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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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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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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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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
-65°C) 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 4°C showed a drop in
viable count to approximately half that of the original culture, while the same
cultures stored at room temperature (22°C) 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 -65°C. In recent years mechanical (electrical) refrigerators
capable of maintaining temperatures as low as -75°C 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 4°C 29°C
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 -196°C 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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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.
Representative terms from entire chapter:
culture collection
