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Sources and Man^pment

of Mîcro-Organîsms

For the Development of

a Fermentation Industry

Agriculture Handbook No. 440

Agricultural Research Service

U.S. DEPARTMENT OF AGRICULTURE
PREFACE

THIS REVIEW of micro-organisms provides a background for the manufacture of

chemicals by fermentation. It was prepared at the Northern Regional Research

Laboratory of the North Central Region, Agricultural Research Service, U.S. Department of Agriculture, Peoria, 111. At this Laboratory, investigations are being conducted

on the industrial utilization of cereal grains, oilseeds, and agricultural v^astes by

fermentation. The ARS Culture Collection, maintained there, is one of the world's

most complete collections of industrially important bacteria, molds, actinomycetes,

and yeasts. It serves as a source of authentic micro-organisms for the fermentative

production of organic acids, vitamins, antibiotics, enzymes, feeds, beverages, and foods.

Inasmuch as the key to success or failure in most fermentative processes is

availability of the proper micro-organisms, the characteristics of suitable microbial

strains are enumerated; the industrial microbial collections of the world—^their locations, their general holdings, and the names of their directors—are listed. The attributes of a good culture collection are emphasized. Various fermentation processes in

use throughout the world are listed, together with the specific micro-organisms needed

to carry them out.

Of these processes, the ones most likely to be beneficial in developing countries

are indicated. Because information is so frequently requested about micro-organisms:

for example, how to maintain stable cultures, how small fermentation plants may

acquire suitable microbial strains, and how micro-organisms can be shipped through

international channels, this publication is being issued to answer the many requests

received yearly by the U.S. Department of Agriculture.

Trade names are used in this publication solely for the purpose of providing

specific information. Mention of a commercial product or company does not constitute

a guarantee or warranty of the product by the U.S. Department of Agriculture or an

endorsement by the Department over other products not mentioned.

11
Sources and Management

of Micro-Organisms

For the Development of

a Fermentation Industry

By C. W. HESSELTINE and W. C. HAYNES

Agriculture Handbook No. 440

Agricultural Research Service

U.S. DEPARTMENT OF AGRICULTURE

Washington, D.C. Issued March 1974

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C. 20402—Price 60 cents

Stock Number 0100-02682
CONTENTS

Introduction _^ *^|

Sources of micro-organisms for industry 2

Characteristics of a good culture collection ^ 21

Location of culture collections 23

Procedures for isolation and selection of micro-organisms from nature 24

Classification of micro-organisms used for production of fermentation

products . 26

Problems in maintaining stable industrial cultures 30

Problems of strain degeneration and loss ^ 32

Contamination by other micro-organisms 33

Infestation by mites 33

Phage infestations 33

Natural selection and mutation 33

Untrained staff ^ ^ 34

Physical conditions affecting micro-organisms . 34

Regulations regarding deposit of cultures for patent purposes 35

Procedures and policies for deposition of cultures for patent purposes in

the ARS Culture Collection . . 35

Shipment of micro-organisms . 36

Literature cited 38

FOLLOW TNK UABfel. ^

«.s. »trAiTMiMT or A«IK«Um

IV
Sources and Management of Micro-Organisms

For the Development of

a Fermentation Industry

By C. W. HESSELTINE and W. C. HAYNíSS, Northern Regional Research Laboratory

North Central Region, Agricultural Research Service,

U.S. Department of Agriculture, Peoría, III.

INTRODUCTION

The micro-organism used in a fermentation

is the key to the success or failure of the

process. It is the catalyst that makes the fermentation work. A microbial culture must have

certain general attributes if the process it

generates is to be operable, regardless of the

nature of the product and the simplicity or

complexity of the engineering process :

1. The strain must be genetically stable.

A culture that constantly and spontaneously

produces one or more different forms is

undesirable.

2. The strain must readily produce many

vegetative cells, spores, or other reproductive

units. Since Basidiomycetes produce only

mycelium, they are rarely, if ever, used in

industrial fermentation.

3. The strain should grow vigorously and

rapidly after inoculation into seed tanks or

other containers used to prepare large amounts

of inoculum before an industrial fermentation.

4. The strain should be a pure culture, not

only free of other microscopically visible microorganisms, but also free of phages.

5. The strain should produce the required

product within a short period of time, preferably in 3 days or less.

6. The strain should produce the desired

product to the exclusion of all toxic substances.

The desired product should be easily separated

from all others.

7. The strain should be able to protect itself

against contamination, if possible. Self-protection might take the form of lowering the pH,

growing at high temperature, or rapidly elaborating a desirable microbial inhibitor.

8. The strain should be readily maintained

for reasonably long periods of time.

9. The strain should be amenable to change

by certain mutagenetic or group of mutagenetic

agents. A mutation program may even be conducted with the object of developing strains

that give enhanced yields of the product.

10. The strain must give a predictable

amount of desired product in a given fermentation time.

Micro-organisms that meet these conditions

may be either isolated from nature or obtained

from a culture collection. Trained microbiologists are required to isolate., purify, screen, and

test a culture from nature. Since, in developing

nations, such trained people are often in shorter

supply even than money, it seems to us that

culture collections would be the best source of

micro-organisms for setting up a fermentation

industry.

Plenty of time and money would still not

guarantee success. To obtain the proper culture, sometimes one must isolate the microorganism from a special, ecological niche that

may not even exist in a particular country. For
AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

example, Blakeslea trispora, which produces

large amounts of )8-carotene, cannot be isolated

in temperate regions of the United States, but

rather one must seek wild strains growing in

the tropics on flowers of certain higher plants.

For such cultures, collections are almost always

the only logical source.

Another source of cultures in the food industry, which should not be overlooked, is the

micro-organisms selected through the centuries

for preparing native fermented food products.

The principal micro-organisms can be obtained

with little difficulty. Since the micro-organisms

have been used in a particular food fermentation for centuries, there has been a constant,

purposeful selection of the best strains. The

yeast strains used in the municipal Bantu beer

breweries of South Africa were acquired in this

fashion. One of the authors (C. W. Hesseltine)

was told that the original strains were isolated

from the better native brews. After a number

of strains were tested, the best were chosen and

are now the ones used in an industry producing

150 million imperial gallons of the beverage

yearly.

In this Handbook, we have tried to be

realistic in our approach to the problem of

obtaining the proper micro-organisms for use

in industrial fermentations. Our views are

based upon first-hand knowledge of the operation of a large industrial culture collection supported entirely by government funds; experience during several years of operating a culture

collection in a large industrial fermentation

company; an understanding of the problems

faced by fermentologists in developing countries; contact with microbiologists working in

our fermentation laboratory from developing

countries; and an acquaintance with some of

the primitive food fermentations of the world.

SOURCES OF MICRO-ORGANISMS FOR INDUSTRY

The ultimate sources of culture and microorganisms for industry are soil; water; fresh,

fermenting, and rotting vegetables; living

plants and animals; sewage; fresh and spoiled

food ; frass and insect droppings ; and the like.

The immediate sources of cultures, however,

are permanent culture collections. Almost all

large industrial firms dealing in fermentations

have their own collections of micro-organisms

secured from a continuous program of isolation. New isolates and variant substrains derived from concurrent mutation studies swell

the numbers of strains so that many of the

proprietary collections are quite large. However, most of their micro-organisms never get

into general circulation, being intended solely

for exploitation by the parent company.

A few cultures from proprietary industrial

collections are in general and private collections

in the United States. In 1949, the U.S. Patent

Office took the position that a culture is an

essential part of a patent process and that the

culture must be disclosed. Hence, it must be

deposited in a recognized culture collection and

be available to the public at the time the patent

issues.

As a result of this practice, two U.S. collections—^the American Type Culture Collection at

Rockville, Md., and the ARS Culture Collection

in Peoria, 111.—are recognized as official depositories for cultures from industrial concerns,

both domestic and foreign. As might be expected, the depositing companies do not advertise that particular strains have been placed

in outside culture collections, and the named

depositories agree not to reveal possession of

patent cultures or to distribute them without

authorization by the depositor, if this is his

wish, until the U.S. patent issues.

The holdings of the companies are supplemented also by accessions from public and

private culture collections whose culture distributions are not so rigidly controlled.
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS

Private collections do not have as a principal

purpose of existence the distribution of cultures. They usually are specialist collections;

that is, their scope is confined to one or a few

taxa of special interest to the scientists who

operate or control them. Generally, private

collections are associated with a university or

research institute. Although their curators decline to distribute cultures far and wide to anyone who asks, they nevertheless often send

cultures to other investigators with like interests, or to research institutes and to industrial

men who might continue research which they

no longer can pursue or who might continue

development of an industrial process. Private

collections generally do not charge fees for

their cultures. Like proprietary collections,

they usually do not publish or distribute lists

of their cultures.

Public collections have as one of their principal reasons for existence the accumulation of

a diverse collection of salable micro-organisms.

They send cultures anywhere in the world to

any bona fide investigator who is willing to pay

their price. As might be expected, they publish

catalogs listing the micro-organisms that are

for sale. They often provide other services, such

as identification of micro-organisms and preservation of cultures by lyophilization or liquidnitrogen refrigeration. Their diversity may be

as wide as that of the American Type Culture

Collection, which maintains actinomycetes,

algae, bacteria, cell lines, molds, protozoa,

viruses, and yeasts.

Among the specialized culture collections,

some concentrate on industrially useful microorganisms. Such micro-organisms are bacteria,

yeasts, molds, actinomycetes, algae, and protozoa that are used in the food, pharmaceutical,

and fermentation industries and in research

and development laboratories to convert selected substrates to products of enhanced nutritional, medicinal, or industrial value or to reduce the biochemical oxygen demand (BOD)

in sewage and industrial effluents. Such collections are of principal interest to the United

Nations Industrial Development Organization

(UNIDO) and its adherent groups and

members.

We concluded that a list of such collections,

giving addresses, names of curators, and types

of micro-organisms contained, would be useful

(Table 1). We are indebted to S. M. Martin of

the Division of Biosciences, National Research

Council, Ottawa 7, Ontario, Canada, for most

of the names of collections and information

about them. Under the aegis of the World Federation of Culture Collections (WFCC, formerly the Section on Culture Collections) of the

International Association of Microbiological

Societies, Dr. Martin published a World Directory of Collections of Cultures of Micro-organisms (11) y^ in which most of the collections in

the world are named and described.

Names and addresses of additional collections

may be found in some of the larger culture

collection catalogs listed at the end of this paper

(2,S,i).

Fees for cultures vary from one collection to

another. In the United States, the American

Type Culture Collection charges $30 per strain

for all cultures to profit-making institutions.

The cost is reduced to $20 for nonprofit institutions (except for some special teaching strains,

which are $10). The Centraalbureau voor

Schimmelcultures in The Netherlands charges

40 guilders for cultures that are to be used for

industrial purposes. There is a reduction in cost

if 10 strains or more are purchased in 1 year.

This collection, like some others, does not

guarantee the production of chemical substances by its cultures.

As a general rule, collections which advertise

their cultures in printed catalogs charge a fee

for their strains. Some collections, such as the

one with which we are associated, do not issue

a catalog, do not charge a fee, but do exert

considerable restraint on the number of strains

sent at any one time to any individual or

institution.

^Italic numbers in parentheses refer to Literature

Cited, p. 38.
AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

TABLE 1.—Collections containing industrially useful micro-organisms

(Includes main collections and those containing at least 500 strains)

Collection, parent organization,

address, and person in charge Contents

ARGENTINA

Centro de Micologiá Fungi

Facultad de Ciencias Médicas Yeasts

Universidad de Buenos Aires

Paraguay 2155, 11°, Buenos Aires

Prof. Dr. P. Negroni

Colección Cátedra Microbiología Agrícola (FAV, Bs. As.) Bacteria

Facultad Agronomía y Veterinaria Fungi

Universidad de Buenos Aires Yeasts

Avenida San Martín 4453 (Suc. 17), Buenos Aires

Prof. Ing. Agr. R. E. Halbinger

Colección de Cultivos Microbianos Bacteria

Facultad de Farmacia y Bioquímica Fungí

Universidad de Buenos Aires Yeasts

Junin 956 Piso. 8°, Buenos Aires

Prof. Dr. R. A. Margni

Instituto de Microbiología e Industrias Agropecuarias Bacteria

Instituto Nacional de Tecnología Agropecuaria Fungí

Villa Udaondo, Castelar FCDFS, Buenos Aires Yeasts

Ing. Agr. E. Schiel

Instituto de Patología Vegetal Bacteria

Instituto Nacional de Tecnología Agropecuaria Fungí

Villa Udaondo, Castelar FCDFS, Buenos Aires

Ing. Agr. C. J. M. Carrera

Cátedra de Microbiología Bacteria

Facultad de Ciencias Agrarias Yeasts

Universidad Nacional de Cuyo

Almiranti Brown 500, Chacras de Coria, Mendoza

Ing. Agr. N. J. Palleroni

AUSTRALIA

Rhízobium Strain Collection Bacteria

Division of Tropical Pastures

Commonwealth Scientific and Industrial Research Organization

Mill Road, 'St. Lucia, Brisbane, Queensland, 4067

Dr. D. 0. Norris

Culture Collection Yeasts

The Australian Wine Research Institute

Private Bag No. 1, Glen Osmond, P.O., South Australia, 5064

W. W. Forest
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

Collection, parent organization,

address, and person in charge Contents

AUSTRALIA—Continued

Soil Microbiology Culture Collection

Division of Soils

Commonwealth Scientific and Industrial Research Organization

Private Bag No. 1, Glen Osmond, South Australia, 5064

Dr. E. G. Hallsworth

Bacteria

Institute of Agriculture

University of Western Australia

Nedlands, Western Australia, 6009

Dr. C. A. Parker

Bacteria

Division of Wood Technology

Forestry Commission of New South Wales

96 Harrington 'Street, Sydney, New South Wales, 2000

E. B. Huddleston

Fungi

Milchwirtschalñiche Bakterienkulturen

Rotholz/Post Jenbach, Tirol

Dipl.-Ing. S. Winkler

AUSTRIA

Bacteria

BELGIUM

Collection of the Laboratory for Microbiology

Laboratorium voor Microbiolgie, Fac. Wetenschappen

Rijksuniversiteitn

Ledeganckstraat, Gent

Prof. J. DeLey

Laboratoire de Mycologie Systématique et Appliquée

Université Catholique de Louvain

92, Avenue Cardinal Mercier, B-3030-Heverlee

Prof. Dr. Ing. G. L. Hennebert

Bacteria

Fungi

Colecâo de Culturas

Instituto Adolfo Lutz

Av. Dr. Arnaldo 355, C.P. 7027, Sâo Paulo

F. de B. M. Jordâo

BRAZIL

Bacteria

Fungi

Protozoa

Animal Viruses

Culture Collection, Instituto Zimotécnico (IZ)

Escola Superior de Agricultura "Luis de Queiroz"

C.P., 56, Piracicaba, Sâo Paulo

Prof. J. Lerne, Jr.

Bacteria

Fungi

Yeasts
AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

Collection, parent organization,

address, and person in charge Contents

BULGARIA

Bulgarian Type Culture Collection Bacteria

Institute for State Control of Medical Preparations Fungi

Ministry of Health Yeasts

Vladimir Zaimov No. 26, Sofia

Prof. R. Ovtcharov/Dr. M. Zheleva

BURMA

Type Culture Collection Bacteria

Burma Pharmaceutical Industry (B.P.I.) Fungi

Industrial Development Corp., Ministry of Industry

BPI Road, Gyogon, Rangoon

Dr. Ko Gyi Ko

CANADA

Mold Herbarium and Culture Collection (UAMH) Fungi

University of Alberta Yeasts

Edmonton, Alberta

Dr. J. W. Carmichael

Atlantic Regional Laboratory Bacteria

National Research Council of Canada Fungi

1411 Oxford Street, Halifax, Nova Scotia Yeasts

Dr. A. C. Neish

University of Western Ontario Culture Collection (UWO) Bacteria

Botany Department Fungi

University of Western Ontario Yeasts

London, Ontario Algae

Dr. J. C. Hickman

Biosciences Division Bacteria

National Research Council of Canada Fungi

Sussex Drive Yeasts

Ottawa 2, Ontario

Dr. G. C. Butler

University of Windsor Culture Collection • Bacteria

Department of Biology Fungi

University of Windsor Yeasts

Windsor, Ontario Animal Viruses

R. J. Doyle

Macdonald College Collection Bacteria

Department of Microbiology, Macdonald College Fungi

McGill University Yeasts

Ste. Anne de Bellevue, Quebec

Dr. R. Knowles
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

Collection, parent organization,

address, and person in charge

CANADA—Continued

Prairie Regional Laboratory

National Research Council of Canada

Saskatoon, Saskatchewan

Dr. R. H. Haskins

Contents

Bacteria

Fungi

CEYLON

Department of Biological Sciences (Microbiology)

Vidyodaya University

Gangodawila

CHILE

Collection Bactéries

Institut Centre National de Microbiologie Bactériologique

Avenida Maratón 1000, Santiago

Prof. E. Dussert

Bacteria

Bacteria

CZECHOSLOVAKIA

(AU but one (*) of these collections are part of the

Czechoslovak Collections of Micro-organisms)

Research Institute for Viticulture and Enology (RIVE)

Matuskova ul. 97, Bratislava

Dr. Ing. A. Veres, C.Sc.

Collection of Cultures of Wood-Rotting Fungi

Laboratory for Anatomy and Physiology of Plants (LAPP)

J. E. Purkyne University

Kotláfska 2, Brno

Prof. Dr. V. Rypácek, Dr. Sc.

Czechoslovak Collection of Microorganisms (CCM)

J. E. Purkyne University

tr. Obráncu Míru 10, Brno

Prof. Dr. T. Martinec, Dr. Sc.

Collection of Rhizobium and Other Soil Microorganisms

Central Research Institute of Plant Production (CRIPP)

Ruzynë, Prague

Dr. E. Hamatová-Hlavácková, C.Sc.

Culture Collection of Entomogenous Bacteria (CCEB)

Department of Insect Pathology

Institute of Entomology, Czechoslovak Academy of Sciences

Na cviciáti 2, Prague 6

Dr. 0. Lysenko, C.Sc.

Fungi

Yeasts

Fungi

Bacteria

Bacteria

Bacteria
AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

TABLE l.—Collections containing industrially useful micro-organisms—Continued

Collection, parent organization,

address, and person in charge Contents

CZECHOSLOVAKIA—Continued

Culture Collection of Fungi (CCF) Fungi

Department of Botany, Faculty of Sciences

Charles University

Benátská 2, Prague 2

Dr. 0. Fassatiova, C.Sc.

Czechoslovak National Collection of Type Cultures Bacteria

Institute of Epidemiology and Microbiology Fungi

ârobarova 48, Prague 10 Yeasts

Dr. J. áourek, C.Sc. Animal Viruses

Bacteriophages

Research Laboratories of the Dairies Bacteria

Association for the Production of Pure Milk Fungi

Cultures Laktoflora (LAKTOFLORA) Yeasts

Ke dvoru 2, Prague 6

Ing. Dr. M. Teply

DENMARK

Bacillus Collection Bacteria

Institute of Hygiçne

University of Aarhus

DK-8000, Aarhus

Prof. G. J. Bonde

Bacteriological Department Unspecified

Lövens kemiske Fabrik

2750 Ballerup

L. Tybring

Antibiotics Department Unspecified

Dumex, Ltd.

Prags Boulevard 37, 2300 Copenhagen S

Dr. K. Andersen

Bacteriological Department Unspecified

H. Lundbeck and Co.

Ottiliaveg 7-8, 2500 Copenhagen Valby

L. Szabo

Statens Fors0gsmejeri Unspecified

3400 Hiller0d

A. M. Madsen

Department of Technical Biochemistry Unspecified

Danmarks tekniske H0jskole

Bygning 223, 2800 Lyngby

Dr. M. Jensen
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS 9

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

Collection, parent organization, P . .

address, and person in charge <^ontents

DENMARK—Continued

Laboratory for Microbiology Unspecified

Danmarks tekniske H0jskole

Bygning 221, 2800 Lyngby

Prof. Dr. J. Hedegaard

FINLAND

Culture Collection Bacteria

Department of Microbiology Fungi

University of Helsinki Yeasts

Helsinki 71

Prof. U. J. Vartiovaara

FRANCE

Centre de Collection de Types Microbiens Bacteria

Centre d'Études et de Recherches Technologiques des Industries Alimentaires Yeasts

62 Boulevard Maréchal VaiUant, Lille Animal Viruses

Dr. H. Beerens Bacteriophages

Institut Pasteur de Lyon: IPL Bacteria

Rue Pasteur, Lyon 69

M. Carraz

Collection de Microorganismes Associes Aux Invertébrés Bacteria

Station de Recherches Cytopathologiques I.N.R.A.-C.N.R.S. Yeasts

MontpeUier 30 Saint-Christol Animal Viruses

Prof. C. Vago

Laboratoire de Cryptogamie Fungi

Museum National d'Histoire Naturelle

Paris

Dr. J. Nicot

Laboratoire des Fermentations Yeasts

Institut Pasteur de Paris

28 Rue du Docteur Roux, Paris 15^

P. Bréchot

Service des Anaérobies Bacteria

Institut Pasteur de Paris

25 Rue du Docteur Roux, Paris 15^

Dr. Rouyer
10 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

TABLE 1.—Collections containing indiistrially useful micro-organisms—Continued

Collection, parent organization,

address, and person in charge Contents

GERMANY, EAST (DEMOCRATIC REPUBLIC)

Fachgebiet Allgemeine Botanik und Pflanzenphysiologie Bacteria

Ernst-Moritz-Arndt-Universität Fungi

Grimmerstrasse 86/88, DDR-22 Greifswald Yeasts

Prof. Dr. H. Borriss Algae

Bacteriophages

Cell lines

Kulturensammlungen Bacteria

Zentral Institutes für Mikrobiologie und Experimentelle Fungi

Therapie (IMET) Yeasts

Deutsche Akademie der Wissenschaften zu Berlin Animal Viruses

Beuthenbergstrasse 11, Jena 69 Bacteriophages

H. Prauser Protozoa

Actinomycetes

Kulturensammlung Bacteria

Institut für Mikrobiologie Fungi

Humboldt-Universität Yeasts

1532 Kleinmachnow, Max-Reimannstrasse 16, Kleinmachnow

^ bei Berlin

Prof. Dr. Jentzsch

Botanisches Institut Fungi

Mykologie Weimar Yeasts

Friedrich-Schiller Universität Jena

Frh.-v-Stein-Allee 2, Weimar

Prof. Dr. R. Trôger

GERMANY, WEST (FEDERAL REPUBLIC)

Mikroorganismensammlung Bacteria

Institut für Garungsgewerbe und Stärkefabrikation Fungi

Seestrasse 13, 1 Berlin 65 Yeasts

Prof. Dr. S. Windisch

Instituts Staamsammlung, Biologische Bundesanstalt Bacteria

für Land und Fortswirtschaft Fungi

Institut für biologische Schädlingsbekämpfung Animal Viruses

Kranichsteinerstrasse 61, Darmstadt

Prof. Dr. J. M. Franz

Sammlung von Algenkulturen Algae

Pflanzenphysiol. Institut

Universität Göttingen

Nikolausberger Weg 18, 34 Göttingen

Prof. Dr. A. Pirson

Food Spoiling Molds Fungi

Deutsche Forschungsanstalt für Lebensmittelchemie

Leopoldstrasse 175, Munich, Bayern

Prof. Dr. S. W. Souci
SOURCES AND MANAGEMENT OP MICRO-ORGANISMS 11

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

Collection, parent organization,

address, and person in charge Contents

GERMANY, WEST (FEDERAL REPUBLIC)—Continued

Bayerische Landensanstalt für Wein-, Obst-u. Gartenbau Yeasts

Residenzpl. 3, 87 Würzburg, Bayern

Dr. I. Benda

GHANA

University Microbial Cultures, Kumasi (U.M.C.K.) Bacteria

Department of Biological Sciences Fungi

University of Science and Technology Yeasts

Kumasi

K. 0. Nyako

HUNGARY

Culture Collection Bacteria

Pedological Inst. Actinomycetes

Hungarian Academy of Sciences

Herman 0.15, Budapest II

Dr. J. Szegi

Diagnostical and Research Laboratory Bacteria

National Institute for Tuberculosis "Korányi" Bacteriophages

Pihenö ut 1, Budapest XII

I. Szabó, M.D., D.Sc. ^~—-

INDIA

BSM Culture Collection Fungi

Botany Department

University of Allahabad

Allahabad

Dr. B. S. Mehrotra

Fermentation Technology Laboratory Bacteria

Indian Institute of Science Yeasts

Bangalore-3

Dr. J. V. Bhat

Department of Microbiology Bacteria

Bose Institute Fungi

93/1 Acharya Prafulla, Chandra Road, Calcutta 9 Yeasts

Prof. P. Nandi Actinomycetes

Bacteriophages

D. R. L. (M) Kanpur Culture Collection Bacteria

Defense Research Laboratory (Materials) Fungi

Research and Development Organization, Ministry of Defense Yeasts

P.B. 320 Kanpur, Uttar Pradesh

Dr. J. N. Nanda
12 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

Collection, parent organization,

address, and person in charge Contents

INDIA—Continued

National Collection of Industrial Microorganisms (NCIM) Bacteria

National Chemical Laboratory Fungi

Council of Scientific and Industrial Research (CSIR) Yeasts

Poona-8

Dr. V. Jagannathan

INDONESIA

Culture Collection, Treub Laboratory Bacteria

National Biological Institute Fungi

The Botanical Garden Yeasts

Bogor Actinomycetrs

Dr. S. Saono

IRAN

Razi Culture Collection Bacteria

State Razi Institute-Hessarak, Ministry of Agriculture Animal Viruses

P.O. Box 656, Karadj, Tehran Protozoa

Dr. F. Entessar

IRELAND

Department of Industrial Microbiology Fungi

University CoUeg^ Actinomycetes

Ardmore, Stillorgan Rd., Dublin, 4

Prof. M. J. Geoghegan

Guinness (Dublin) Culture Collection Bacteria

A. Guinness Son and Co. (Dublin) Ltd. Yeasts

St. James's Gate, Dublin, 8

Dr. C. E. Dalgliesh

Johnstown Castle Collection Bacteria

Soil Laboratory

The Agricultural Institute

Wexford

C. L. Masterson

ITALY

Centro di Studio dei Microorganismi Autotroñ Bacteria

Instituto di Microbiologia Agraria e Técnica Fungi

Università di Firenze Yeasts

Piazzale délia Cascine, 27 Firenze Algae

Prof. G. Florenzano
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

13

Collection, parent organization,

address, and person in charge Contents

Instituto di Patologia Végétale

Université di Milano

Via Celoria 2, Milano 20133

Dr. E. Baldacci

ITALY—Continued

Actinomycetes

Fungi

Lepetit S.p.A.

Via Durando 38, Milano 20158

Prof. P. Sensi

Bacteria

Fungi

Yeasts

Actinomycetes

Protozoa

Collezione dei Lieviti Vinari

Instituto di Microbiologia Agraria e Técnica

Université di Perugia

Bg. XX Guigno, Perugia 06100

Prof. T. Castelli

Bacteria

Yeasts

Collezione Micróbica Agraria, Marina e Industriale (COMAMI)

Instituto Microbiologia Agraria e Técnica

E. de Nicola, Sassari

Prof. A. Capriotti

Bacteria

Yeasts

JAMAICA

Department of Microbiology

University of West Indies

Mona St., Kingston 7

Prof. L. S. Grant

Bacteria

Animal Viruses

JAPAN

Department of Fermentation Technology (HUT)

Faculty of Engineering

Hiroshima University

3-Chôme, Senda-machi Hiroshima

Dr. T. Nehira

Bacteria

Fungi

Yeasts

Culture Collection (IFO)

Institute for Fermentation

Juso Nishinocho 4-54, Higashi-yodogawa-ku, Osaka

Dr. T. Hasegawa

Bacteria

Fungi

Yeasts

Bacteriophages

Department of Fermentation Technology (OUT)

Faculty of Engineering

Osaka University

9-Chôme Higashinoda-machi, Miyakoshima-Ru, Osaka

Dr. G. Terui

Bacteria

Fungi

Yeasts
14 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

Collection, parent organization,

address, and person in charge Contents

JAPAN—Continued

ATU Culture Collection

Department of Agricultural Chemistry

Faculty of Agriculture

University of Tokyo

l-Chome, Yayoi, Bunkyo-ku, Tokyo

Dr. K. Arima

Bacteria

Fungi

Yeasts

Actinomycetes

Institute of Applied Microbiology (lAM)

University of Tokyo

l-Chome, Yayoi, Bunkyo-ku, Tokyo

Prof. Dr. H. lizuka

Bacteria

Fungi

Yeasts

Algae

Actinomycetes

Nagao Institute (NI)

96, Kinuta-machi, Setagaya-ku, Tokyo

K. Nagao

Fungi

Yeasts

Algae

The Research Institute of Fermentation

Yamanashi University

1 Kitashin-machi, Kofu, Yamanashi

Prof. Dr. I. Yokotsuka

Bacteria

Fungi

Yeasts

KOREA

Korean Federation of Culture Collections of Microorganisms

Ministry of Science and Technology

2nd Chongro, Seoul

Prof. Dr. Lee Zoo Shik

Bacteria

Fungi

Yeasts

Algae

Protozoa

Plant Viruses

Animal Viruses

Bacteriophages

MALAYSIA

Pathology Division

Rubber Research Institute of Malaya

P.O. Box 150, Kuala Lumpur, Selangor

Mr. B. S. Rao

Bacteria

Fungi

Yeasts

NETHERLANDS

Centra albureau voor Schimmelcultures (CBS)

Oosterstaat 1

Baarn

Dr. J. A. Von Arx

Fungi

Yeasts

Actinomycetes
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS 15

TABLE 1. Collections containing industrially useful micro-organisms—Continued

Collection, parent organization, r-r.^for^+c

address, and person in charge i^oni^enxs

NETHERLANDS—Continued

Culture Collection Bacteria

Laboratory of Microbiology

Technical University

Julianalaan 67A, Delft

Prof. Dr. T. 0. Wiken

NEW ZEALAND

Culture Collection Bacteria

Plant Diseases Division v "^^

Department of Scientific and Industrial Research Yeasts

Private Bag, Auckland Pl^^^ Viruses

Mr. J. D. Atkinson

NIGERIA

Fungus Culture Collection Fungi

University of Lagos Yeasts

University Road, Yaba, Lagos

Prof. S. H. Z. Naqvi

NORWAY

Norges Tekniske Hagskoles Collection (NTHC) Bacteria

Department of Biochemistry ^^^^}

Technical University of Norway Yeasts

Trondheim MTH

Dr. K. Eimhjellen

Department of Microbiology Fungi

Agricultural College of Norway

Box 40, Vollbekk

Dr. G. G. Lindeberg

PHILIPPINES

Rhizohium japonicum and Rhizobium spp. from other Bacteria

tropical legumes

College of Agriculture

University of the Philippines

College, Laguna

M. E. Raymundo

POLAND

Special Centre of Industrial Cultures Bacteria

Department of Industrial Microbiology v ^^

Technical University of Lodz ^ Yeasts

Wólczañska 171/173, Lodz

Prof. dr. J. Jakubowska
16 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

TABLE 1.—Collections containing industrially icseful micro-organisms—Continued

Collection, parent organization,

address, and person in charge Contents

POLAND—Continued

Culture Collection of Industrial Microorganisms Bacteria

Institute of the Fermentation Industry Fungi

Rakowiecka 36, Warsaw Yeasts

Dr. J.N.Z.T. Golébiewski

Central Centre of Microorganisms Collections Bacteria

Microbiologie Committee

Polish Academy of Sciences

Commission of Taxonomy and Storage of Microorganisms

ul. Chalubinskiego 4, Wroclaw

Prof. dr. S. álopek

PORTUGAL

Colleccao de Culturas de Fungos Fungi

Micologia, Faculdade de Ciencias

Lisbon

Prof. J. Pinto-Lopes

Institute Gulbenkian Ciencia Yeasts

Laboratorio de Microbiologia

Centro de Biología

Rua da Quinta Grande, Oeiras

Dr. N. van Uden

RHODESIA

Grasslands Rhizobium Collection Bacteria

Grasslands Research Station

P.B. 701, Marandellas

T. C. D. Kennan

ROMANIA

Microbiological Laboratory Bacteria

Biosynthesis Department Fungi

Institutu de Cercetari Chicico-Farmacentice

112, Soseaua Vitan, Bucharest

Mr. T. Slave

SOUTH AFRICA

Microbiology Research Group Bacteria

South African Council for Scientific and Industrial Research Fungi

Pretoria Yeasts

Dr. J. P. van der Walt
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS

TABLE 1.—Collections containing industrially useful micro-organisms—Contmued

17

Collection, parent organization,

address, and person in charge

SWITZERLAND

Culture Collection of Microorganisms

J. R. Geigy S. A.

Schwarzwaldallee, CH 4021 Basel

Dr. W. A. Vischer

Botanische Sammlungen der Eidg. Technische Hochschule

Institut für spezielle Botanik

Universitätstrasse 2, 8006 Zürich

Dr. E. Müller

Mikrobiologisches Institut der Eidg. Technische Hochschule

Swiss Federal Institute of Technology

Universitätstrasse 2, CH 8032 Zürich

Prof. Dr. L. Ettlinger

UNITED ARAB REPUBLIC

Agricultural Microbiology Division (A.M.D.)

Ministry of Agriculture

Soils Department Building

University Street, Orman, Giza

Mr. Abou El-Fadl

Microbic Collection

College of Agriculture

Ain Shams University

Shoubra El-Khaima, Kalubia

Dr. S. M. Taha

UNITED KINGDOM

The Wellcome Bacterial Collection

Wellcome Foundation Ltd.

Langley Court, Beckenham, Kent, England

Dr. J. Cameron

Culture Collection of Algae and Protozoa

36 Storey's Way

Cambridge University

Cambridge CB3 ODT England

Dr. E. A. George

Glaxo Research Ltd.

Greenford, Middlesex, England

Dr. P. W. Muggleton

Contents

Bacteria

Fungi

Yeasts

Protozoa

Fungi

Bacteria

Fungi

Yeasts

Actinomycetes

Bacteria

Fungi

Yeasts

Bacteria

Fungi

Yeasts

Algae

Bacteria

Algae

Protozoa

Bacteria

Fungi

Yeasts

Protozoa
18 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

Collection, parent organization,

address, and person in charge Contents

UNITED KINGDOM—Continued

The Commonwealth Mycological Institute (CMI) Fungi

The Ministry of Technology

Ferry Lane, Kew, Surrey, England

Mr. A. Johnston

Akers Culture Collection Bacteria

Pharmaceuticals Division Fungi

Imperial Chemical Industries Ltd. Yeasts

P.O. Box 25, Alderley Park, Macclesfield, Cheshire, England

Dr. D. Broadbent

British National Collection of Yeast Cultures Yeasts

Brewing Industry Research Foundation

Nutfield, Surrey, England

Dr. A. H. Cook

National Collection of Dairy Organisms Bacteria

National Institute for Research in Dairying Bacteriophages

Shinfield, Reading, Berkshire, England

Dr. L. A. Mabbitt

National Collection of Industrial Bacteria Bacteria

Ministry of Technology Bacteriophages

Torry Research Station

P.O. Box 31,135 Abbey Road, Aberdeen AB9 8DG, Scotland

Dr. J. M. Shewan

National Collection of Marine Bacteria Bacteria

Ministry of Technology Yeasts

Torry Research Station

P.O. Box 31, 135 Abbey Road, Aberdeen AB9 BDG, Scotland

Dr. M. Shewan

UNITED STATES OF AMERICA

Chas. Pfizer & Co., Inc. Bacteria

Groton, Connecticut 06340 Fungi

Dr. J. Routien Actinomycetes

IMC Culture Collection Bacteria

Growth Sciences Center Fungi

International Minerals & Chemical Corporation Bacteriophages

P.O. Box 192, Libertyville, Illinois 60048

Dr. M. H. Rogoff
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

19

Collection, parent organization,

address, and person in charge Contents

UNITED STATES OF AMERICA—Continued

ARS Culture Collection (NRRL)

Northern Regional Research Laboratory

Agricultural Research Service

U.S. Department of Agriculture

1815 N. University St., Peoria, Illinois 61604

Dr. T. G. Pridham

Bacteria

Fungi

Yeasts

Actinomycetes

The Culture Collection of Algae

Department of Botany

Indiana University

Bloomington, Indiana 47401

Dr. R. C. Starr

Algae

Microbiology Research

Eli Lilly and Company

307 East McCarty St., Indianapolis, Indiana 46206

Dr. D. H. Lively

Bacteria

Fungi

Actinomycetes

Grain Processing Corporation

1600 Oregon St., Muscatine, Iowa 52761

Mr. C. Smith

Bacteria

Fungi

Yeasts

Algae

American Type Culture Collection (ATCC)

12301 Parklawn Drive, Rockville, Maryland 20852

Dr. R. Donovick

Bacteria

Fungi

Yeasts

Actinomycetes

Algae

Protozoa

Animal Viruses

Bacteriophages

Cell lines

Culture Collection of the U.S. Army Material Command

U.S. Army Natick Laboratories

Natick, Massachusetts 01762

Dr. E. G. Simmons

Bacteria

Fungi

Yeasts

The Upjohn Stock Culture Collection

The Upjohn Company

301 Henrietta Street, Kalamazoo, Michigan 49001

Dr. G. B. Whitfield, Jr.

Bacteria

Fungi

Yeasts

Algae

Actinomycetes

Protozoa

Culture Collection

Squibb Institute for Medical Research

E. R. Squibb & Sons, Inc.

Georges Road, New Brunswick, New Jersey 08901

Mrs. F. Arnow

Bacteria

Fungi

Yeasts

Actinomycetes
20 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

Collection, parent organization,

address, and person in charge Contents

UNITED STATES OF AMERICA—Continued

IMRU Collection

Institute of Microbiology

Rutgers—The State University

New Brunswick, New Jersey 08903

Dr. R. E. Gordon

Bacteria

Actinomycetes

Merck Sharp & Dohme Research Laboratories

Culture Collection

Merck & Co., Inc.

Rahway, New Jersey 07065

Dr. T. H. Stoudt

Bacteria

Fungi

Yeasts

Algae

Actinomycetes

Lederle Microbiology Research Collection

Lederle Laboratories Division

American Cyanamid Company

No. Middletown Road, Pearl River, New York 10965

Dr. E. J. Backus

Bacteria

Fungi

Yeasts

Algae

Actinomycetes

Bristol Laboratories

P.O. Box 657, Syracuse, New York 13201

Dr. C. A. Claridge

Actinoplanaceae (Actinomycetales)

Botanical Department

University of North Carolina

Coker Hall, Chapel Hill, North Carolina 27514

Dr. V. A. Greulach

Bacteria

Fungi

Actinomycetes

Bacteriophages

Wyeth Laboratories Collection

Wyeth Laboratories

P.O. Box 8299, Philadelphia, Pennsylvania 19101

Dr. G. H. Warren

Bacteria

Fungi

Yeasts

Department of Plant Pathology and Botany

Agricultural Experiment Station

P.O. Box H, Rio Piedras, Puerto Rico 00928

Dr. J. E. Pérez

Department of Bacteriology

College of Agricultural and Life Sciences

University of Wisconsin

Madison, Wisconsin 53706

Dr. K. B. Raper

Bacteria

Fungi
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS

TABLE 1.—Collections containing industrially useful micro-organisms—Continued

21

Collection, parent organization,

address, and person in charge Contents

UNION OF SOVIET SOCIALIST REPUBLICS

All-Union Collection of Type Cultures

Institute of Microbiology

U.SJS.R. Academy of Sciences

Profsouznaya Str., 7a, Moscow, B-133

Prof. V. Kudriavtsev

Institute for Research of New Antibiotics

U.S.S.R. Academy of Medical Sciences

Bolshaia Pirogovskaia 11, Moscow

Dr. G. F. Gauze

Centro de Microscopía Electrónica

Edeficio Central

Universidad de los Andes

Apartado 163, Calle Vargas, Merida

Dr. J. A. Serrano

Bacteria

Fungí

Yeasts

Actinomycetes

Actinomycetes

VENEZUELA

Bacteria

Fungi

CHARACTERISTICS OF A GOOD CULTURE COLLECTION

Although much has been spoken and written

about culture collections, to our knowledge no

one has ever laid down the characteristics of a

good applied or industrial culture collection.

Many of the following points apply equally well

to other types of collections :

1. The collection must be part of, or closely

related to, a fermentation research laboratory

or to a fermentation plant, or both. At the

Northern Regional Research Laboratory, the

ARS Culture Collection is the responsibility of

one of the four research units in the Fermentation Laboratory.

Interactions between fermentologists and

culture-collection staff work to the mutual

benefit of both. The fermentologists, being

aware of general trends in fermentation research, are able to anticipate future areas of

interest and to provide guidance as to what

micro-organisms the culture collection should

accession to meet future needs. The microbiologists, with their knowledge of the relationships among genera and the physiological

requirements of various micro-organisms, can

make valuable suggestions regarding screening

programs. In their detailed studies on individual strains, they may make observations leading to new fermentation products or higher

yields of known products. Their ready recognition of contamination or degeneration of the

culture being used in the development of a

process acts as a type of quality control.

2. A culture collection must be well funded,

and this funding must be at a relatively uniform level each year. In many other research

operations, a program may be increased or

decreased readily with changes in the amount

of budgeted money. Personnel can be shifted

easily from one project to another. On the other

hand, a culture collection is a continuing operation which must be sustained without great

ñuctuations in budget or people from year to

year. Many of the culture-collection projects

become long-term studies of a genus or family,

and years are required to assemble wild cultures and known type materials in order to do

a first-rate job.

3. A culture collection must have adequate
22 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

facilities and equipment, including transfer

rooms, refrigerator space, incubators, microscopic and photographic equipment, autoclaves,

and lyophilizers. Usually these facilities should

be separate from those of other research

groups.

4. Library facilities are necessary so that

personnel may have access to the taxonomic

and fermentative literature being published,

not only in the region or country of location,

but also in the world.

5. The collection should have an active and

continuous program of isolating new strains of

micro-organisms from nature. This goal will

lead to the discovery of new products and

reactions. New material will also add to the

understanding of the classification of special

groups of micro-organisms. New material

makes it possible to discover species and genera

new to science.

6. The collection must have an adequate

staff to support the curators. By this requirement, we mean technical help to prepare media,

sterilize glassware, and perform routine techniques ; secretarial help to keep the voluminous

records and to handle correspondence; and

shops to construct special apparatus. At the

Northern Laboratory, our glassblower devised

an automatic machine to make lyophil tubes.

Reliable sources of supplies are also necessary.

We have had experience in setting up a lyophil

apparatus in a developing nation. Although the

lyophil equipment was readily made to our

specifications, not a culture could be processed

for 2 years because there was no dry ice.

Optimally, each curator should have a careful, intelligent, and dedicated assistant with

some microbiological training. In our experience, technicians need not be specialized because they always must be trained in the special

techniques required for the collection. These

assistants should handle periodic transfers,

lyophilization and associated records, inoculation of cultures for study by the curator, seeding of flask cultures for preliminary surveys

for new products, and making and recording

routine observations on all cultures.

7. The curator (s) must do research, as well

as maintain the collection. Each must have an

active research program either in taxonomy or

genetics, with preference to the former. Thus a

curator will have an intimate knowledge of the

strains he is maintaining and will develop

a reputation as an expert in his field.

Consequently, important material will be sent

to him for safekeeping, for identification, and

for other purposes. Other microbiologists will

know from whom they may get expert advice,

cultures, and information. This point is an

important one that we have stressed before

8. College-trained members of a culturecollection staff must be aware of the field of

applied microbiology, appreciate the work

being done in fermentation research and development, and understand the operation of fermentation plants. They must comprehend the

problems of geneticists, fermentologists, engineers, biochemists, and organic chemists.

Probably the most difficult job from an administrator's standpoint is indoctrinating the

curators of a culture collection. They must

understand fully the point of view of other

scientists and must realize that they are part

of a team. They must be made aware of the

needs of other research people. Anyone in a

culture collection who does not appreciate other

areas of work should never be in the position of

decisionmaking.

In turn, the members of a culture collection

should be informed of developments in associated fermentation research areas and of problems in a fermentation plant. Currently, all

reports and papers from our Fermentation

Laboratory are circulated to all the other senior

scientists. Before papers and reports are given

at scientific meetings, the authors present them

before other members of the Fermentation

Laboratory for review and criticism. By this

means errors are detected, speaking time is

adjusted, and lastly, the staff is kept informed

on progress in related areas.

9. Although the training of curators should

be in taxonomy, the overall background of the

staff should have balance. If the collection has

more than one senior man, then the broader the

interests of the group, the better. It does no

good to have three specialists on bacteria and

yet have no mycologists, or vice versa.

In the ARS Culture Collection, we currently
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS 23

have a zymologist, two bacteriologists, two

mycologists, a plant pathologist, and a biochemist. Although a geneticist is not part of

the Collection, a microbial geneticist works

closely with the Collection members.

10. At least in larger collections, young people with new ideas and knowledge of new techniques should be brought into the group periodically. This means of rejuvenation may be

supplemented with postdoctoral fellows and exchange of personnel from other institutions.

They should not necessarily be people from

other collections. In turn, the resident staff

needs periodically to travel or study in other

laboratories.

11. Members of a culture collection should

be like playmakers in a basketball game. They

should spot profitable new ideas of fermentation research. They should be the originators of

new processes and products. On the other hand,

once a profitable research area has been discovered, they should become advisors to other

groups who are responsible for developmental

research. They should not do developmental

research beyond this point.

12. Culture-collection people should not only

be engaged in research, but they should be actively reporting their research in the form of

papers published in scientific journals, giving

lectures, and occasionally taking out initial

patents.

LOCATION OF CULTURE COLLECTIONS

The questions of how many kinds of culture

collections (agricultural, general, medical, or

reference) should be sponsored and where they

should be located are under study by the WFCC.

Our concern, which overlaps theirs, is the narrower one of where collections of industrial

micro-organisms should be situated so that they

will be accessible to, and do the most good for,

people in developing nations. Should each nation have its own collection? Will the existing

ones suffice? We think the answer to both questions is "No.''

It seems unrealistic to support the idea of

national collections of any sort because scientists trained in culture-collection science in developing countries are scarce. It seems to us

that the source of cultures of micro-organisms

should be limited to a few well-equipped collections located at various places around the

world. They need to be adequately staffed and

financially supported on a long-term basis. A

culture collection in each country would be

wholly unrealistic and unworkable. Money

spread over so many places would be utterly

wasted. On the other hand, public collections

that distribute industrial micro-organisms are

rare and often are located too far from the

emerging nations to furnish the expert assistance that is needed in handling the cultures.

Also, the cost of the cultures is prohibitive because hard currency is diflficult to obtain in

many developing areas. These are our conclusions based upon practical experience and on

frank discussions with a number of knowledgeable people from various countries.

We think that it would be good if regional

collections could be set up in strategic places

where governmental stability would allow

proper development of a collection and where

political considerations would not restrict the

free fiow of cultures and information to fermentologists in the service areas. Certainly, the

existing ones must suffice for now, and in certain

regions they can provide satisfactory service.

Thus culture collections in existence in the

United States, U.S.S.R., United Kingdom,

Netherlands, Japan, Canada, and South Africa

serve many areas. The excellent Dutch collection would, and does, fill the needs of Central

Europe. The U.S.S.R. All-Union Collection can

adequately meet the requirements of the

U.S.S.R., Poland, Romania, and Bulgaria. Wellstaffed and financed collections need to be established in South America (perhaps in Brazil or

Argentina), India, Central Africa, and the Middle East and North Africa. We do not mean to

imply that none exist in these regions, but

rather we suggest that those that do should be

enlarged in size, better equipped, and more adequately staffed. We believe that if these pro-
24 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

posed collections were established, the fermentation industry in these areas would be adequately backstopped with sources of cultures,

culture information, and technical expertise.

Existing collections, which have gained stature over the years, are associated with either a

research institute or with a university that is

famous for its fermentation studies. For example, the University of Tokyo collection is housed

in the Departments of Agricultural Chemistry

and Applied Microbiology. The famous Dutch

collection of yeasts is housed with the Institute

of Microbiology at Delft. Governments or organzations establishing new collections should

bear this kind of location in mind. A culture

collection of fermentation micro-organisms not

placed in close proximity with an active institution of fermentation and applied microbiological research would be like planting a seed on a

rock.

The question can then be justly asked, "What

should one do with a small plant producing a

given product, say a fermented food destined

for human consumption?" In this instance, the

developmental work should be done in some

central research laboratory. To ensure reliability, inoculum should be prepared and supplied

in a dry, stable form which the workers in the

plant can use to seed the fermentation to the

degree that the process will reach completion

despite contaminants. For example, in the

Bantu beer process, even the larger plants do

not keep cultures or prepare inoculum. Instead,

it is supplied to them in 1-pound packages

which a technician uses to inoculate a given

quantity of media in full confidence that he can

depend on obtaining a certain type of product

at a certain time. Little or no formal microbiology needs to be known by the plant operator.

The original starter culture can be kept in a

central culture collection and supplied to a company that makes, packages, and distributes the

inoculum.

PROCEDURES FOR ISOLATION AND SELECTION OF

MICRO-ORGANISMS FROM NATURE

Innumerable techniques for isolation of microorganisms are described in the literature. No

attempt will be made here to give specific details because they vary from group to group,

and sometimes even a special technique is required for a single species. Information can

usually be found in textbooks on microbiology

or taxonomic monographs. Currently, the Mycological Society of America has a project involving the preparation of a manual on methods for the isolation and study of all the groups

of fungi. An expert on each fungus family or

genus has prepared a section on how to isolate

and to study his group. The material was written more than 2 years ago and now is being

evaluated by graduate students to discover

which parts are workable and which must be

revised. The entire text will be edited for uniform style. Eventually, the whole will be published as a source book of information.

Techniques for isolating micro-organisms,

which can grow free of other living things, can

be classified into several general categories.

Micro-organisms are here interpreted to include

bacteria, fungi, algae, and protozoa. One of the

oldest techniques is culture enrichment. It involves the transfer of soil, sewage, or some

other material with a large and diverse population of micro-organisms into a selective medium, followed by incubation of the culture

under such conditions of temperature, aeration,

and pH, among others, that the growth of desired forms is favored. A small amount from

the initial culture is transferred to a second

culture that is established in the same manner

as the first, and this procedure is continued

until the desired flora predominates and can be

isolated in pure culture.

If a strain of Clostridium that would produce

acetone on cornmeal is wanted, a sterile corn

mash would be inoculated with soil, sewage, and

other material containing large bacterial populations. The corn mash would be kept under

anaerobic conditions at a desirable fermentation temperature, for example, 37° C. A large

number of flasks would be started. Certain ones
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS 25

that gave the appearance of vigorous anaerobic

grov^th and that had a solvent odor would be

used to inoculate new flasks. These, in turn,

would be incubated until a culture of exceptional solvent-producing ability emerged. Finally, the selected Clostridium would be plated

out on a cornmeal medium under anaerobic conditions, and many strains would be isolated as

pure cultures. Each would then be tested in the

proposed industrial fermentation for which

cornmeal is the basic ingredient. Assays of the

solvent yields would be determined, and the

best strain selected for evaluation in the scaleup of the fermentation.

Sometimes, the isolation of pure cultures is

not even necessary. In the fermentation of

cucumbers for the manufacture of pickles, conditions are established in the fermentation

tanks that favor part of the natural microbial

flora on the cucumbers. Certain bacteria grow

to the practical exclusion of all other microorganisms, and the fermentation goes to completion without resort to a pure culture. The

efficacy of pure culture starters in picklemaking is being studied.

In the native African fermentation of corn,

called magou, conditions of anaerobiosis and

temperature are so adjusted that a high-temperature, lactic acid fermentation occurs without the use of pure culture starter. Part of a

previous batch is used as starter for each new

batch.

Another general approach is to isolate a

microbial strain from the natural flora of a

choice sample of material and to use it to make

a uniformly good product. For example, during

the past 12 years in the United States a culture

of Pediococciis has been used to inoculate commercial sausage. According to information supplied by the manufacturer, a search was made

for samples of superior quality sausage. From

these sausages, a strain was selected that produced the desired fermentation ; that also could

be grown under conventional fermentation conditions and then be preserved by freeze-drying

without undue loss of vitality; and that could

initiate growth rapidly in fresh sausage despite

competing bacteria.

However, these various approaches cannot

always be used because we do not know where

to look for suitable strains or because the actual

nature of the product desired is not known. The

search for antibiotics is an example. In this instance, initial searches indicated that Streptomyces had excellent possibilities. From earlier

studies on soil microbiology, it was known that

soil, particularly grassland soil, had a great

number and variety of species. The method used

in this research was to get many different samples of soil from various geographical and ecological areas. The soils were plated out on

media suitable for the growth of StreptoMyces

and other bacteria, and myriads of strains of

actinomycetes were isolated. Selection of colonies for making pure cultures was often influenced by observation of their inhibition of adjacent bacterial or mold colonies. A multitude of

strains so selected were then tested on plates

against the target pathogen or, more often,

against a harmless micro-organism known to

be closely related to the pathogen.

Ultimately, the antibiotic from a particular

culture had to be tested against strains of the

pathogen either in vitro or in vivo. As an aside,

a search of this sort for new antibiotics now appears to be a useless activity except by possibly

the most highly skilled and experienced researchers in industrial laboratories. A better

approach is the modification of known antibiotic compounds.

The plating technique can be used to isolate

single cells or spores of practically all microorganisms that grow in laboratory media. This

technique involves the dilution and separation

of propagules of the micro-organism. These

then grow into colonies of sufficient size to be

seen with the naked eye or under a dissecting

microscope on, or in, the agar medium. Using

aseptic techniques, one can pick some or all of

a colony off the substrate and establish a pure

culture. However, with many fungi, a more

rapid and efficient technique is the isolation of

a few spores from one fructification.

Routinely in our laboratory, mold growth

that has fruiting heads on the substrate (this

growth may be on medium in petri dishes) is

used to start pure cultures. This technique can

readily be done by placing the material on

which the mold is growing under a dissecting

microscope and selecting a well-isolated fruiting
26 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

head containing mature spores. A transfer

needle with a fine straight wire (a filament

from an electric light bulb mounted in a holder

works very well) is flame-sterilized, cooled, and

moistened in sterile agar. The tip is carefully

brought into contact with the fruiting head.

The adhering spores may then be transferred

to suitable nutrient agar and a pure culture

established in a matter of a few seconds. This

technique requires considerable hand dexterity

and practice. It works well for all fungi that

produce spores on stalks. We use it routinely

for the isolation of Mucorales, Aspergillus,

Pénicillium, and the Fungi Imperfecti—including genera such as Alternaría, Cladosporíum,

and Gliocladium.

A modification of this technique is the use of

a micromanipulator to isolate single spores

from the surface of agar. It is particularly useful when all the ascospores in an ascus or the

basidiospores on a basidium are to be isolated

for genetic studies. In some yeasts and fungi,

spores are forcibly discharged, and these can

be isolated from an agar surface placed above

growing colonies.

For some fungi, especially those of the class

Basidiomycetes, spores are only produced in, or

on, large macroscopic fruiting structures.

Basidiospores may be allowed to discharge on

nonnutrient agar; then a few are transferred

to nutrient agar, and cultures become established. However, one often encounters basidiospores that fail to germinate. Frequently cultures can be made, if the fruiting body is large

enough, by carefully dissecting a small fragment of tissue from inside the sterile fruiting

body. The tissue is transferred to an appropriate medium. Since colonies will produce mycelium but no fruiting structure, extreme care

must be taken to ensure that one is not isolating

mold contaminants. There is no way of positively identifying a culture that produces sterile

mycelium.

One last technique, often combined with one

or more of the general methods described, is the

use of specific inhibitors in either liquid or solid

medium to eliminate other groups of microorganisms. Routinely, in making yeast and

mold counts of cereal products, tetracycline is

incorporated into the medium to inhibit almost

all bacteria. It has no adverse effect on the

growth of molds or yeasts. On the other hand,

actidione, an antifungi antibiotic, can be incorporated into nutrient media to inhibit both

yeasts and fungi without affecting the growth

of the bacteria. We are not aware of any combination of materials that will inhibit all bacteria and fungi but will permit the exclusive

growth of actinomycetes.

CLASSIFICATION OF MICRO-ORGANISMS USED FOR

PRODUCTION OF FERMENTATION PRODUCTS

Since this topic is reviewed in great detail in

various texts on fermentation (11, 12, IS), no

attempt will be made to discuss the microorganisms involved except to summarize the information in tabular form (table 2). Even this

summary cannot be complete because some fermentation products were, or are, made on a

limited custom basis and are not regular articles of commerce. When one considers all the

types of fermented food made all over the world,

some of which are exclusively local, it becomes

quite impossible to enumerate even a small portion of them. Also, some products once made by

fermentation are now made by chemical synthesis, especially from petrochemicals. Thus

ethanol for industrial uses is made exclusively

via this method in the United States. Other

countries with a shortage of oil, but with enormous amounts of molasses, still make ethanol

by fermentation.
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS 27

TABLE 2.—Alphabetical listing of commercial products by gentes and

kind of micro-organisms

Kind of

Product Genus micro-organism

AMINO ACIDS

Aspartic acid Pseudomonas Bacteria.

Glutamic acid (monosodium glutamate) Bacillus Do.

Do Brevibacterium Do.

Do MicrococcMs Do.

Isoleucine Pseudomonas Do.

Lysine Micrococcus Do.

Phenylalanine do Do.

Threonine Bacillus Do.

Valine Micrococcus Do.

ANTIBIOTICS

Various products Aspergillus Mold.

Do Bacillus Bacteria.

Do Cephalosporium Mold.

Do Fusidium Do.

Do Micromonospora Actinomycetes.

Do Nocardia Do.

Do Pénicillium Mold.

Do Streptomyces Actinomycetes.

BEVERAGES

Beer Saccharomyces Yeast.

Distilled spirits do Do.

Sake Aspergillus Mold.

Do Saccharomyces Yeast.

Wine do Do.

ENZYMES

Amylase (EC 3.2.L1)^ Aspergillus Mold.

Do Bacillus Bacteria.

Do Endomycopsis Yeast.

Do Rhizopus Mold.

Amyloglucosidase {see Glucoamylase)^

Asparaginase (EC 3.5.1.1) Erwinia Bacteria.

Do Escherichia Do,

Catalase (EC 1.11.1.6) Aspergillus Mold.

Do Pénicillium Do.

Cellulase (EC 3.2.1.4) Aspergillus Do.

Do Myrothecium Do.

Do Trichodenna Do.

Dextranase (EC 3.2.1.11) Pénicillium Do.

/3-Fructofuranosidase (EC 3.2.1.26) Saccharomyces Yeast.

j3-Galactosidase (EC 3.2.1.23) do ^__Do.

Glucoamylase (EC 3.2.1.3) Aspergillus Mold.

Do Endomycopsis Yeast.

Do RhizopTis Mold.

See footnotes at end of table.
28 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

TABLE 2.—Alphabetical listing of commercial products by germs and

kind of micro-organisms—Continued

Kind of

Product Genus micro-organism

ENZYMES—Continued

Glucose isomerase (see Xylose isomerase)

Glucose oxidase (EC 1.1.3.4) Aspergillus Mold.

Do Pénicillium Do.

a-Glucosidase (EC 3.2.1.20) Aspergillus Do.

/3-Glucosidase (EC 3.2.1.21) do Do.

Hemicellulase (see Xylanase)

Invertase (see /8-Fructofuranosidase)

Lactase (see /3-Galactosidase)

Laundry enzymes (see Proteases, alkaline)

Lipase (EC 3.1.1.3) Aspergillus Mold.

Do Candida Yeast.

Do Mucor Mold.

Milk-clotting enzymes (see Rennin)

Pectinase (see Polygalacturonase)

Penicillinase (EC 3.5.2.6) Bacillus Bacteria.

Penicillin amidase (EC 3.5.1.11) Many micro-organisms

Pentosanase (see Xylanase)

Plasmin (EC 3.4.4.14) Streptococcus Bacteria.

Polygalacturonase (EC 3.2.1.15) Aspergillus Mold.

Proteases (EC 3.4.4) do Do.

Do Conidiohohis Do.

Do Mucor Do.

Do Streptomyces Actinomycetes.

Proteases, alkaline Bacillus Bacteria.

Rennin (EC 3.4.4.3) Endothia Mold.

Do Mucor Do.

Streptokinase (see Plasmin)

Xylanase (EC 3.2.1.8) Many micro-organisms

Xylose isomerase (EC 5.3.1.5) Streptomyces Actinomycetes.

FOODS

Ang-kak Monascus Mold.

Bacterial starters (fermented dairy

products and sausage) Lactohacillus Bacteria.

Do Leuconostoc Do.

Do Pediococcus Do.

Do Propdonibacterium .— Do.

Do Streptococcus Do.

Bantu beer Lactohacillus Do.

Do Saccharomyces Yeast.

Blue-cheese flavor Pénicillium Mold.

Bread (bakers' yeast) Saccharomyces Yeast.

Cheese and fermented dairy products Lactohacillus Bacteria.

Do Pénicillium Mold.

Do Propionihacterium, Bacteria.

Do Streptococcus Do.

Chinese yeast Chlamydomucor Mold.

Do Hansenula Yeast.

Do Rhizopus Mold.

Do Saccharomyces Yeast.
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS 29

TABLE 2.—Alphabetical listing of commercial products by genus and

kind of micro-organisms—Continued

Product Genus

Kind of

micro-organism

FOODS—Continued

Fermented fish Aspergillus

Do Unidentified

Hamanatto AspergilltLs _.

Koji do

Do Rhizopus

Magou Lactobacillus

-Mold.

_ Halophilic

bacteria.

-Mold.

-Do.

_Do.

_ Bacteria.

Miso

Do Saccharomyces

Nata Acetobacter

Natto Bacillus

Ontjom Neurospora

Pickles and sauerkraut Lactobacillus _

Do Streptococcus _

Shoyu Aspergillus

Do Saccharomyces

Do Torulopsis

Sufu Actinomucor __

Do Mucor

Tempeh Rhizopus

Yeast ..Candida

Do Saccharomyces

-Aspergillus Mold.

-Yeast.

-Bacteria.

-Do.

-Mold.

_ Bacteria.

-Do.

-Mold.

-Yeast.

-Do.

-Mold.

-Do.

-Do.

-Yeast.

_Do.

INDUSTRIAL SOLVENTS

Acetone Clostridium ^

Butanol do

2,3-Butanediol Aerobacter

Do Bacillus

Dihydroxy acetone Acetobacter _ Ethanol . Clostridium

Do Saccharomyces

Glycerol do

Do Torulopsis

-Bacteria.

-Do.

Do.

-Do.

Do.

-Do.

-Yeast.

-Do.

-Do.

MISCELLANEOUS

Alkaloids Claviceps

Bioinsecticides Bacillus

Dextran Leuconostoc

Gibberellin Gibberella (Fusarium)

Inosinic acid Bacillus

Do (Several genera)

Nucleotides Brevibacterium

Do Candida

Rhizobium cultMYQ Rhizobium ...

Silage Lactobacillus

Sorbose Acetobacter _

Steroid transformations . Aspergillus

Do Corynebacterium

Do Curvularia .

Do Rhizopus

Do Streptomyces

-Mold.

- Bacteria.

-Do.

-Mold.

-Bacteria.

- Yeast.

_ Bacteria.

-Yeast.

-Bacteria.

Do.

-Do.

-Mold.

- Bacteria.

Mold.

_Do.

- Actinomycetes.
30 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

TABLE 2.—Alphabetical listing of commercial products by genus and kind of micro-organisms

—Cont

Product Genus

Kind of

micro-organism

ORGANIC ACIDS

Acetic Acetobacter Bacteria.

Citric Aspergillus Mold.

Erythorbic Pénicillium Do.

Fumaric Rhizopus Do.

Gluconic Aspergillus Do.

Itaconic do Do.

Itatartaric do Do.

2-Ketogluconic Pseudomonas Bacteria.

5-Ketogluconic Acetobacter Do.

a-Ketoglutaric Pseudomonas Do.

Kojic Aspergillus Mold.

Lactic Lactobacillus Bacteria.

PROTEIN

Various products Chlorella Algae.

Do Saccharomyces Yeast.

Do Torula Do.

VITAMINS

Ascorbic acid (vitamin C) (in part) Acetobacter Bacteria.

Bia Bacillus Do.

Do Propionibacterium Do.

Do Streptomyces Actinomycetes.

/3-Carotene , Blakeslea Mold,

Riboflavin Ashbya Yeast.

Do Eremothecium Do.

1 Enzymes are identifíed by their code numbers, the key to classification as assigned by the Commission on Enzymes, International Union

of Biochemistry (6).

^ Included in this alphabetical list of enzymes are those names faigiiliar to microbiolosrists but no longer recommended by the "Enzyme

Commission for use. Recommended and familiar names are cross-referenced.

PROBLEMS IN MAINTAINING STABLE INDUSTRIAL CULTURES

From years of experience in handling cultures of all kinds, we believe that certain steps

should be taken as soon as a micro-organism is

isolated in pure culture to ensure it remains in

a stable state. These steps need to be taken as

soon as the culture is brought into the laboratory or isolated, long before its potential or lack

of potential is known.

1. The culture is examined under a dissecting microscope to determine : (a) That the culture is growing uniformly; (b) that it is free

from other micro-organisms; (c) that if it is a

mold, mature spores are present; and (d) that

the culture appears to be the genus and perhaps

species isolated or named when received. This

examination will then determine the next step

to be taken.

2. If the culture appears to be pure, shows

vigorous and uniform growth, and has mature

spores, three to five ampules of the microorganism should be lyophilized immediately.

8. If the strain is known to be a member of

a species or genus in which lyophilization is

always successful, no viability check is needed.

However, it is good practice, even with such

strains, to sacrifice one lyophil tube and to di-
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS 31

lute or streak the culture out onto a suitable

growth medium. The check on viability will

show three things : (a) If the propagules have

survived in large numbers; (b) if the lyophil

preparations are free of other micro-organisms; and (c) if the regenerated culture is still

uniform in growth and sporulation. If the

strain being preserved has not been lyophilized

before, the viability check is a must because

some micro-organisms fail to survive this process. In case of failure, an alternative method

must be found while the first generation culture

is still available and in a healthy state.

4. If the culture is recovered successfully

from lyophil or from a preparation preserved

by another method, such as freezing in liquid

nitrogen, records should be kept of the proper

medium for growth and sporulation, as well as

for any special requirements, such as temperature of incubation, length of incubation, or pH.

For example, the mold Blakeslea trispora will

sporulate rapidly (3 to 4 days at 25° C), but

often within 10 days the spores will germinate

in place. Lyophilization subsequent to this

occurrence will be a complete failure. The

process works beautifully if the spores are processed when they have just reached maturity.

5. The lyophil tubes should be stored at 4°

to 10° C. and, perhaps, checked for viability at

the end of each 10 years of storage.

6. At the time of lyophilization, the culture

should be examined in the appropriate way to

determine its identity. Sometimes this examination may lead to species and variety identification, but other times it leads only to the approximate species and genus. The records

should certainly show its approximate identity

because (a) it allows the person reviving the

culture, perhaps years later, to know what was

preserved and (&) it makes the records more

complete and, therefore, more useful. In some

collections of fungi, a microphotograph is made

of the fungus at the time of identification. This

is an excellent type of record.

7. At the same time that lyophil preparations are made, records should be completed

showing the following items : (a) The name of

the organism; (b) where obtained—whether it

was isolated in a laboratory or received from

another microbiologist and, if the latter, his

name and address; (c) accession number assigned and any other designation given to it,

such as a temporary number or other laboratory or collection number; (d) location and

original source of the material (where was the

organism found in nature) ; (e) special requirements—medium of maintenance, optimum temperature, and other conditions ; (/) products or

unique properties and approximate yields; (g)

number of cultures made; and (h) references

if strain is cited in a paper or patent. Rarely

can all this information be assembled. With

time, additional information will be needed for

the record. The data can be placed on? cards,

which should be cross-indexed so that one can

find a culture by number, source, product, and

name. Some collections are being indexed for

computer sorting. With a large collection, such

as ours (35,000+ strains), to put this data

onto cards now would be a Herculean task.

Records should be kept showing who uses a

given strain in the laboratory and to whom it

is sent in laboratories at other institutions. This

information will be useful if it should become

necessary to obtain a replacement of a culture

which died or degenerated, but which may still

be in the original state in someone else's laboratory. Written and dated records are also very

essential for industrial strains that may become involved in patent and legal cases. For

this reason, we ask anyone requesting cultures

from our collection to put his request in writing. When the culture is sent, a letter to the

requester is prepared by the appropriate curator as a matter of written record.

8. If the culture does not meet the requirements set forth in item 2, the following steps

are taken : If the culture is pure, but shows sectoring, then the nonuniform culture is lyophilized. Also, the various forms are isolated separately, and each type is lyophilized individually.

Sometimes a heterogeneous culture cannot be

separated into its components. The philosophy

of lyophilizing the sectoring culture is to try to

preserve all the component parts because typically, at this stage, you do not know which part

you actually may need later.

If the culture is impure, methods must be

used to rid the culture of its contaminants

either by dilution and picking an isolated col-
32 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

ony or by picking one or a few spores from a

fruiting head. Occasionally, two or more organisms are associated (mixed culture) as, for instance, in koji starters, where it may be necessary to lyophilize the total starter as well as its

components.

9. Frequently, two different methods of

preservation should be used at the same time.

For instance, we still carry some of our fungus

cultures on agar slants with periodic transfers,

as well as in lyophil. Oiled and soil cultures are

other possibilities. The former consists of agar

slant cultures covered with sterile mineral oil.

In the latter, spores are placed in sterile soil or

sand and allowed to dry. Details of preservation techniques require too much space to describe them here.

10. If a culture that has degenerated is received, certain steps must be carried out to obtain a better one. Perhaps the ability to sporulate is deteriorating; often a series of dilution

plates will produce some colonies that grow

more vigorously or sporulate more heavily in a

natural manner. In some cultures, especially

molds, the isolation of spores from individual

heads may lead to better cultures. In others, the

fault may be the medium or the growth conditions. Many Aspergilli and Penicillia grow normally and vigorously on a synthetic medium.

However, even though some cultures grow and

fruit on a synthetic medium, they will do much

better if the medium contains organic nitrogen

and growth factors in the form of malt or yeast

extract. This response may represent a better

form of nitrogen, or it may reflect a partial

vitamin deficiency that has been overcome.

We believe that it is appropriate to list a number of principles which we consider important

for the cultivation of micro-organisms in order

to ensure vigorous, healthy, and stable starters :

1. For the maintenance of stock cultures, a

chemically undefined, but reproducible, stock

medium is better than a synthetic one. A microorganism, as it occurs in nature, is almost always growing on an undefined substrate. A

defined medium will more likely select a certain

part of the genetic population. The result may

well lower the yield of the desired product.

2. In general, a stock culture medium should

be no more nutritionally rich than is required

to perpetuate the culture without change. Thus

glucose (or other sugar) is customarily excluded, or if glucose is essential (as it is for

lactic acid bacteria), a buffer is incorporated

to control the pH. If the pH were allowed to

drop, the longevity of cells might be endangered. Worse yet, the population imbalance

mentioned in number 1 might reduce or destroy

the usefulness of the culture. Appropriate

media for use with a variety of micro-organisms are given in a paper entitled "Maintenance

of cultures of industrially important microorganisms" (8).

3. Stock cultures are usually subjected to

two different sets of conditions. First, they are

encouraged to grow rapidly and vigorously for

a relatively short time by incubating them at or

near their optimum temperatures and, if they

are aerobic, allowing them free access to air.

Then they are induced to slow down metabolically by storing them for a comparatively long

time in a refrigerator and sometimes also by

limiting their access to air by stoppering test

tubes and flasks and sealing petri dishes. Stoppering also hinders loss of moisture from the

culture. These variations are not too much

different from those they encounter in nature.

Regardless, they seem not to harm the microorganisms.

The pH of the medium is also important.

Generally, bacteria are grown in neutral media ;

molds are grown in media that have a pH between 6 and 7; and yeasts, in the vicinity of

pH6.

4. When new cultures are started, inoculum

is taken from a mature culture. It consists of a

small amount of growth of yeasts or bacteria

or, for molds, a few spores without mycelium.

PROBLEMS OF STRAIN DEGENERATION AND LOSS

In looking back on our experiences involving

the loss of pure cultures, five causes come to

mind: (1) Contamination by other microorganisms, (2) infestation by mites, (3) phage

infestations, (4) natural selection and mutation, and (5) untrained staff.
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS 33

Contamination By Other Micro-organisms

We have encountered many cultures reputedly pure, but they carried a second microorganism never separated from the original

culture at the time of isolation. This situation

is particularly true when colonies are picked

from dilution plates in which an inhibitor was

placed in the medium to control bacterial

growth. Often colonies growing on the surface

of the agar plates with tetracycline as a bacterial inhibitor appear to be well isolated and

pure. When the colony is picked off, however,

dormant bacterial cells are removed with it.

When placed on a medium free of the inhibitor,

they grow again. A bacterial culture producing

a thin growth may be obscured by the more

luxuriant growth of a mold.

A common cause of contamination is the storage of cotton-plugged, agar slant cultures in

refrigerators. Often in the summer the air is

warm and moist. When the refrigerator door is

opened, moist air enters and upon cooling condenses on labels and cotton plugs. Some Penicillia can grow on moist cotton and the labels

at 4° to 5° C. in a few weeks. When they do, the

conidia present on the tubes make pure culture

transfers all but impossible. If sufficient time is

allowed, mycelial growth of the Penicillia will

penetrate the cotton plug where they sporulate,

and conidia will then drop onto the surface of

the agar and develop new colonies.

Infestation By Mites

Certain species of mites feed on fungus

spores. These mites are extremely small and

barely can be seen with the naked eye. They

occur in nature in decaying plant material and

are worldwide in distribution. When an active

program of isolating fungi from soil, humus,

or moldy plant material is going on, these

animals are often present as adults or eggs.

If care is not taken, the mites will travel from

the contaminated material into petri dishes and

test-tube cultures. They invariably carry various mold spores on them, and they appear to

be attracted by the odor of certain mold species.

Even though cotton plugs are a good barrier

to mold spores, the fungus mites traverse the

cotton plugs into pure cultures unless the cotton

plugs are poisoned. Besides contaminating a

culture, they will lay eggs that hatch in a few

days, and the young will migrate into new

cultures. In a short time, hundreds of cultures

are contaminated, and a whole collection may

be lost. If infestation has not spread too far, the

contaminated cultures may be destroyed, but

many other cultures that appear to be pure will

contain mites, and the contamination will

reoccur.

Once mites are introduced from natural

material or have been introduced from cultures

deposited in the collection, they are difficult to

control. The best solution is to prevent mites

from becoming free in the laboratory by

quarantining all suspected, contaminated material in a location away from stock cultures.

The same precaution should be taken with

respect to cultures received from outside the

culture collection. The further precaution of

poisoning all cotton plugs of stock cultures

should be taken.

Phage Infestations

Some strains of bacteria and actinomycetes

carry phage in one form or another. Generally,

these are difficult to detect, and it is even harder

to free the culture from them. At one time,

yeasts and mold species were believed never

to be infected with phage, but this belief is now

known to be false.

Natural Selection And Mutation

Changes in the genetic population in a culture will occur in all micro-organisms. Our

personal belief is that some of these changes

may be prevented by the use of more natural

media. For example, in our collection of

Mucorales, no change appears to occur if the

cultures are maintained on a potato-dextrosesalts medium. Some of the Mucorales will develop sectoring and sterile growth if cultures

are carried on a synthetic medium.

Culture rundown frequently occurs in some

species of Pénicillium and Aspergillus. Once

this process has gone to a certain phase, it is

impossible to regain the original form. For
34 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

example, some three culture lines of the type

strain of A. parasiticus NRRL 502 are poor

aflatoxin producers, but the same strain carried

under different cultural conditions in two other

laboratories over many years is still a good

producer of the mycotoxin.

Untrained Staflf

Probably as serious as any cause of culture

failure is the handling of cultures by untrained

persons. Often the media are improperly

sterilized by people who do not comprehend

that some complex media require more heat

than others and that the larger the amount of

medium, the more sterilization is needed. Sometimes inadequately trained people just do not

know how to transfer cultures to avoid contamination. Often they transfer a large mass

of spores and mycelium of a mold causing the

whole work area in the transfer room to be

filled with spores suspended in the air and on

the table tops. Only a few spores attached to

sterile agar on a transfer needle are needed to

start new stock cultures. Some microbiologists

do not recognize even the micro-organisms they

are working with. One prominent microbiologist has estimated that from one-third to onehalf of all the work published on bacterial

physiology has been done with contaminated

cultures or with the wrong species !

PHYSICAL CONDITIONS AFFECTING MICRO-ORGANISMS

The physical conditions that aflfect the

growth and longevity of micro-organisms are

the same as those that inñuence other forms of

life ; viz., pH, temperature, light, humidity, pressure, oxidation/reduction potential, surface

tension, and radiations. In the context of this

discussion, we are interested in the effect of

these factors on the survival of microbes in

culture collections and while the organisms are

in transit.

In most modern culture collections, stock

strains are carried as lyophilized (freeze-dried)

cultures. Essentially to lyophilize a culture,

microbial cells, spores, or, sometimes, portions

of mycelium are suspended in a protective colloid—such as blood serum or skim milk—

quickly frozen at about —40° C, and dehydrated by allowing sublimation of moisture

in vacuo. The dried preparation is sealed under

vacuum and stored, usually in a refrigerator

at 5° to 10° C.

In the lyophilized state, microbes take on

some of the properties of bacterial endospores

becoming less suspectible to extremes of temperature, dryness, and radiations. They are

safe from contamination, changes in pressure,

pH, humidity, oxidation/reduction potential,

and surface tension. They can be shipped by

land, sea, and air in temperate, tropical, or

frigid climates without loss of life or change in

character. (See reference 5 for a review of

methods.)

Some micro-organisms cannot withstand

lyophilization and must be maintained by other,

less convenient, means. One that has come into

use in recent years, and that has some of the

advantages of lyophilization, is preservation by

freezing and storage in and over liquid nitrogen

(-165° to -195° C). The full range of microbial types that can be preserved in this manner

is not yet known, but many fastidious forms

that fail to survive lyophilization have remained viable for long periods in liquidnitrogen refrigerators. For instance, some

fungus cultures are reported still viable and

apparently unchanged after 5 years in a liquidnitrogen refrigerator (10).

Ultra-low-temperature frozen cultures are

sealed in glass vials or ampules so that they

have essentially the same protection as lyophilized preparations against contamination and

changes in the physical environment. However,

they must be shipped in special trucks, freight

cars, or in portable, liquid-nitrogen refrigerators because it is only while they are kept at

-165° to -195° C. that they are guarded

against damage. Although this method is less

convenient than lyophilization, it is becoming

more common because it is still better than

alternative methods.
SOURCES AND MANAGEMENT OP MICRO-ORGANISMS 35

Use of alternative methods is still inescapable

for microbial cells that cannot be preserved by

either of the two techniques already discussed.

These alternatives have been in use for many

years although more time consuming and subject to hazards that are minimal or absent in

the other two methods. Basically, there is a

single technique but with modifications. It is

the serial transfer method by which some

growth (vegetative cells, spores, mycelium,

tissue) is transferred from one culture (agar

slant, agar stab, agar plate, broth, tissue culture) to fresh medium, allowing the new culture to grow under optimum conditions to

maturity, storing the new stock culture for a

time, and then repeating the cycle.

Storage usually is in a refrigerator (5*^ to

10° C), but sometimes it is at room temperature. The length of time between transfers

varies, depending upon the nature of the strain,

from one or a few days to several months or

even years. Often the interval may be lengthened by preventing dehydration of stock cultures by covering them with mineral oil (oiled

cultures) or by closing the cultures with rubber

stoppers, corks, or by impregnating the cotton

plugs with paraffin. The rate of growth is

slowed by refrigerating the cultures. This step

minimizes changes in pH and in oxidation/

reduction potential, and it also reduces the

danger that predominance in the population

will be gained by the progeny of cells mutated

by stray radiations (cosmic rays).

These are the principal methods of maintaining and preserving cultures, and they all

succeed to some degree in minimizing damage

to, or loss of, life of cultures by inimical physical conditions.

REGULATIONS REGARDING DEPOSIT OF CULTURES FOR PATENT PURPOSES

One activity in which many of the larger

culture collections become involved is the handling of cultures of micro-organisms deposited

in connection with patent applications. In some

countries it is desirable to deposit a culture,

not necessarily a high producer of a product,

with a recognized culture collection. This deposit is to ensure that a process being patented

is fully disclosed to the public. In other words,

a fermentation process is not considered fully

operable until a culture is available for use in

the process.

Over the years, the Northern Regional

Research Laboratory (NRRL) has developed

guidelines regarding this culture-collection

activity. They are based on considerable experience and also on consultations with inventors,

companies, and patent lawyers. These guidelines are updated from time to time and are

not to be construed as being final. The latest

revision was on November 9, 1971.

PROCEDURES AND POLICIES FOR DEPOSITION OF CULTURES FOR

PATENT PURPOSES IN THE ARS CULTURE COLLECTION

The ARS Culture Collection serves as a

depository for cultures that are involved in

fermentation patents and, therefore, will be

glad to receive such a culture in connection

with a patent application. When such a culture

is received, it is assigned a number in the collection and is maintained thereafter in a living

state. Immediately after receipt, a letter is

written to the depositor advising of the

number assigned and including the following

statement :

". . . Furthermore, insofar as is practicable in carrying out the business of

the Department of Agriculture, we

shall refrain from distributing this

culture pending the issuance of the

United States patent to your company, with the exception, however,

that access to this culture by other

parties will be granted upon receipt

of written authorization from your

company specifying the name and the
86 AGRICULTURE HANDBOOK 440, U.S. DEPT. OP AGRICULTURE

ARS Culture Collection designation

(NRRL number) of the culture and

identifying the party who is to receive it."

More recently, some depositors have requested replacement of the paragraph above

by a simple statement such as :

"As of this date, the subject culturéis) will be made available to

anyone who requests the same."

It is suggested that advice be sought from an

attorney as to which type of statement should

be used. Either one of these statements will be

written, depending upon the wishes of the

depositor. The ARS Culture Collection letter

then can be attached to the patent application

for the Patent Examiner.

Curators in the ARS Culture Collection do not

attempt to make an identification or to name

any organism that has been deposited in connection with a patent application, nor do they

carry out research work with such deposits

until a U.S. patent issues or cultures are otherwise released. It is not necessary, of course, to

provide a precise identification, but the microbiologist concerned should at least state to what

genus the micro-organism belongs. Also, if special media are required for its maintenance,

the curators need to know this. Ordinarily, one

or two agar slant cultures, one lyophilized

preparation, or both, are received from depositors. Depositors also are responsible for resupplying material should the need ever arise.

The depositor has the option of sending

cultures for deposit in the ARS Culture Collection in three ways :

1. Thirty lyophilized preparations, clearly

labeled with the depositor's original strain

designation and in tubes no longer than 2

inches. One of these is checked for viability, the

NRRL designation is placed on each tube, and

the supply of tubes is stored at 3° to 5° C.

Bona fide letter requests for the culture would

be shipped from this stock.

2. One lyophilized preparation, clearly

labeled with the depositor's original strain

designation. On receipt, the micro-organism is

cultivated on appropriate agar media and 30

lyophilized preparations made. One of these is

checked for viability, the remainder handled

as in option 1.

3. One, or preferably two, agar slant cultures of the micro-organism growing on an

appropriate medium. SuflScient material is prepared by our curators to make 30 lyophilized

preparations; one is checked for viability, and

the remainder are handled as in options 1 and

2. When the initial agar slant cultures deposited

appear suitable, lyophilizations often are made

from that material.

There is no charge for the deposit or maintenance of cultures.

Cultures deposited in connection with patent

applications may be obtained, free of charge, by

letter request stating the name of the microorganism and the ARS Culture Collection

strain designation (NRRL number).

The ARS Culture Collection does not issue a

catalog or list. It has no regulations imposing

restrictions on the use of such cultures deposited for patent purposes. Such materials are

distributed according to the depositor's wishes

which, in turn, generally are based on his interpretation of Patent Office requirements. Use

of such materials, once distributed, are the

responsibility of the requestor. Cultures are

automatically removed from any restrictive

category, once a U.S. patent issues in which

the particular micro-organism is involved.

SHIPMENT OF MICRO-ORGANISMS

Living cultures of micro-organisms are items

of international commerce. Every year many

thousands of strains are transported by land,

sea, and air to scientists on every continent,

with the possible exception of Antarctica. Most

cultures are dispatched from large culture collections, such as the American Type Culture

Collection in the United States, the various

national collections in England, and the Institute for Fermentation in Japan. Many strains

are also distributed by small, specialized collections and by individual scientists who maintain
SOURCES AND MANAGEMENT OF MICRO-ORGANISMS 37

a few micro-organisms, primarily for their own

research. In the course of time, the large collections have learned to solve the problems associated with packaging and shipping living cultures so that they arrive at their destinations

whole, alive, and unchanged.

Except for micro-organisms used in the

pharmaceutical industry to produce vaccines

and antisera, very few of the microbes used in

the food, feed, and fermentation industries and

carried in the mails endanger public health or

agriculture. In the United States, and presumably in other nations, nonpathogenic cultures are virtually immune from legal restrictions on their movement from laboratory to

laboratory. However, in the United States and

very likely in other countries, a number of laws

apply to the import, export, and internal transport of "etiological agents and vectors." These

are discussed in a brochure published in 1970

(1) by the American Type Culture Collection.

Six Departments of the U.S. Government—

Agriculture ; Commerce ; Health, Education,

and Welfare; Transportation; Treasury; and

the U.S. Postal Service are listed as those concerned with regulating potentially dangerous

micro-organisms.

In the U.S. Department of Agriculture, the

Animal Plant Health and Inspection Service's

(APHIS) Animal Health Programs are

charged with responsibility to see that "no

organisms (which may introduce or disseminate any contagious or infectious diseases of

animals, including poultry) . . . shall be

imported into the United States or transported

from one State ... to another State . .. without

a permit issued by the Secretary and in compliance with the terms thereof." APHIS'

quarantine inspection programs require a permit for the movement of any plant pest into or

through the United States or any of its territories and possessions. "Plant pest," as defined

in the Federal Plant Pest Act, includes microbial cultures which can directly or indirectly

injure or cause disease or damage in plants.

The primary concerns of the Department of

Commerce, as it relates to distribution of living

cultures, are safeguarding our national security

and furthering our foreign policies. It has

published a "general license" that authorizes

the export of living cultures to most destinations. Special "validated" licenses are required

for the export of living cultures to a few

nations.

The Public Health Service of the U.S.

Department of Health, Education, and Welfare,

through its Foreign Quarantine Program

(Center for Disease Control), promulgates and

enforces regulations to "prevent the introduction and spread of communicable disease from

foreign countries into the United States . . .,

or from one state to another." It operates under

a law which states, "A person shall not import

into any place under the control of the United

States, nor distribute after importation, any

etiologic agent ... of human disease . . . unless

accompanied by a permit issued by the Surgeon

General."

Shipments of living cultures are free of

customs duty. The principal reasons that the

Bureau of Customs of the United States

Department of the Treasury is involved are :

1. To determine from documentation and

inspection at ports and airports of entry if

shipments are controlled by Federal law and

regulations enforced by other Government

agencies.

2. To select required samples for referral to

enforcement agencies.

3. To withhold release of shipments during

examination of referred samples and pending

issuance of permits or licenses that are required

as a condition of release.

Regulations of the U.S. Postal Service and

the Department of Transportation specify how

pathogenic micro-organisms shall be packaged.

The intent of these instructions is the same as

those governing permits and licenses; that is,

to ensure that etiological agents do not escape

and endanger public health and agriculture.

The reasons we have detailed the United

States agencies and their requirements are that

we suspect other nations either have similar

regulations or will ultimately pattern theirs

after those of the United States. It behooves

culture collection curators to be cognizant of

such laws, if they wish to avoid difficulty and

delay in receiving and sending cultures.

In perusing culture catalogs from several of

the larger collections, we found only three that
88 AGRICULTURE HANDBOOK 440, U.S. DEPT. OF AGRICULTURE

make any mention of possible need for licenses

and permits. One is the Catalogue of Strains of

the American Type Culture Collection (2).

Another is the Catalogue of the Culture Collection of the Commonwealth Mycological Institute (i). The third is the List of Cultures of

the Centraalbureau voor Schimmelcultures (S).

Inasmuch as no mention is made in catalogs

of other collections (Argentina, Czechoslovakia,

England, Germany, India, Indonesia, Japan,

Netherlands, Scotland, U.S.S.R.) about the

need for licenses, permits, or customs arrangements, we suspect that either such requirements in countries other than the United States

and Canada do not exist or else enforcement is

ineffective. If true, curators need concern themselves only with safe packaging and labeling

of cultures destined for most nations.

Problems to be overcome in packaging are

the selection of a sturdy mailing tube or carton

that will remain intact, despite rough handling

and possible exposure to moisture, and that will

protect the enclosed cultures from breakage.

Additionally, the container must be so made

that, if despite all precautions breakage does

occur, the released micro-organisms cannot

escape to the outside.

Although the requirements delineated may

seem formidable to the uninitiated, curators

ordinarily have little or no trouble in obtaining

cultures from anywhere in the world or in

sending cultures to anyone who has a legitimate

need for them.

LITERATURE CITED

(1) ALEXANDER, M. T., and BRANDON, B. A.

1970. THE PACKAGING AND SHIPPING OF LIVING

REFERENCE CULTURES. ATCC Pub. No. 1,

26 pp., American Type Culture Collection,

Rockville, Md. 20852.

(2) AMERICAN TYPE CULTURE COLLECTION.

1970. CATALOGUE OF STRAINS. Ed. 9, 243 pp.

Rockville, Md. 20852.

(3) CENTRAALBUREAU VOOR SCHIMMELCULTURES.

1968. LIST OF CULTURES. Ed. 27, 262 pp. Baarn,

Netherlands.

(4) COMMONWEALTH MYCOLOGICAL INSTITUTE.

1968. CATALOGUE OF THE CULTURE COLLECTION OF

THE COMMONWEALTH MYCOLOGICAL INSTITUTE. Ed. 5, 162 pp. Kew, Surrey, England.

(5) FENNELL, D. I.

1960. CONSERVATION OF FUNGOUS CULTURES. Bot.

Rev. 26: 79-141.

(6) FLORKIN, M., and STOTZ, E. H., eds.

1965. ENZYME NOMENCLATURE. Ed. 2, VOl. 13.

In Comprehensive Biochemistry. New

York.

(7) HAYNES, W. C.

1963. DISCUSSION II. (on "The organization of a

type culture collection,'* by Shewan, J. M.,

Torry Research Station, Aberdeen.) In

Martin, S. M. (ed.). Culture Collections:

Perspectives and Problems. Proc. Specialists' Conf. on Cult. Collect., August 1962,

pp. 36-39, Ottawa.

(8) HAYNES, W. C, WICKERHAM, L. J., and

HESSELTINE, C. W.

1955. MAINTENANCE OF CULTURES OF INDUSTRIALLY IMPORTANT MICROORGANISMS.

Appl. Microbiol. 3: 361-368.

(9) HESSELTINE, C. W., HAYNES, W. C,

WICKERHAM, L. J., and ELLIS, J. J.

1970. HISTORY, POLICY, AND SIGNIFICANCE OF

THE ARS CULTURE COLLECTION. In lizuka,

H. I., and Haseg;awa, T. (eds.). Culture

Collections of Microorganisms. Proc. Internatl. Conf. on Cult. Collect. October

7-12, 1968, pp. 21-38. University of Tokyo

Press, Tokyo.

(10) HWANG, S. H.

1966. LONG-TERM PRESERVATION OF FUNGUS CULTURES WITP LIQUID NITROGEN REFRIGERATION. Appl. Microbiol. 14: 784-788.

(11) MARTIN, S. M., ed.

1972. WORLD DIRECTORY OF COLLECTIONS OF CULTURES OF MICROORGANISMS. 560 pp. NeW

York.

(12) PEPPLER, H. J., ed.

1967. MiCROBiAL TECHNOLOGY. 454 pp. New York.

(13) PRESCOTT, S. C, and DUNN, C. G.

1959. INDUSTRIAL MICROBIOLOGY. Ed. 3, 945 pp.

New York.

(14) SMITH, G.

1969. AN INTRODUCTION TO INDUSTRIAL MYCOLOGY. Ed. 6, 390 pp. New York.

1VU.S. GOVERNMENT PRINTINQ OFFICE: 1973—0—478-751