitable for others. Not only do the requirements of fungi differ from those of bacteria, algae, protozoa or animal and plant cells, but there may be substantial differences between different strains of the same species. This little

Table 2. Some major international culture collections useful to biotechnologists 200* --Culture collection

Address

American Type 12301 Parklawn Drive, Culture Collection Rockville, Maryland 20852, USA Total 29 650

~Anticipated holding.

maintenance of new strains places substantial demands on the resources of busy service collections. The use of cryopreservation and freeze-drying, together with miniaturized storage methods, alleviates the problem to some extent since cultures may be preserved as soon as they are received and stored with some degree of safety until there is time to complete all the procedures involved in integrating the new strains into the collection. It is essential, however, that strains are properly documented on arrival and their availability made generally known. This approach allows service collections to act as safety nets for important departmental collections that are endangered through changes in staffing or projects. The loss of valuable

One strain that was a minor and insignificant component of a traditional brewing yeast was found to be particularly successful in the new fermenters and is now the major production strain in a large brewery. The natural instinct of curators to keep all cultures is increasingly justified by experiences of this kind.

microbial collections is of concern to the UKFCC which has appointed an officer to consider difficulties of this kind whenthey arise. The size of collections could readily be controlled if seemingly redundant cultures were culled. However, it is the experience of industrial microbiologists that the redundant cultures of today become the production strains of tomorrow, as company targets change and new engineering developments lead to the need for strains with different properties. For example, changes in fermenter design in the brewing industry led to failure of traditional yeast species and a search was made to find alternative strains that could tolerate the different, oxygenlimited conditions in deep fermenters.

Deutsche Sammlung Grisebachstrasse 8, C,-6ttingen 3400, FRG yon Microorganismen Centraalbureau voor Oosterstraat 1, PO Schimmelcultures Box 273, 3740 AG Baarn, The Netherlands Czechoslovak Collection of Microorganisms

J. E. Purkyne University, 662 43 Brno, Czechoslovakia

Collection Nationale Institut Pasteur, 28 Rue de Cultures de du Docteur Roux, Microorganismes 75724 Paris Cedex 15, France Culture Collecuon of Institute for the Institute for Fermentation, 17-85 Fermentation Jugo-Hohmachi 2-chome, Yodogawa-ku, Osaka, Japan USSR All-Union Collection of Microorganisms

Institute of Microbiology, USSR Academy of Sciences, Profsojuznaja 7, Moscow B-133, USSR

Trends :n Blotechnology, VoL 1, No. 1, 1983

understood phenomenon poses fascinating questions for the cryobiologist, but enormous problems for the industrialist who may be faced with partial or total loss of product, 'sticky' fermentations or qualitative changes that are unacceptable. Curators have learned through experience and empirical investigations the most reliable preservation methods for the strains in their collection, although for particularly sensitive cells - leptospira, for example - reliable methods have yet to be developed. There is an urgent need for research into the molecular events which occur during preservation so that the genetic stability of strains can be assured. The problem becomes more urgent with the increasing need to maintain genetically manipulated microorganisms that are often unstable; moreover it is important that cultures deposited for patent purposes should be maintained in such a way that strain drift is minimized. Maintenance methods are very diverse and the choice of the most suitable to use for the preservation of a particular strain is often difficult. The U K F C C is aware of this problem and arranges courses on culture preservation; in addition a laboratory manual of methods is currently in preparation 2 which provides practical details oftried methods and compares the suitability of different methods for different types of microorganism. In general, methods fall into four classes: subculturing (active transfer), drying, freeze-drying and cryopreservation. Subculturing is convenient and cheap and requires little in the way of equipment and skills. Moreover, many 'cultures' remain alive after storage for months or even years. However, the proportion of cells surviving within the original population may be very low and there is growing evidence that substantial changes take place. To retain strain stability subculturing must be used in conjunction with selection procedures and this diminishes the simplicity of the method. Culture collections, while recognizing the convenience of the method in the short term, prefer other procedures. The three other types of preservation method arrest cell multiplication and thus halt strain drift. Cells can be dried on soil, sand, silica gel or paper, but changes in strain characteristics may occur. Freeze-drying is widely practised because it is convenient to distrib-

RESEARCH INSTITUTE~

•~

OVERSEAS:

SCHOOLS

UNIVERSITIES 27% HOSPITALS Public Health Labs.

22%

UNIVERSITIES RESEARCH INSTITUTES INDUSTRY

22%

15%

uK:[--] Fl~,. 1. Distribution of cultures to different users by UK service collections (1981). ute cells in this form and it is possible to prepare large batches of microorganisms. However, while this method is very useful for most bacteria, it is unsuitable, or gives very low survival levels, for a number of other microorganisms. In addition it has been implicated in several reports of genetic change. Nevertheless, freeze-drying provides substantially greater stability than drying or subcuhuring for a wide range of microorganisms and cells that withstand the process survive storage for long periods. Cryopreservation at temperatures between - 7 0 ° C and - 1 9 6 ° C is generally accepted as the preservation method conferring maximum stability to cells. A wide range of microorganisms and cell types can be stored successfully at low temperatures and, in general, survival levels are significantly higher than those obtained with other methods. Preliminary experimentation is needed to establish the best growth parameters, cryoprotectants and freezing and cooling rates for particular cells. It is hoped that research into the molecular changes which occur during freezing and thawing may lead to the establishment of protocols that can be

applied to cell preservation. Reports on genetic change in cryopreserved strains are few, but the costs of the process are relatively high. Distribution,

documentation

and

information retrieval Cultures are distributed* to a wide range of users including universities, technical colleges, schools, research institutes, hospitals and industry (Fig. 1). The collection, maintenance, characterization and distribution of cultures generates a great deal of information that must be effectively documented. As the activities of service collections increase, the storage and retrieval of information assumes greater importance and collections are turning to computers as the only method that

*The despatch of non-infectious or mfecuous substances is governed by national and international regulations laid down by postal, customs and airline authorities and collection staffand the UKFCC havebeenmstrumentalm mak,ng reeommendations to the appropriate author:ties. Difficulties remain in certain countries; pressure from microbiologistscould help rationalizethe regulations.

Trends m Btotechnology, VoL 1, No. 1, 1983

can meet the growing requirements. The biotechnologicat importance of culture collections can be exploited only if information on the properties of holdings is known, properly documented and easily accessible. Information relating to the day to day running of collections must be recorded (accession, maintenance, quality control, literature references, stock holdings, customers, income) and strain data stored in a form that can be readily retrieved. Most service collections now use computers to produce their catalogues and since the catalogues can be printed directly from computer tapes or disks it is much easier to update these essential publications. The extent to which information is available varies between collections, depending on the industrial use to which the cultures have been put in the past. Yeasts, for example, have been used for several thousand years while the industrial potential of algae has only recently been realized. This is reflected in the extent to which physiological information is available on the two groups of organism. Several governments have now recognized that collection strains should be screened more extensively to determine their biotechnological value and ways of assessing such characteristics as the tolerance of strains to environmental extremes, the ability to flocculate or perform basic chemical transformations are being considered. In the UK, the Department of Industry is looking at ways in which information in the UK service collections can be made more readily available to industry; the development of on-line computer access is under review. Internationally the computerization of catalogue and strain data has been under consideration by a number of agencies (the World Federation for Culture Collections; the EEC; national organizations in Scandinavia, Japan, Europe; MIRCENS in the developing countries) and it is clear that great progress will be made over the next decade. Making the most of culture collections In addition to providing cultures, collec[ions act as advisory and information centres. Through the daily use of cultures and close contact with users, collection staff accumulate expertise on taxonomy and identification, preservation and storage, the physiology and

morphology of strains and the choice of organisms for specific purposes. Staff spend much time answering enquiries by letter and telephone and although they do not know all the answers, they usually do know appropriate authorities to whom questions may be directed. The exchange of information between collections and users should be encouraged. For example, it is valuable to industry to know which strain in a collection could best ferment a specific substrate, but it is just as valuable for collections to learn subsequently that the strain of choice did not perform adequately at the temperature required by the company. It would be even more valuable to industry if this basic information had been available from the collection records initially and there is clearly a strong case for directing government resources to screening work of this kind. Again it is of great benefit to culture collections if users report problems they have experienced with collection strains. Much of the expertise in culture collections is of a taxonomic nature and collections generally offer an identification service. This provides important support for biotechnology since identification depends on experience obtained through frequent access to large numbers of related organisms and may in addition be a lengthy procedure requiring sophisticated equipment. Rather than develop expert knowledge in a number of laboratories for a function that is only required intermittently it is clearly sensible for culture collections to provide this service. A further requirement of great importance to biotechnology is access to collections with deposit facilities for patent strains. It is a legal requirement that if a process involving the use of a microorganism is to be patented, a culture of the microorganism must be deposited with a recognized culture collection. Different countries have different patent laws and in the past the regulations governing the deposit and release of patent strains have been confusing for depositors. In addition, patents taken out in more than one country required the culture to be deposited in each country. The Budapest Treaty, which came into effect in 1977, simplifies proceedings by allowing a single deposit made in an accepted International Depository Authority

(IDA) to serve for patents taken out in any number of member states. To qualify as an IDA a collection must meet requirements concerning stability, facilities and scientific competence. Many service collections have now been accepted and a list of IDAs and member states can be obtained from the World Intellectual Property Organization in Geneva 3. Culture collections provide a number of other services which directly support industry. For example, production strains may be deposited for expert preservation with subsequent access only to the depositor; alternatively strains may be preserved and returned to the depositor for safe-keeping. Some collections develop strain selection programmes for industry; others carry out specific services on a contract basis. The ways in which collections and industry may collaborate have not yet been fully explored and it would be to the benefit of both to consider further interactive programmes. Research and development Nearly all culture collections have their own research interests and in the UK it has always been felt that close association with new developments is of great benefit to the services that collections provide. Moreover collections are uniquely placed to take maximum advantage of access to large numbers of authenticated microorganisms. Research naturally leans towards basic taxonomic studies (comparative studies, DNA homology, electron microscopy, detection of plasmids), developments in identification methods or work in the field of culture preservation. These are areas not immediately justifiable in an industrial laboratory or in laboratories concerned with specialized aspects of microbial behaviour, but are particularly appropriate for study in culture collections. Charges and costs Most service collections make a charge both for cultures and services. In general, costs are of the same order throughout the world, with higher charges being made for profit-making organizations than research and educational establishments. Fixing the level of charges for cultures is difficult since, unlike chemicals, they are re-usable and charges that are levelled too high encourage microbiologists to obtain

Trends tn Biotechnology, VoL 1, No. 1, 1983

cultures second h a n d - a practice which can lead to poor microbiology through the use o f contaminated or unauthentic cultures. The service collections facilitate the use of reliable microorganisms by keeping charges as low as possible. The U K network of collections earns about ten per cent of the cost of their service functions and research, and it is recognized that they must be subsidized in the same way as libraries, botanical gardens and zoos. T h e future Culture collections exist to support all microbiology - the future requirements of education and research as well as industry must be safeguarded. There is clearly a need for a more extensive examination of present holdings for useful properties, for improved access

to existing information by the development of computer links and for the development of research into the area of strain stability. In addition, there is a need to develop culture collections as banks not only for microorganisms but for animal and plant cells, viruses and hybridomas, genetically marked strains, genetically manipulated strains, plasmid-bearing strains and perhaps even plasmids and gene fragments. It is clear that culture collections provide many services that support the infrastructure of biotechnology. It is vital that the infrastructure of the collections themselves is protected; the fundamental work of microbiology the comparative studies, the examination and identification of new isolates, the basic taxonomy from which other microbiological studies develop - must

be fostered. It is encouraging that there are at present many international developments with this end in view. References and notes 1 McGowan, V. F. and Skerman, V. B. D., eds (1982) lVorldDirectory of Collections of Cultures of Microorganisms. World Data Center, University of Queensland. Available in book form (US$25.00) or microfiche (US$15.00) from the UNEP/UNESCO/ICRO panel on Microbiology, Swedish University of Agricultural Sciences, S-75007, Uppsala, Sweden Kirsop, B. E. and Snell, J., eds, Maintenance of Microorganisms: a methods manual. Academic Press (in preparation) World Intellectual Property Organization, 34 Chemin des Colombettes, 1211 Geneva 20, Switzerland