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Chapter 10 Pure Cutfures for Microbial Processes Mankind has utilized microbial fermentations to prepare foods and bever- ages for thousands of years. Two types of inocula have traditionally been used to initiate such fermentations: the natural flora associated with the products being fermented, such as yeasts on grapes in wine making, or a small amount of the previous fermented material containing the active micro- organisms, as in yogurt culture. In both types of fermentations, the inocula usually consist of a mixture of microorganisms. Occasionally conditions may favor the growth of undesirable organisms normally present in small numbers, as may occur with acetic-acid-producing bacteria, contaminating a fermenta- tion designed to produce an alcoholic beverage. Techniques for producing a pure culture, that is, one containing a single type or strain of microorganism, were first developed by Robert Koch in the mid-19th century. These methods were immediately adopted by the micro- biologists of the time, who were principally concerned with microorganisms as causes of disease. Such pure culture techniques led eventually to the defini- tion and characterization of the bacteria that cause anthrax, tuberculosis. scarlet fever, and other major diseases. Similar procedures were developed for fungi, algae, and protozoa. The discovery of viruses came later, and because of their obligatory parasitism and submicroscopic size, they were much more difficult to characterize. Pure culture techniques were in turn applied to commercial fermentations, which provided more consistent yields of the products desired. Such cultures have been used to make alcohol, yogurt, and citric and lactic acid and other useful products. The techniques also made possible the development of vac- cines and antibiotics. Development of Pure Culture Collections Pure culture collections are important for a number of reasons. First they provide a source of reference to enable microbiologists to verify more easily the organisms with which they are working. They also provide a readily accessible source of cultures of known organisms and a means of preserving genetic resources of such organisms. 177

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178 MICROBIAL PROCESSES As pure cultures came into general use, they began to be collected on a systematic basis in a number of countries. The collections vary greatly in size, and some are quite specialized. In many cases, bacteria and protozoa are not kept in the same collection. There are a few exceptions, which will be noted below. Pure culture collections have gained an important role as resources for authentic, reliable microbial cultures for both research and-practical use. The need for international cooperation in establishing such culture collections has become increasingly evident with the development of important microbial biosynthetic processes. As a result, the International Federation of Type Cul- ture Collections and the British Commonwealth Collection of Microorganisms were established in 1947. The International Association of Microbiological Societies approved the formation of a "Section on Culture Collections" in 1963, and in 1970 the Section was reorganized as the World Federation of Culture Collections (WFCC). National federations of culture collections exist in a number of countries, and at the urging of the Japanese federation, UNESCO brought together a group of culture collection specialists in Paris in 1966 to consider various problems relating to such collections. The train- ing of culture collection personnel and the establishment of collections in developing nations were among the topics discussed. It became evident in the discussion that more information was needed on the location, content, and personnel of culture collections throughout the world. Major Pure Culture Collections A World Directory of Collections of Microorganisms was prepared in 1972 by S. M. Martin, of Canada, and V. B. D. Skerman, of Australia, with support from UNESCO, the World Health Organization, the Australian Common- wealth Scientific and Industrial Research Organization (CSIRO), and the Canadian National Research Council. The directory provides a relatively complete list of collections throughout the world, but it will obviously require periodic updating. The location and na- ture of a few of the major collections are given in Table 10.1. The dates of publication of the most recent catalogues of cultures issued by these collec- tions are shown in parentheses. It should be noted that most service collec- tions charge a fee for providing cultures, in order to support the maintenance, characterization, and preservation of the cultures and to recover the costs of shipping them. World Data Center and Microbiological Resource Centers Although the importance of pure culture collections for the preservation of microbial germ plasm has long been recognized internationally, problems

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PURE CULTURES FOR MICROBIAL PROCESSES 179 frequently arise when a comparison is made of the fermentative properties or other characteristics of microbial cultures listed under the same species name in different culture collections. About 15 years ago V. B. D. Skerman, Professor of Microbiology, Univer- sity of Queensland, Brisbane, Australia, began preparing a computerized list of the strains maintained by various institutions and their characteristics. This cataloging operation has been expanded over the years and is now known as the World Data Center (WDC). Recently, the WDC has taken on the task of documenting the characteristics of viruses on behalf of the International Committee on the Taxonomy of Viruses, as well as cataloging specialized collections such as those with strains of Rhizobium species for legume inocu- lation. Information concerning the WDC may be obtained from Professor Skerman. Activities similar to those of the WDC have been carried on by other groups. For example, because of the importance of microorganisms in the production of antibiotics, the International Streptomyces Project was ini- tiated in 1958. In this remarkable collaborative effort strains of important organisms were carefully studied and deposited in several of the culture col- lections (Table 10.1~. Similar data for higher fungi are maintained in the Fungal Genetics Stock Center, Humboldt State University, Arcata, California. In 1974 the concept of Microbiological Resource Centers (M IR CEN S) was proposed to a group of microbiologists by M. K. Tolba, Executive Director, United Nations Environment Programme (UNEP). Financial support for put- ting this concept into action was provided by UNEP and UNESCO. One of the several aims of the MIRCENS iS to provide the infrastructure for a net- work that will incorporate regional and interregional units geared to the management, distribution, and utilization of microbial gene pools. The first step in initiating the concept was the establishment of the WDC as a MIRCEN in close collaboration with the World Federation for Culture Collections. This MIRCEN serves as a pivotal unit for the formation of culture collections in developing countries and for providing data services to the centers acting in liaison with the WDC. A regional MIRCEN at the Applied Scientific Research Corporation of Thailand in Bangkok serves microbiologists of Southeast Asia through the exchange of economically important microbial strains in the region, and by offering training and fellowship programs and promoting research on organ- isms relevant to the region. In the specialized area of microbial nitrogen fixation, similar MIRCENS have been developed in the Department of Soil Science and Botany, University of Nairobi, Kenya, and at the Instituto de Pesquisas Agropecuarias in Porto Alegre, Rio Grande do Sul, Brazil. The MIRCEN at Ain Shams University, Cairo, Egypt, serves the Middle East, and the one at the Central American Research Institute for Industry in Guatemala will function in the Central American region.

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180 MICROBIAL PROCESSES TABLE 10.1 A Representative List of Major Culture Collections Australia Fungi, yeasts, and actinomycetes {1973 cataloged Australian National Reference Laboratory in Medical Mycology The Institute of Medical Research The Royal North Shore Hospital of Sydney St. Leonards, N.S.W., Australia Bulgaria Pathogenic bacteria and fungi (19 70 catalogue) Bulgarian Type Culture Collection India Institute for State Control of Medical Preparations Sofia, Bulgaria Czechoslovakia Bacteria, mycoplasmas, viruses, and fungi (197S catalogueJ Czechoslovak Collection of Micro- organisms U.K. Purkyne University Enda OBRANCU Miru 10, 66243 Brno, Czechoslovakia France Bactena (19 75 catalogue) Collection of the Institut Pasteur 25, rue du Docteur Roux Pans, France 75015 Germany Bactena, fungi, yeasts, and plant viruses (19 74 catalogue) Deutsche Sammlur~g van Mikro- organ~slr~en Germany Continued D-3400 Gottingen Grisebachstrasse 8, West Germany Hungary Bacteria, fungi, and virus (19 7S catalogueJ Microbiological Gene Bank Microbiological Department Group of the Department of Food Technology and Microbiology University of Horticulture 1064 Budapest, Izabella U. 46, Hungary Bactena and fungi {19 71 catalogueJ Indian Type Culture Collection Indian Agricultural Research Institute New Delhi 12, India Japan - Bactena, fungi' viruses, bactenophages, algae, protozoa, and nckettsza (1968 catalogue) Japanese Federation of Culture Collections of Microorganisms c/o Institute of Applied Microbiology University of Tokyo, Bunkyo-ku Tokyo, Japan Fungi, yeasts, bacteria, and bacteno- phages (1972 catalogue) Institute for Fermentation 4-54 Juso-nishinocho Higashiyodogawaku Osaka, Japan Bacteria and fungi (19 71 catalogueJ Laboratory of Culture Collection of Microorganisms Although the MIRCEN concept applies mainly to the less-developed coun- tries, a technological MIRCEN has been established at the Karolinska Insti- tute in Stockholm. This MIRCEN collaborates actively with the WDC in mapping metabolic characteristics of microorganisms and in helping other culture-collection personnel organize their specialized research projects. Preservation Methocis A description of the maintenance of a small collection of microorganisms has been provided by Skerman (1977), including a method of scheduling the transfer of cultures. Skerman notes that, while a wide variety of culture

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PURE CULTURES FOR MICROBIAL PROCESSES 181 Japan Continued Faculty of Agnculture Hokkaide University Sapporo, Japan Actinomycetes (1976 catalogueJ Kaken Chemical Company, Ltd. 642, Jujodai 1-chome, Kita-ku Tokyo 114, Japan The Netherlands Centraalbureau voor Schimmel Culture Oostexstraat 1 Baarn, The Netherlands United Kingdom Algae and protozoa (19 76 catalogue) Culture Centre of Algae and Protozoa Institute of Terrestrial Ecology 36 Storey's Way Cambridge, CB3 ODT, England Bactena of industrial importance {197S catalogueJ National Collection of Industrial Bacteria Torry Research Station PO Box 31, 135 Abbey Road Aberdeen AB9 SDG, Scotland Bactena of medical and veterinary importance {19 72 catalogue) National Collection of Type Cultures Central Public Health Laboratory Colindale Avenue London NW9 SHT, England Bactena pathogenic for plants (1971 list) National Collection of Plant Patho- genic Bacteria United Kingdom Continued Plant Pathology Laboratory Hatching Green Harpenden, Hertfordshire AL5 2BD, England Fungi (other than animal pathogens and wood-rotting fungi) (19 75 catalogue) Collection of Fungus Cultures Commonwealth Mycological Institute Ferry Lane Kew, Surrey TW9 3AF, England United States of America Bacteria, fungi, algae, protozoa, bacte- nophages, cell cultures, viruses, anti- serllm, nckettsiae and chlamyciae (19 78 and 19 79 catalogues) American Type Culture Collection 12301 Parklawn Drive Rockville, MD 20852, U.S.A. Bactena and fungi (no catalogue) Northern Regional Research Laboratory U.S. Department of Agriculture, Sci- ence and Education Administration 1815 North University Street Peoria, IL 61604, U.S.A. World List Rhizobium Collections (1973 catalogueJ International Biological Program World Catalogue of Rhizobium Collections 7 Marylebone Road London N.W. 1, England Source: S. M. Martin, and V. B. D. Sherman, eds. 1972. World directory of collections of cultures of microorganisms. New York: John Wiley and Sons. media may be required for growth of organisms held in a collection, it is desirable that the number of media be kept to a minimum. In the collection that Skerman describes, involving about 1,200 strains of bacteria and fungi plus a few algae, 68 percent of the organisms could be grown on two kinds of media. Yet for the entire collection 57 different types of media were re- quired. For a much larger collection (approximately 25,000 strains), the American Type Culture Collection (ATCC) stores over 1,000 culture media arid these are listed in the ATCC Catalogue of Strains published in 1978. As with the

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182 MICROBIAL PROCESSES smaller collection designed by Skerman, many of the bacterial strains can be grown on relatively few media. Nevertheless, a wide variety of media are needed for the more fastidious organisms in a collection, making a culture collection somewhat expensive to maintain, depending ore the types and number of cultures in it. In the early days of culture collections, the cultures were maintained by serial transfer, that is, from culture grown in laboratory tubes or dishes to fresh medium. This method maintains the viability of a colony of micro- organisms, but it is frequently ineffective in maintaining genetic integrity and ensuring that the important biosynthetic characteristics will not be lost or modified. Thus a strain of a microorganism developed to yield high levels of an antibiotic may gradually lose that important capability during continuous transfer in the laboratory, despite the ability of the microorganisms to multi- ply. Ironically, the ability to grow may often be enhanced as the loss of the ability to produce the desired compound occurs. This problem led micro- biologists to seek other means of maintaining cultures. Some of the methods that have been developed are quite simple and have proved useful for many strains of microorganisms. They include drying the culture on sterile sand or soil, sterile filter paper strips, plastic spheres, or glass beads. Regardless of the method used, however, extensive laboratory studies of every highly developed strain are necessary to assure against loss of any economically important biosynthetic characteristic. The development of freeze-drying (lyophilizing) procedures during the past 30-40 years represented a large step forward in preserving cultures. Lyophilization involves freezing a culture at very low temperatures (about -65C) in a mixture of dry ice and alcohol, after which the water is removed by sublimation from the solid state under a high vacuum. Although freeze-drying will significantly stabilize the characteristics of many types of microorganisms, not all species will survive lyophilization. With continued improvements, however, a larger number and variety of microorganisms are surviving the process. Even when an organism survives freeze-drying, the freeze-dried culture must be stored under controlled conditions. Some results of studies carried out at the ATCC using relatively hardy organisms are given in Table 10.2. Even those organisms listed in Table 10.2 that were freeze-dried and stored for 11 months in a refrigerator at a temperature of 4C showed a drop in viable count to approximately half that of the original culture, while the same cultures stored at room temperature (22C) dropped to less than 1 percent of the original count. The advantage of the freeze-dried method, despite the loss, is that transfer is not necessary and the culture can be kept stored for years. Nevertheless, while viable cultures could be retrieved from either group, the question remains whether the survivors will retain the biosynthetic capability that is important. Therefore, where proper facilities are available, even freeze-

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PURE CULTURES FOR MICROBIAL PROCESSES 183 dried cultures should be stored at as low a temperature as the laboratory can provide. For many years dry ice was used to lower the temperature of heavily insulated boxes to -65C. In recent years mechanical (electrical) refrigerators capable of maintaining temperatures as low as -75C have been developed and are widely employed in modern laboratories. TABLE 10.2 Survival of Freeze-Dried Cultures BactenalCountsX 1~6 Original Storage Temperature Count Count after 11 months Organism 4C 29C Streptococcus faecalis 620 310 9.0 Pseudomonas aeruginosa 200 120 7.6 EscherichZa colt 680 290 1.2 Enterobacter aerogenes 980 440 3.6 Average 620 290 5.3 Source: American Type CultureCollection, CatalogueofStrains,I. 1978.13thedition. Rockville, Maryland: American Type Culture Collection. The ultimate in present-day refrigerators are units cooled by liquid ni- trogen. Storage temperatures as low as -196C can be maintained. Liquid nitrogen storage units are excellent for maintaining almost all types of micro- organisms, including algae, protozoa, and even mammalian tissues, in viable form. Since this type of equipment is not likely to be available in smaller collections, many smaller laboratories arrange to store key strains under liquid nitrogen in the larger culture collections. In addition to the storage of cultures, culture collections are often respon- sible for research and education in culture maintenance, storage and charac- terization. Taxonomic studies are invaluable to culture collections since the material held in a collection must be properly identified and classified. The culture collection is a most appropriate location for taxonomic research, with many major contributions to scientific knowledge made in connection with culture collections. Culture collections exist in many places apart from the major collection centers. Many organizations using microorganisms in agriculture and industry maintain small collections of organisms for their particular purposes. In devel- oping countries, cultures, and the microbiologists who maintain and use them, may represent important resources that are not fully appreciated or utilized for national development objectives. Brought together, they could at a minimum serve as an expert source of advice and insight for development

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184 MICROBIAL PROCESSES authorities into alternative ways in which microorganisms can be exploited for development objectives, such as those described in this report, in the context of local resources and constraints. Mixed Microbial Cultures The preoccupation over many decades with pure culture techniques is giving way to a second look at mixed microbial cultures. It has been clearly shown, for example, that Chlorella, among the green algae, can be cultivated effectively under nonsterile conditions. Bacteria are present, to be sure, but appropriately compounded nutrient media permit a growth pattern favoring Chlorella and prevent bacterial overgrowth. Likewise, many foodstuffs customarily used in less-developed countries contain substantial numbers of mixed species of microorganisms. Mixed cul- tures of lactic acid bacteria are prominent in fermented foods derived from milk. Cheeses, curds, and cakes of various descriptions for human consump- tion have evolved in many parts of the world and are found even among . . . . . primitive societies. The subject of mixed-culture microbial technology is a fascinating one, and major portions of some international meetings are now devoted to this subject. The Symposium on Indigenous Fermented Foods presented as part of the Fifth International Conference on Global Impacts of Applied Micro- biology (Bangkok, November 1977) covered many processes of this nature. Patenting of Processes Involving Microorganisms In many countries it is possible to obtain patents for products and processes involving microorganisms. To file for such a patent, it is usually necessary to deposit the microorganisms) involved in a culture collection recognized for the purpose by the local patent authority. Both of the collec- tions listed in Table 10.1 for the United States are recognized by the U.S. Patent Office for this purpose. (The Northern Regional Research Laboratory, however, will not accept pathogenic organisms, and in general restricts its collection to bacteria and fungi.) In case of multinational filing of patent applications, it is necessary to deposit the cultures in each of the countries in which a patent is sought, except in those cases where countries have reached international agreements recognizing each other's depositories. Since this has been both troublesome and somewhat costly, the World Intellectual Property Organization (WIPO), which deals with international agreements on patents arid copyrights, sought to develop an international treaty to make it possible to recognize the deposi-

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PURE CULTURES FOR MICROBIAL PROCESSES 185 tion of such organisms in a singe depository as fulfillment of this require- ment for all signatories to the treaty. Such a treaty was finally completed in Budapest, Hungary, in May 1977, and a number of the major nations have already signed it. Although it may take some time before this treaty is activated, it would be well for microbiologists in all nations interested in seeking patents to be aware that it exists. For further information, inquiry can be made to the Secretary General, w1Po, Geneva, Switzerland. References and Suggested Reading American Type Culture Collection, Catalogue of Strains, I. 1978. 13th edition. Rock- ville, Maryland: American Type Culture Collection. Colwell, R. R. 1975. The role of culture collections in the era of molecular biology. Washington, D.C.: American Society for Microbiology. Martin, S. M., and Skerman, V. B. D., eds. 1972. World directory of collections of cultures of microorganisms New York: John Wiley and Sons. Skerman, V. B. D. 1977. The organization of a small general culture collection. In Proceedings of the International Conference on Culture Collections-II, July 15-20, 1973, Sdo Paulo, Brazil. A. F. Pestana de Castro, E. J. Da Silva, V. B. D. Skerman, and W. W. Leveritt, eds., pp. 20-40. Bowen Hills, Queensland, Australia: Courier-Mail. Steinkraus, K. H., ed. 1977. Papers presented at the Symposium on indigenous fer- mented foods, Fifth International Conference on Global Impacts of Applied Micro- biology, November 21-27, 1977, Bangkok, Thailand. (Will be published under title Handbook of tropical indigenous fermented foods.) 21 U.S. Code 111. Research Contacts P. Atthasampunna, Thailand Institute of Scientific and Technological Research, Bang- ken, Bangkok 9, Thailand. J. R. Jardim Freire, IPAGRO, Caixa Postal 776, 90000 Porto Alegre, Rio Grande do Sul, Brazil. C.-G. Heden, Karolinska Institutet, Solnavagen 1, S-104 01 Stockholm 60, Sweden. S. O. Keya, University of Nairobi, P.O. Box 30197, Nairobi, Kenya. V. B. D. Skerman, University of Queensland, St. Lucia, Brisbane, Queensland 4067, Australia.