Most of the biochemical characterization tests described in this Manual should be controlled by organisms that are known to give either positive or negative reactions under appropriate conditions. For this purpose, we list in Table Dl the species recommended together with the accession numbers of suitable strains available from the National Collection of Type Cultures, Central Public Health Laboratory, Colindale Avenue, London NW9 5HT and, in many instances, also from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA. As controls for the common tests shown in Table D2, the set of four strains given is adequate. For other bacterial characters, the test strains shown in Table D3 may be used. We emphasize, however, that most of the organisms shown in these tables are pathogenic so that due regard must be paid to safety and hygiene in the laboratory.

For the maintenance and storage of test-control strains, freeze-dried (lyophilized) cultures which retain their characters and keep virtually indefinitely are ideal. However, for those who wish to keep frequently used strains for immediate use, we give in Table D4 suggestions for the nutrient media, cultural conditions and short-term storage period in order to be reasonably sure of their survival before they need replacement from stock cultures.

Nature of the resource

Living and authenticated cultures of bacteria are essential in virtually all practical applications of bacteriology, whether for routine work or for research. Such cultures are needed as controls, sources of special products, indicators of particular reactions or interactions; as representatives of their kind, or species (important for comparative identifications); as standards. Far from detracting from these ‘classical’ needs, the advent of the biotechnology of today has simply added more user needs, often, however, with a demand for greater amounts of information about the cultures.

There are three principal elements that convert a culture, or collection of cultures, into a ‘resource’. First is the culture itself, successfully preserved in a viable state. If viability cannot be maintained, either technically or simply through default, then only the information that may have been published is a resource: to exploit this limited resource a new isolate has to be obtained. Second is the information recorded about the culture. This includes such documentation as the history of the culture, original source, and also a record of the properties of the culture. The information may be little or exhaustive, but the preservation of cultures without information means they form a substantially less useful resource. Third is the availability of the culture, to enable its further application. However well preserved and well documented, a culture cannot be regarded as a ‘resource’ if it is not available: at best, it is a resource solely for its owner. Availability may, of course, be subject to restrictions or conditions; for example, hazardous bacteria (see Chapter 4) or patent strains (see Chapter 6) may not be universally available.

Introduction

This chapter is intended to give the reader who is unfamiliar with patents an introduction to the patent system as it applies to biotechnology, and a general guide to the procedures and pitfalls involved in obtaining patent protection for biotechnological inventions. For a detailed discussion of the whole subject of patents in biotechnology and a review of the variety of national patent systems the reader is referred to the excellent texts by Crespi (1982), Crespi & Straus (1985) and Straus (1985). It is not possible here to provide a step-by-step guide to getting a patent in every country in the world, for, despite an overall similarity, variations between different national patent laws are manifold, and professional help is necessary to guide even the experienced inventor through their complexities. The present account does no more than skim the surface of what is a complex and often fascinating subject; for this reason a short list of selected publications which illustrate in more detail many of the points raised here is given in Section 6.6, Further reading.

Basis of the patent system

Principles

The principle (if not the practice) of the patent system is straightforward: the inventor of a new product or process publicly discloses the details of his invention and in return he is granted for a limited period a legally enforceable right to exclude others from exploiting it. In this way the inventor's ingenuity is acknowledged and rewarded, while at the same time further technical progress is encouraged by the public dissemination of information about the invention.

Introduction

In response to the needs of users, many culture collections provide a range of services to the scientific, technological and commercial world. This chapter provides an introduction to the types of services available from culture collections, but it is beyond its scope to give a comprehensive list of such services. As the range of work that can be undertaken is increasing at many of the collections, the reader should contact individual collections to find out whether they can offer particular services.

Types of services

Directly associated and customer services

The two major services which are intrinsically part of culture collection work are those concerning the identification and preservation of organisms. Collections of necessity need expertise in these fields to be able to function, and many provide comprehensive services in these areas. Aspects of culture identification methods (Chapter 5), sales of cultures (Chapter 3), preservation techniques (Chapter 4) and patent deposits (Chapter 6) are covered elsewhere in this volume.

Safe-Deposits. Many collections hold organisms which are not listed in their catalogues. These cultures are held for a variety of reasons: the organisms may not be fully identified, their taxonomic status may be unclear, their stability in preservation may be suspect or they may be held at the request of the depositor who wishes to have back-up material and yet retain ownership and confidentiality, not releasing the strain to other parties. Many collections have introduced safe-deposit services as a back-up to the depositor's working collection, providing a service intermediate between an open collection deposit and a deposit for patent purposes.

Culture

The variety of nutritional requirements covering the whole spectrum of known bacteria is wide, and aspects of initial culture of bacteria that will be considered here will be restricted to those relating to the subsequent process of diverse preservation techniques. Reference should be made to standard textbooks for information on how best to grow different species, details of media formulations, pH, gaseous conditions and optimum incubation temperatures and times. However, a number of general factors must be borne in mind regarding culture for preservation.

Primary isolation

An obvious first requirement is to ensure that the culture is pure. Wherever practicable, the use of solid media is to be preferred to liquid media, since these allow plating out and subsequent single-colony isolations. In medical bacteriology, one plating out and single-colony isolation is usual for immediate investigations (for example, identification or determination of antibiotic sensitivity), where speed of obtaining an answer is the over-riding factor. For less urgent requirements and for preservation, however, it is advisable to go through two or even three successive platings out and single-colony isolations to ensure purity of the culture.

Enrichment

Primary isolation may sometimes be preceded by enrichment of the source material and usually this will be done in liquid media. Indeed, liquid media may be essential if the required bacterium requires, for example, good aeration or fluxing with special gases. Plating out from appropriate dilutions will yield single colonies and again, wherever practicable, further culture should be carried out on solid media.

The rapid advances taking place in biotechnology have introduced large numbers of scientists and engineers to the need for handling microorganisms, often for the first time. Questions are frequently raised concerning sources of cultures, location of strains with particular properties, requirements for handling the cultures, preservation and identification methods, regulations for shipping, or the deposit of strains for patent purposes. For those in industry, research institutes or universities with little experience in these areas, resolving such difficulties may seem overwhelming. The purpose of the World Federation for Culture Collections' (WFCC) series, Living Resources for Biotechnology, is to provide answers to these questions.

Living Resources for Biotechnology is a series of practical books that provide primary data and guides to sources for further information on matters relating to the location and use of different kinds of biological material of interest to biotechnologists. A deliberate decision was taken to produce separate volumes for each group of microorganism rather than a combined compendium, since our enquiries suggested that inexpensive specialised books would be of more general value than a larger volume containing information irrelevant to workers with interests in one particular type of organism. As a result each volume contains specialised information together with material on general matters (information centres, patents, consumer services, the international coordination of culture collection activities) that is common to each.

The WFCC is an international organisation concerned with the establishment of microbial resource centres and the promotion of their activities.

Introduction

Individual resource and information centres provide valuable services to biotechnology, but their role can be substantially enhanced if their activities are effectively co-ordinated. This has been recognised in the past, and a number of committees, federations and networks have been set up for this purpose at the national, regional and international levels. Although the origins and composition of existing organisations differ and their geographical locations are widespread, their common purpose is to support and develop the activities of resource and information centres for the benefit of microbiology.

International organisation

World Federation for Culture Collections

There are fewer difficulties in setting up national and regional co-ordinating mechanisms than international systems, and yet one of the first developments in this area was the formation of the World Federation for Culture Collections (WFCC). In 1962 at a Conference on Culture Collections held in Canada it was recommended that the International Association of Microbiological Societies (IAMS) set up a Section on Culture Collections. The Section was established in 1963. Five years later, at an International Conference on Culture Collections in Tokyo, the formation of the WFCC was proposed and an ad hoc committee, together with the Section on Culture Collections, drew up statutes which were agreed at a congress in 1970. Following the conversion of the IAMS to Union status, the WFCC is now a federation of the International Union of Microbiological Societies (IUMS) and an Organisation of resource centres 161 interdisciplinary Commission of the International Union of Biological Sciences (IUBS).

Introduction

Microbiologists are faced with consideration of exponential growth in their laboratories on a daily basis. As users of a chapter on information resources for biotechnology they are exposed to a double dose of exponential growth. First, the explosion of information technology itself is due to the massive amounts of computing power available at ever diminishing cost. In turn, a population of computer aware and computer literate microbiologists represent a growing demand for more sophisticated access to modern information technology. The community of information technologists in concert with microbiologists are responding to this demand with a multiplicity of initiatives using various strategies.

The resulting activity induces feelings of inadequacy in the authors of such chapters as this, since at the moment of delivery to the editors the information is out of date. Resources previously known only by rumour are tested. Simple facilities being tested as pilot projects are quickly made available to the community. Local data banks open their doors to regional and even world-wide participation. Databases on databases spring up because of the need to discover available resources. The net result is an ever increasing base of information resources for biotechnologists.

In some cases, useful resources fall by the wayside, as have at least two of the resources listed. They have been discontinued in the interval between the first and present versions of this chapter. The root cause of such discontinuing of effort is lack of appreciation by the initial funding bodies of the complexity and time scale involved in database initiatives of this sort.

Introduction

Bacteria are ubiquitous and although the most comprehensive compendium, Bergey's Manual, lists only two to three thousand named species, the diversity of habitats means that no single identification scheme can be devised for the whole spectrum. With bacteria, the species definition itself is pragmatic, since a species is that which, for practical purposes, we find useful to consider as a species. A listing of only two to three thousand species names – a small number compared with, say, fungi, or insects – perhaps tells us more about the restraint of bacterial taxonomists rather than about bacterial diversity. It is certain that a unit currently considered to be ‘species’ in one of those parts of the total bacterial spectrum that has come under much study (e.g. pathogens of the human gut; antibiotic producing soil organisms) does not correspond in taxonomic rank with a ‘species’ in a little-studied part of the spectrum.

Identification to species level, however broadly or narrowly defined, is a major part of bacteriological practical work. However, it may be important to know not only to what species the organisms belong, but whether two or more isolates are, in fact, the same strain. For example, from multiple isolates in a hospital, can it be deduced whether a particular strain is spreading?

Administration

Supply of cultures

The aim of service culture collections is to supply authenticated cultures to bona fide scientists, on request, promptly and without restriction on their ultimate use. The supply of certain bacteria such as pathogens (plant, animal or human) or patent strains will, however, be subject to statutory regulations and collections may impose further conditions. These may include, for example, proof that the requesting scientist holds the appropriate licence or permit to work with the cultures requested; that the request or order for cultures bears an authorised signature; and that an appropriate import licence is held. Supply of cultures to and from different countries, if pathogenic to man or animals, is subject to the International Air Transport Association (IATA) regulations whether sent by post or by air-freight. While the culture collection will effect the despatch of cultures as quickly as possible, delay can occur if those requesting the cultures are unaware of, or attempt to ignore, regulations. The collections, however, can only operate within the regulations.

Location of strains

The primary information about cultures available from a particular collection will be found in its printed catalogue. These, however, are not published as frequently as, say, catalogues of commercial suppliers of chemicals or equipment and so are never fully up-to-date. Many catalogues are now held on computers, and this enables quick and continual up-date by the collection and, in some cases, can be made available on-line (see Chapter 2).

Bacteria have a daily impact upon human activities. The emergence of the new biotechnology has increased the awareness by scientists of the long recognised need for reliable, permanent, culture collections which safe-keep viable exemplars of the many known bacterial species and varieties. There is now an increased awareness too that what is in fact conserved in service collections represents but a small part of the bacterial gene pool. Outside the recognised and long-established ‘service-supply culture collections’ there are many other centres whose holdings of cultures add to overall microbial, living resources available to scientists. There is an emphasis in this book on what defines a useful microbial resource: the cultures themselves, their documentation, and increasingly wide knowledge of their existence.

Today we are also in an age of developing information technology. Progress here enhances the existing resources, making it increasingly easy for individual scientists to access the great body of technical information associated with holdings of cultures. An additional benefit from the use of information technology to improve wider access to known information is to bring more clearly into focus gaps in our present knowledge and shortfalls in the presently conserved ranges of organisms available.

This book is an introduction to these resources, to culture collections, their holdings, and to the ways and means scientists responsible for their upkeep are exploiting information technology in the service of science. Hopefully, it will act as a stimulus to both research scientists and those engaged even in focused applied work. Reality dictates that often the distinction between research and applied science is blurred, but the extremes of each have need for authenticated, documented exemplars of the known microbial gene pool.

Studies of the physical properties of exopolysaccharides involve the application of a wide range of techniques; the interpretation of the results also requires a thorough knowledge of the chemical structure of the polymer. Electron microscopy has recently been applied to materials like xanthan to determine the persistence length of the molecule and to ascertain whether it is in a single- or double-stranded form. However, there are many limitations on interpreting the data thus obtained, owing to the possible introduction of artifacts both in the initial recovery of the polysaccharide and in sample preparation. Provided that the exact primary sequence and structure are known, X-ray fibre diffraction can supply information on polysaccharide conformation; circular dichroism provides a sensitive probe of the local environment of cation-binding sites.

Conformation

Determination of the molecular structure and conformation of a bacterial polysaccharide can be accomplished by X-ray diffraction of crystalline samples in the form of fibres. The techniques for orienting and crystallising the polymer use stress fields and annealing in the same way as they are applied to synthetic materials. The detail determined depends on the quality of the fibre diffraction pattern. The periodicity along the polysaccharide chain is visible in the approximately horizontal layer lines of the diffraction pattern. The spacing of the layer lines gives the pitch of the helical structure. Molecular model building can then be used, based on the known chemical repeating unit structure and standard values for bond angles and lengths and ring structures.

Polysaccharides are incorporated into foods to alter the rheological properties of the water present and thus change the texture of the product. Most of the polysaccharides used are employed because of their ability to thicken or to cause gel formation (Table 9.1). Advantage is also taken of the ability of some mixtures of polysaccharides to exhibit synergistic gelling: basically, for the two polymers to yield a gel at concentrations of each which will not in themselves form gels (Chapter 8). Associated with these readily measurable properties are others, such as ‘mouth feel’, which are more difficult to define but which also show some correlation with physical properties. ‘Mouth feel’ has been related to viscosity and, in particular, to non-Newtonian behaviour. This also relates to the masking effect of viscosity on the intensity of taste. There is also a specific relationship between the polysaccharide and flavours present in any food. Thus, corn starch and xanthan both provide good perception of sweetness and flavour when compared with gum guar or carboxymethylcelluose. In addition, polysaccharides are used because of their capacity to control the texture of foods and to prevent or reduce ice crystal formation in frozen foods; they may also influence the appearance and colour as well as the flavour of prepared foodstuffs (Table 9.2). It must also be remembered that many foodstuffs already naturally contain polysaccharides such as starch or pectin. Thus, addition of any further polysaccharide or polysaccharides will in all probability involve interactions with these compounds as well as with proteins, lipids and other food components.

Exopolysaccharides have a number of industrial applications which relate either directly or indirectly to medicine. One such is the use of dextran and its derivatives on both a laboratory and an industrial scale for the purification of compounds of medical interest, including Pharmaceuticals, and of enzymes for diagnostic purposes. Polysaccharides may also be used to encapsulate drugs for their gradual delivery and they may be used to immobilise enzymes employed for diagnosis or for the chemical modification of pharamaceutical products. These applications clearly utilise the functional properties of the polysaccharides, such as their rheology or capacity for gel formation. Alternatively, the pharmacological or other biological properties of the polymers may be employed. There are essentially three types of direct application of the biological properties of exopolysaccharides to medicine. The exopolysaccharides may be used as vaccines in preference to whole microbial cells or cultures. Thus side-effects due to other cell components such as lipopolysaccharides or proteins are avoided. On the other hand, not all polysaccharides are good immunogens, nor are the exopolysaccharides necessarily the major factors in the specific disease syndromes caused by the polysaccharide-producing microbial pathogens.

Several exopolysaccharides mimic eukaryotic polymers in their structural details. For this reason, they may be associated with certain specific diseases such as meningitis (in the case of sialic acid-synthesising bacteria). Some of these polysaccharides are, however, useful as substrates for determining enzyme specificity, and others are used as substitutes for the eukaryotic polymers. Finally, a number of exopolysaccharides having antitumour or antiviral activity have been identified.

Introduction

The biosynthesis of most exopolysaccharides closely resembles the process by which the bacterial wall polymers peptidoglycan and lipopolysaccharide are formed. Indeed, the three types of macromolecule share the characteristic of being formed of carbohydrates and associated monomers, being synthesised at the cell membrane and exported to final sites external to the cytoplasmic membrane. The only exceptions are the exopolysaccharide levans and dextrans, which are synthesised by a totally extracellular process and whose formation will be discussed later in this chapter.

Formation of the precursors for polysaccharide synthesis occurs within the cytoplasm. This is probably a necessity to ensure that they are thus readily available, as in many cases they are utilised for several different polymer-synthesising systems. As they are freely soluble in the cytoplasm, they can be readily channelled to the appropriate biosynthetic process occurring at or within the cytoplasmic membrane. Elucidation of the initial stages of polysaccharide synthesis has proved more difficult than was the case for polymers found in microbial and particularly bacterial walls. This has been mainly because of the lack of suitable selection systems for obtaining mutants and of antimicrobial agents specifically inhibiting polysaccharide biosynthesis. Even the preparation of cell-free systems or of membrane fragments is rendered more difficult by the presence of the viscous extracellular polysaccharides. Various cell-free systems, including membrane fragments from ultrasonically lysed cells, solvent-extracted cells or cells permeabilised with solvents or chelating agents, have been used.

Mutants have proved useful in studies where it has been possible to obtain microorganisms deficient in precursor synthesis (UDP-glucose pyrophosphorylase or UDP–galactose-4-epimerase, etc).