VOL. 5 SUMMER 1969 No.2 ,, .'

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I -·,~ ~

MICROBIOLOGICAL. CORROSION OF METALS- MARINE WOOD .r· BORERS- RODENT ATTACKS ON STORED PRODUCTS- FOULING OF !' SHIPS BY BARNACLES- DETERIORATION OF STONE BY BACTERIA­ , ., ... ROTTING OF WOOD BY FUNGI- BACTERIAl:: BREAKDOWN OF

~ ,. ~ ASPHALT- MILDEWING OF LEATHER -INSECT DAMAGE TO BOOKS ' -BIRD HAZARDS TO AIRCRAFT- FUNGI IN JET FUEL TANKS­ • I TERMITES IN TIMBER- MICROBIOLOGICAL ATTACK ON RUBBERS ~• .. ~ ·~' PLASTICS AND PAINTS,- FUNGAL ETCHING OF GLASS ....

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I • ~ • 'f I I NTERNAT/ONAL

I ' l •j •, BIDDETERIORAT/ON I .. ! t i; ~ i BuLLETIN l L ' _, '·~: •• if.' , ?..' • •

, . ~ # A QUARTERLY JOURNAL OF BIODETERIORATI ON '!

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r . BIODETERIORATION INFORMATION CENTRE THE UNIVERSITY OF ASTON IN BIRMINGHAM

ENGLAND ,. . i ' CATOMANCE LIMITED manufacturers of mystox* for the preservation of , timber, textiles paper, cordage plastics and specialised applications

*mysmx is the registered trade mark of Catomance Limited 94 BRIDGE ROAD EAST, WELWYN GARDEN CITY, HERTS., ENGLAND. Telephone: Welwyn Garden 24373/8 '

I VolumeS Summcr1969 Number2

INTERNATIONAL

BIODETERIORATION

BuLLETIN I ' t l I (

I CONTENTS I t I I 37-38 j NEWS AND COMMENT THE USE OF ANTIBIOTICS IN FOOD PRESERVATION 39-61 I B. Jarvis and M. D. Morisetti

EXPERIENCES WITH BIOLOGICAL TESTS IN THE FIELD OF BIODETERIORATION OF MATERIALS. 3. SOME PRINCIPLES OF THE TESTING OF MATERIAL PROTECTANTS. 63-66 H. J. Hueck

THE INCIDENCE AND EFFECTS OF BIRD IMPACTS WITH AIRCRAFT 67-75 W. J. Forse

USEFUL METHODS FOR ISOLATION AND IDENTIFICATION OF FUNGI ON WOOD 77-94 C. Grant and J. G. Savory

LETTERS 95-96

BOOK REVIEWS 96-98

i

l \ TABLES DES MATIERES Nouvelles et commentaires L'UTILISATION DES ANTIBIOTIQUES POUR LA PROTECTION DES ALIMENTS B. Jarvis et M. D. Morisetti EXPERIENCE AVEC DES ESSAIS BIOLOGIQUES DANS LE DO MAINE DE LA DETERIORA- 63-66 TION BIOLOGIQUE DES MATERIAUX. 3. QUELQUES PRINCIPES DES ESSAIS DES PRODUITS DE PROTECTION DES MATERIAUX H. J. Hoeck L'INCIDENCE ET LES EFFETS DU CHOC CAUSE SURLES AVIONS PAR LES OISEAUX 67-75 W. J. Forse METHODES POUR L'ISOLEMENT ET L'IDENTIFICATION DES CHAMPIGNONS SUR LE BOIS 77-94 C. Grant et J. G. Savory Lettres 95-96 Revues des Livres 96-98 INHALT Nachrichten ond Kommentar 37-38 DIE ANWENDUNG VON ANTIBIOTIKA IN DER LEBENSMITTELKONSERVIERUNG 39-61 B. Jarvis ond M. D. Morisetti ERFAHRUNGEN MIT BIOLOGISCHEN PR0FVERFAHREN AUF DEM GEBIET DER 63-66 ZERSTORUNG VON WERKSTOFFEN DURCH ORGANISMEN. 3. EINIGE GRUNDSATZE 1 FOR DIE PR0FUNG VON SCHUTZMITTELN FOR WERKSTOFFE H. J. Hucck HAUFIGKEIT UNO EINFL0SSE VON VOGELAUFSCHLA.GEN AUF FLUGZEUGE 67-75 W. J. Forse N0TZLICHE VERFAHREN ZUR ISOLIERUNG UNO BESTIMMUNG VON PILZEN AUF 77-94 HOLZ C. Grant und J. G. Savory Briefen 95-96 Bochbesprechungcn 96-98 CONTENIDO Noticias y comcntarios 37-38 EL EMPLEO DE LOS ANTIBI6TJCOS EN LA CONSERVACI6N DE LOS COMESTIBLES 39-61 B. Jarvis y M. D. Morisetti EXPERIENCIAS CON PRUEBAS BIOL6GICAS EN EL CAMPO DEL LA BIODETERIORACI6N 63-66 DE LOS MATERIALES. 3. CIERTOS PRINCIPIOS DE LA PRUEBA DE LOS PROTECTORES DE LOS MATERIALES H. J. Hoeck FRECUENCIA Y EFECTOS DES CHOQUE DE LOS PARAJOS' CONTRA LOS AVIONES 67-75 W. J. Forse METODOS UTILES PARA AISLAR E IDENTIFICAR LOS HONGOS EN LA MADERA 77-94 C. Grant y J. G. Savory Cartas 95-96 Revistas de Libros 96-98 ii ACKNOWLEDGMENTS TO SUSTAINING ORGANISATIONS Financial support for the International Biodeterioration Bulletin from the following organisations is gratefully acknowledged: ALBRIGHT & WILSON (MFG) LTD., Oldbury Walden, Essex; makers of agricultural chemicals. Division, P.O. Box 3, Oldbury, Warley, Worcs. Research on control of weeds, plant diseases, AVON RUBBER COMPANY LIMITED, Melk­ spoilage organisms, and agricultural and livestock sham, Wiltshire; manufacturers of tyres and mis­ pests. cellaneous rubber products. FORESTAL INDUSTRIES (UK) LTD. Incorporating BEECHAM GROUP LIMITED, Toiletry Division, Dearborn Pittam and Feedwater Specialists. Both Beecham House, Great West Road, Brentford, companies are suppliers of industrial water treatment Middlesex. service and chemicals, including a wide range of BORAX CONSOLIDATED LIMITED, Borax biocides for control of micro-organisms in cooling House, Carlisle Place, London, S.W.l. (Tel. VIC­ water. _ toria 9070); miners, refiners and suppliers of borax, GAGLIARDI RESEARCH CORPORATION, East boric acid, boron ores, boron products and allied Greenwich, Rhode Island, U.S.A.; sponsored in­ chemicals. dustrial research in textile chemical dyeing and B.D.H. CHEMICALS LIMITED, Laboratory Chemi­ finishing products and processes. cals Division, Poole, Dorset; manufacturers of GALLOWAY & BARTON-WRIGHT, Haldane laboratory chemicals, biochemicals, industrial fine Place, London, S.W.18.; consultants in industrial chemicals and microbiocides. microbiology and microbiological deterioration. BRITISH INSULATED CALLENDERS CABLES GEIGY (U.K.) LIMITED, Simonsway, Manchester, LIMITED, 38 Wood Lane, London, W.l2. 22. THE BRITISH PETROLEUM COMPANY J. R. GEIGY S.A., Basle, 21, Switzerland; manu­ LIMITED. facturers of dyestuffs, industrial chemicals, pharma­ B.X.L. PLASTICS MATERIALS GROUP ceuticals and agricultural chemicals. LIMITED, Tyseley, Birmingham. GLAXO LABORATORIES LIMITED, Greenford CADBURY BROTHERS LIMITED, Bournville, Road, Greenford, Middlesex; manufacturers of Birmingham; manufacturers of cocoa, chocolate, pharmaceuticals, industrial enzymes and fine chemi­ cake and milk products. cals. CATOMANCE LIMITED, Welwyn Garden City, ARTHUR GUINNESS SON & COMPANY Hertfordshire; manufacturers- of speciality chemi­ (DUBLIN) LIMITED. cals for the textile, paper, timber, leather industries, HICKSON & WELCH (HOLDINGS) LTD., Jugs etc., including fungicides, bactericides and insecti­ Lane, Castleford. cides. IMPERIAL CHEMICAL INDUSTRIES CENTRAL LABORATORY TNO, Delft, The LIMITED, Agricultural Division, Billingham, Co. Netherlands; research, analysis and testing facilities Durham. in materials science and technology, including hie­ ARTHUR D. LITTLE LIMITED, with offices at deterioration and marine research. Sponsored by Berkeley Square House, Berkeley Square, London, government agencies and by international industries. W.l., and offices and laboratories at Inversk Gate, CHRIS FOREIGN FOODS WHOLESALERS LTD., Musselburgh, Midlothian; independent sponsored Stories Mews, Stories Road, Camberwell, London S.E.5.; tropical foodstuffs importers. research organisation. CIBA LIMITED, 4000 Basle 7, Switzerland. MAY & BAKER LIMITED, Dagenham, Essex; COALITE AND CHEMICAL PRODUCTS chemical manufacturers. LIMITED, P.O. Box No. 21, Chesterfield, Derby­ RECKITT & SONS LIMITED, Hull. shire; manufacturers of chlorinated and alkylated RENTOKIL LABORATORIES LIMITED, East phenols, phosphate plasticisers and bactericides. Grinstead, Sussex. Extensive research work and development carried REVERTEX LIMITED, Harlow, Essex. out in all these fields and customer service facilities SCIENTIFIC CHEMICALS INC., 1637 South Kil­ are available for bactericides. bourne Ave., Chicago, lllinois, 60623, U.S.A.; COMMERCIAL PLASTICS GROUP OF COM­ leading manufacturers of industrial fungicides and PANIES, Industrial Division, Berkeley Square bactericides who maintain substantial research and House, Berkeley Square, London W.l.; specialists development facilities to assist customers in the in the production of film and sheeting in PVC, development of final products geared to meet polythene and high impact polystyrene for all government and industry standards. types of applications. SHELL INTERNATIONAL CHEMICAL COM­ COURTAULDS LIMITED, Coventry. PANY LIMITED. CUPRINOL LIMITED, Adderwell, Frome, Somer­ STAUFFER CHEMICAL COMPANY RICH­ set; manufacturers of preservatives for wood, tex­ MOND RESEARCH CENTER, 1200 South 47th tiles, cordage, paper, adhesives, paints, leather, Street, Richmond, California 94804, U.S.A. plastics, and paint driers. VINYL PRODUCTS LIMITED, Butter Hill, THE DUNLOP COMPANY LIMITED. Carshalton, Surrey; manufacturers of synthetic FARBENFABRIKEN BAYER A.G., Leverkusen, resins, specialising in emulsion polymers for ad­ Germany; manufacturers of dyestuffs, industrial hesives, building, paper, surface coating and textile chemicals, synthetic fibers, pharmaceutical and applications. agricultural chemicals and preservatives for wood, YARSLEY LABORATORIES LIMITED, Clayton foodstuffs and technical products. Road, Chessington, Surrey; independent research FISONS LIMITED, Cambridge Division, Saffron and testing facilities. iii INTERNATIONAL BIODETERIORATION BuLLETIN

Editor Dr. H. J. Hueck, Central Laboratory TNO, Sch~ makerstraat 97, P.O. Box 217, Delft, The Nether­ Dr. H. 0. W. Eggins, Biodeterioration Information lands. Centre, Department of Biological Sciences, The University of Aston in Birmingham, 80 Coleshill Dr. A. M. Kaplan, Fungicides and Germicides Street, Birmingham 4, England. Laboratory, Pioneering Research Division, U.S. Army Natick Laboratories, Natick, Mass. 01760, U.S.A. Editorial Board Monsieur Y. Le Grand, Centre d'etudes du Bouche!, Mr. G. Ayers!, Department of Biology, College of Vert-le-Petit, (S & 0), France. Technology, Wolverhampton, Staffordshire, Mr. A. 0. Lloyd, Catomance Limited, 94 Bridge England. Road East, Welwyn Garden City, Herts., England. Professor G. Becker, Bundesanstalt fUr Material­ Professor R. W. Traxler, Department of Microbiology, prufUng, Berlin~Dahlem 45, Unter den Eichen 86/87, University of Southwestern Louisiana, Drawer 1007, Germany. USL Station, Lafayette, La. 70501, U.S.A. Dr. D. S. Belford, Hickson & Welch (Holdings) Dr. R. Zinkernagel, J. R. Geigy, S.A., CH-4000, Limited, Ings Lane, Castleford, Yorkshire, England. Basel 21, Switzerland. Mr. D. G. Coursey, Tropical Products Institute, 56/62 Dr. B. J. Zyska, Glowny Instytut Gornictiva, Kato­ Gray's Inn Ro?-d, London, W.C.I., England. wice, Plac Gwarkow I, Poland. Mr. J. J. Elphick, Admiralty Materials Laboratory, Holton Heath, Poole, Dorset, England. Publications Editor Dr. J. Garrido, Departmento de Fermentaciones Industriales, Castello 25, Madrid, Spain. Mr. M. J. D. Willsher, Biodeterioration Information Centre, Department of Biological Sciences, The Dr. N. E. Hickin, Rentokil Research Laboratories, University of Aston in Birmingham, 80 Coleshill Felcourt, East Grinstead, Sussex, England. Street, Birmingham 4, England.

NOTES FOR CONTRffiUTORS

The International Biodeterioration Bulletin is pub­ The bibliographic references are to be indicated in lished four times per year (Spring, Summer, Autumn the text as, e.g.: and Winter). Typescript contributions in triplicate should be sent to the Editor, Dr. H. 0. W. Eggins, Reese and Levison (1952). at the above address. The Bulletin acts as a vehicle for the publication of and in the bibliography: works on all aspects of biodeterioration, i.e. the deterioration of materials of economic importance by Reese, E. T. and Levison, H. G. (1952) Comparative micro-organisms, insects, rodents, etc. study of the breakdown of cellulose bymicroorganisms. Contributions may be in English, French, German Physiol. Plant., 5, 35~366. or Spanish and should be submitted in triplicate on international A4 size paper (21.0 em x 29.7 em or Authors are requested to abbreviate journal titles 8.27 in. x 11.69 in.); typewritten on one side of the according to the conventions of the World List of paper only. A summary of 25-100 words should Scientific Periodicals. accompany each contribution. Illustrations should be clearly drawn in Indian ink Proofs will not be sent to authors before final or should be photographed. The reduction desired publication. should be clearly indicated and illustrations when reduced are not to exceed 17 em X 26 em. Where 25 reprints will be sent free of charge to each figures are to be inserted in the text the approximate author. Additional reprints are obtainable on applica­ position for each one should be clearly marked in tion to the Publications Editor at a charge of £5 ($12) the typescript. per 100. iv NEWS AND COMMENT

For a period of five years, 1963 Applied Scientific Research in the built. Alternative feeding grounds to 1967, the Organisation for Netherlands (TNO) provides the could if necessary be artificially Economic Cooperation & Develop­ Secretariat for the, International created elsewhere in the Thames ment (OECD) through its Commit­ Biodegradation Research Group. Estuary. tee on Scientific Research spon­ For information concerning mem­ sored an Expert Group on the bership and activities of the Group, Dr. Schaefer is at present work­ Biodeterioration of Materials. This inquiries should be directed to the ing on a radar system that could Group conducted cooperative Secretary General of the Interna­ enable aircraft pilots to change research in limited areas which tional Biodegradation Research course to avoid flocks of birds. had been determined to be of Group, Mrs. Drs. E. H. Hueck-van Accurate bird identification is primary significance to its mem­ der Plas, Economical Technical possible because the radar "signa­ bers. At various times representa­ Department TNO, c/o P.O. Box tures" of different species differ tives of approximately twelve 217, Delft, The Netherlands. according to their wing beat nations participated in the several pattern. Insects can also be distin­ programmes. During its tenure the guished by this system and it has Group investigated problems con­ another possible application for cerned with the deterioration of We should like to congratulate the tracking of locust swarms. textiles and plastics, anaerobic Mr. H. J. Bunker who received (Abstracted from The Times.) metal corrosion, enzymatic the Honorary Degree of Doctor of deterioration, ecology and taxon­ Technology of Brunei University omy. The results of the collabora­ on 19th July. The Department of Microbio­ tive research studies were logy, University of Southwestern published in the open literature Louisiana will be in new and and the findings of some of these expanded laboratory facilities iu studies have been incorporated Dr. Arthur M. Kaplan, U.S. the fall of 1969. These facilities into various official test procedures Army Natick Laboratories, has include a large well equipped and documents found in the been appointed Adjunct Professor laboratory for the investigation of member countries. With the in the Department of Plant Patho­ biodeterioration problems. Group's encouragement two jour­ logy and Entomology, University nals, Material und Organismen of Rhode Island, Kingston, Rhode Graduate study positions are and the International Biodeteriora­ Island, with all privileges of available at both the M.S. and tion Bulletin, were established. professorial rank. This appoint­ Ph.D. levels as well as space for Two issues of an International ment is in addition to Dr. Kaplan's post-doctoral students in the fun­ Directory of Biological Deteriora­ activities as Head, Applied Micro­ damental aspects of microbiology tion Research were published and biology Group, Pioneering Re­ and biodeterioration. In addition, the formation of the Biodeteriora­ search Laboratory, U.S. Army a special masters program is tion Information Centre at the Natick Laboratories. offered in conjunction with the University of Aston in Birming­ Department of Chemical Engineer­ ham, England, was encouraged. ing in the field of Applied Micro­ OECD also aided in sponsoring biology. Interested persons should the First International Biodeterio­ As a result of recent discussions write to: ration Symposium held during on the suitability of Foulness as a site for London's third airport, Dr. R. W. Traxler, September 1968 at the University Department of Microbiology, fears have been expressed about of Southampton, Southampton, University of Southwestern England. the hazards which might be caused to aircraft by the birds frequenting Louisiana, Lafayette, Louisiana 70501, With former members of the the Thames Estuary sands on U.S.A. OECD Group as a nucleus, a which the airport would be built. successor organisation has now been formed that is open for At an enquiry into the propo­ membership to qualified indivi­ sals early in May, however, Dr. We should like to congratulate duals and laboratories on a broad G. W. Schaefer of Loughborough those of the International Bio­ basis. The new group, the Inter­ University gave the opinion that deterioration Bulletin's Sustaining national Biodegradation Research the frequency of bird strikes would Organisations who have recently Group, is an independent organisa­ be no greater than at Heathrow received the Queen's Award to tion which still maintains ties with airport, but that the birds at Industry. They are Imperial Chemi­ OECD. Its purposes are to con­ Foulness included many geese and cal Industries (export achievement tinue biodegradation research swans, which, because of their size and technical innovation); Albright undertaken by the OECD Group and weight, would be particularly and Wilson, Glaxo Laboratories and to undertake new international dangerous to aircraft. Between and Hickson and Welch (export cooperative studies in biodegrada­ November and March up to achievement) and Courtaulds and tion research of interest to its 10,000 brent geese visited the area May and Baker (technical innova­ members. The Organisation for on which the airport would be tion.) 37 Mr. T. A. Oxley, Director of the Continuing the expansion of candidate for trial as a preserva­ Forest Products Research Labora­ their overseas pest control services, tive in jet fuels, heating fuel oils, tory (U.K. Ministry of Techno­ Rentokil Group Limited have now etc. Since no metallic atoms are logy), has recently announced the formed P.T. Rentokil Indonesia, a present in the molecule no ash internal reorganisation of the La­ new subsidiary company based in remains after combustion. boratory. In place of the two Djakarta, the capital city of the divisions of Science and Techno­ Republic. Rentokil decided to Vancide PA (Trans-1,2-Bis (N­ logy, there will be three divisions: invest in Indonesia as a result of propylsulfonyl) ethene) is receiving the Anatomy and Preservation the Indonesian Government's new wide acceptance because ofits long­ Division, under Dr. E. J. Gibson, laws offering incentives to en­ lasting effectiveness and durability. who also becomes the laboratory's courage foreign capital investment, Paint protection from mould has Deputy Director; the Structures and the current promising econo­ been achieved where 0.3-0.5% has and Building Division, under Mr. mic climate in Indonesia. been used in non-alkaline emul­ J. G. Sunley and the Production sion paints, or 0.5-1.0% in oil­ and Processing Division under R. J. Orme, director and general based paints. Mr. J. F. S. Carruthers. manager of Rentokil Malaysia Vancide PA's low order of Pte. Ltd., who is based in Singa­ toxicity for warm-blooded animals The FPRL has recently estab­ pore will assume over-all respon­ lished a termite colony in order to and its water insolubility make it sibility for the Group's operations safe for use proximal to both be able eventually to provide a in these territories. termite resistance testing service plants and animals. Water in­ to manufacturers and exporters of Manager of operations in Indo­ solubility means no leaching by wood-based goods. The colony nesia will be Nigel Fisher-Jaine rain water, and consequently, no was obtained from the French who is experienced in Rentokil's effect on plants when water Institute of Tropical Timber and pest control activities in the United reaches the soil. the species is Reticu/itermes luci­ Kingdom, East Africa and South Many other potential uses are fugus var. santonensis. The high East Asia, and Assistant Service suggested, such as a paper mill tolerance to insecticides of this Manager is Sulaeman Hassan, a slimicide, for industrial paper species makes it particularly suit­ biology graduate from the Uni­ mould-proofing, and in controlling able for testing purposes. versity of Djakarta who has been slime in industrial water cooling trained in Singapore. towers. New buildings have recently Most of the Company's work been opened at the U.K. Agri­ will be concerned with the fumiga­ W. H. Mayes & Son have cultural Research Council's Food tion of shipping and warehouses to recently announced the addition of Research Institute at Norwich. protect cargoes and stored produce the electronic Light Capacity Uni­ Under the directorship of Pro­ but a full service against insects versal Testing machine to their fessor S. R. Elsden research is and rodents in Government, Com­ range of materials testing machines currently being carried out on mercial and domestic premises and and instruments. This model has aspects of spoilage of fruit, vege­ on ships is now available from been designed to meet the demands tables and poultry meat. (Abstrac­ Rentokil in Djakarta. of industry, research and teaching ted from The Times.) Mr. Fisher-Jaine was previously establishments for a low priced with Rentokil's Pest Control Divi­ versatile electronic force and strain sion in Bristol, England. measuring system. Further details The U.S. National Bureau of may be obtained from: Stundards has proposed that a W. H. Mayes & Son (Windsor) separate institute for corrosion Ltd., research be established with an R. T. Vanderbilt Company, Inc. Vansittart Estate, Arthur Road, annual budget of $2- $3 million have recently announced two new Windsor, Berkshire, England. aud a staff of about 185. microbicidal products. Telephone 0753 5 65103. Corrosion in the U.S.A. is Vancide Til (Hexahydro-1,3,5- estimated to cost SlO billion per triethyl-s-triazine) is a newly dis­ annum in preventive maintenance covered chemical which preserves An International Congress on and wasted materials. The precise alkaline systems. Synthetic rubber Cybernetics is to be held at mechanisms of chemical and bio­ latex susceptible to mould and Imperial College London from logical corrosion are not fully bacteria is protected by suitable 2nd to 6th September, 1969 under understood and more research on levels of Vancide TH (0.02-0.05 %). the aegis of the International the subject is needed. The Institute Adhesives not to contact food are Cybernetic Congress Committee. of Materials Research of the protected by addition of 0.01-0.05 Further information and applica­ Bureau of Standards already oper­ per cent Vancide TH. Starch­ tion forms can be obtained from: ates a small programme of basic based as well as soya flour adhe­ Dr. J. Rose, and applied corrosion research, sives are protected from spoilage Hon. Sec., r.c.c.c., with a staff of about 20 and an caused by mircroorganisms. College of Technology and annual budget of S250,000 and Design, the proposed Institute would be Solubility in kerosene and re­ Blackburn BB2 lLH, and enlargement of this. lated hydrocarbons make it a Lancashire, England. 38 Jarvis, B. and Morisetti, M. D. Int. Biodetn Bull., 5, (2), 39 - 61, (1969). The use of antibiotics in food preservation.

THE USE OF ANTffiiOTICS IN FOOD PRESERVATION B. Jarvis and M. D. Morisetti1

Sununary The use of antibiotics in foods is considered in Dcr Anwendung von Antibiotika in der Lebensmittelkonsenierung. relation to their effectiveness as preservatives, current legislation Die Anwendung von Antibiotika flir Nahrungsmittel wird and the "ideal criteria" for food preservatives. Legislation on untersucht in bezug auf ihre Wirksamk.cit als Konservierungs­ mittel, auf die giiltige Gesetzgebung und auf die "idealen the use of antibiotics in foods is summarized in an appendix. Kriterien'' flir Konservierungsstoffe fUr Nahrungsmittel. Gesetze L'Utilisation des Antibiotiques pour Ia Protection des Aliments. tiber die Anwendung von Antibiotika in Lebensmitteln werden L'utilisation des antibiotiques pour les aliments est etudiee en in einem Anhang zusammengesfasst. fonction de leur efficacite en tant qu'agents de protection, de Ia El empleo de los nntibi6ticos en Ia consenaci6n de los comestibles. legislation courante et des "critCres ideals" pour les agents de Se considera el empleo de los antibi6ticos en los comeStibles protection des aliments. On trouvera, en appendice, un respecto de su eficacia como preservatives, Ia legislaci6n vigente resume Y los criterios ideales de los preservatives para comestibles. relatif a Ia legislation sur l'utilisation des antibiotiques pour Se resume en un apC:ndice Ia legilaci6n sobre el empleo de los les aliments. antibi6ticos en los comestibles.

Introduction Anon, 1963; Ingram, Buttiaux and Mossel, 1964). The principle precautionary arguments may be summarized Raw foods provide an ideal substrate for the growth as follows:- of many kinds of microorganisms. For many centuries man has utilised processes such as drying, smoking, a) At the concentration of use the antibiotic should salting, fermentation, refrigeration and freezing in an. preferably be bactericidal (fungicidal) rather than attempt to delay the microbial spoilage of food. These bacteriostatic (fungistatic) and after exerting its methods have been improved by modern technology effect the antibiotic should decompose into atoxic and their application has been extended to a greater degradation products. variety of foods. Chemical preservatives are frequently b) Where an antibiotic is bacteriostatic (fungi­ used to increase the storage life of both raw and static), at the concentration of use, it should persist processed foods and, in this context, antibiotics can be in the food throughout the period of storage, but considered to be a specific class of food preservatives. should preferably be destroyed during cooking of The era of antibiotics in food preservation probably the food. An antibiotic used in conjunction with a had its inception in 1948 with the discovery of chlor­ thermal process should be heat stable. tetracycline (Duggar, 1948). Since this time other c) The use of an antibiotic should not result in the non-medical applications of antibiotics have been selection of naturally resistant organisms, nor induce investigated, including their use in agriculture as feed the emergence of resistant strains, nor of strains additives and in the control of bacterial and fungal with cross-resistance to antibiotics approved for plant diseases. Detailed reviews of these applications therapeutic use. have been published (see for example Farber, 1959; d) the antibiotic should be active against all Woodbine, 1962; Marth, 1965; Sarkisov, 1966). organisms including food poisoning organisms, Much concern has been expressed about the presence which are capable of developing in the food, under of antibiotics in foods for human consumption. In all conditions of use. 1966 the report of an ad hoc committee on the veteri­ e) Antibiotics used in food preservation should not nary and non-medical uses of antibiotics in the United have a therapeutic use either in human or in veteri­ States was published (Anon, 1966). Many current nary medicine. practices were strongly criticised and the immediate f) An antibiotic should not be used both as a feed reaction of the Federal Food and Drug Administration additive and in the preservation of food derived (FDA) was to revoke the established residue toler­ from animals which have been fed on feed contain­ ances for tetracyclines in meat and poultry' and to ing the particular antibiotic. reject petitions for the use of nisin and tylosin in heat­ g) The antibiotic and its degradation products processed canned foods and for pimaricin in orange juice. Whilst the FDA were concerned about the uses should be atoxic and its use should not result in the development of hypersensitivity to the antibiotic in of antibiotics in animal feeds and in veterinary medi­ any individual consumer. cine they were not opposed to their use provided more stringent controls were introduced. h) Antibiotics must not be used to conceal in­ sanitary practices in the food industry. In their report (Anon, 1966) the FDA proposed certain "guidelines" for the use of antibiotics as food The purpose of the present review is to consider the additives. In general these "guidelines" are similar to antibiotics presently used in food preservation, or for recommendations made previously (Deatherage, 1955; which petitions for use have been filed, in relation to Ingram, Barnes and Shewan, 1956; Bacharach, 1957; • their efficacy as preservatives, current legislation and

'National College of Food Technology, University of Reading, St. George's Avenue, Weybridge, Surrey, England. (Copy received 31st March, 1969).

39 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti. the recommendations outlined above. The presence in upon the numbers and age of the organisms, the pH foods of antibiotic residues arising from agricultural or value and composition of the medium and the con­ veterinary applications and the presence of naturally centration of the antibiotic; (b) that nisin acts in a occurring antimicrobials are outide the scope of this manner similar to that of cationic detergents and review, although such residues may influence the surface-active polypeptide antibiotics; (c) that nisin is growth of both spoilage and pathogenic organisms and neither sporostatic nor sporicidal, but acts on germina­ cause changes in the spoilage patterns of particular ted spores. Earlier Hirsch and Grinstead (1954) re­ foods. ported that nisin was sporicidal for C/. butyricum, but not for C/. sporogenes or C/. bifermentans. NISIN Jarvis (1967 a) observed that nisin was bactericidal Nisin is the family name for a group of closely for both lag phase and logarithmic phase vegetative related polypeptide antibiotics (Berridge, Newton and forms of several species of Bacillus, but had little effect Abraham, 1952) produced by Streptococcus /actis and on stationary phase cells. Jarvis and Byrom (1968, isolated by Mattick and Hirsch (1944). Early investiga­ unpublished data) observed lysis in cultures of staphy­ tions showed nisin to be active against many gram­ lococci, micrococci and streptococci treated with positive bacteria, but attempts to use nisin clinically, nisin at all stages of growth. Since the lytic effect is notably in the treatment of tuberculosis and bovine observed immediately on addition of nisin, it is con­ staphylococcal mastitis, were unsuccessful largely due cluded that nisin probably acts on the cell membrane. to the insolubility of nisin at the pH of blood and to the localised irritant effects which developed on intramam­ Many reports on the mode of action of nisin have mary infusion (Hirsch and Mattick, 1949; Taylor, been concerned with its use as an antibacterial agent Hirsch and Mattick, 1949; Gowans, Smith and Florey, in the processing of canned foods. 1952). Early reports suggested that nisin (and also subtilin) Unlike many other peptide antibiotics, the consti­ decreased the heat resistance of bacterial spores (Lewis, tuent amino acids of nisin are all in the L-configuration Michener, Stumbo, and Titus, 1954; O'Brien, Titus, (Berridge, Newton and Abraham, 1952) and nisin Devlin, Stumbo and Lewis, 1956). That this effect was contains the unusual S-amino acids lanthionine and more apparent than real was shown by several workers B-methyllantionine (Newton, Abraham and Berridge, (O'Brien et a/., 1956; Campbell and Sniff, 1959; 1953) in which respect it resembles the antibiotic Denny, Sharpe and Bohrer, 1961); the effect resulted subtilin (Lewis and Snell, 1951). Based on their results from carry over of antibiotic from the heating men­ with amino acid composition and diffusion studies, strum into the recovery medium, and Thorpe (1960) Cheeseman and Berridge (1959) proposed a molecular concluded that the carry-over occurred as a result of weight of 7,000 for nisin A. Recently, Gross and adsorption of nisin onto the spore-coat. Heinemann, Morrell (1967) have shown the minimum M.W. to be Voris and Stumbo (1965) showed that spores damaged 3300 and this has been confirmed by Jarvis, Jeffcoat by severe heat treatment had an increased sensitivity to and Cheeseman \1968) who have also shown that nisin, but Tramer (1964) observed no difference be­ nisin normally exists as a dimer. tween the nisin sensitivity of spores of B. stearother­ Nisin is only sparingly soluble in water at neutral mophi/us heated with nisin and those to which an equal pH, and has maximum solubility at pH 2. On auto­ concentration of nisin was added after heating. Hawley claving, nisin is stable at pH 2 but over 40% is in­ (1961 a) studied the effect of nisin on sublethally­ activated by autoclaving in buffer at pH 5 and over heated spores of Cl. botulinum and C/. sporogenes. He 90% is inactivated at pH 6.8 (Tramer, 1964). The concluded that both nisin-sensitive and nisin-resistant addition of milk solids or broth largely protects nisin strains were equally sensitive to the combined effect of on autoclaving at neutral pH, but loss of activity occurs heat and nisin, but that use of nisin alone was in­ on prolonged storage in neutral solution at tempera­ sufficient to prevent germination and outgrowth of tures over 37°C. Nisin is inactivated by alkali, by spores from nisin-resistant strains. chymotrypain and by certain bacterial enzymes The stage of development of spores at which nisin (Berridge, 1949; Jarvis, 1967 a, b; Jarvis and Mahoney, acts is still uncertain, although there is general agree­ 1969). ment that nisin prevents outgrowth of vegetative forms. The unit of biological activity (Reading unit) was Tramer (1964) reported that spores of B. stearother­ originally defined by Berridge (1949), but has since mophilus did not germinate in the presence of nisin, been redefined by Tramer and Fowler (1964) in terms but Hitchins, Gould and Hurst (1963) reported that of the activity exhibited by I pg. of a standard batch nisin permitted germination (i.e. phase darkening and of commercial nisin (Nisaplinl). loss of heat resistance) and post-germination swelling in spores of B. cereus and B. subtilis, but prevented Mode of Action pre-emergence rupture of the spore-coat. Gould (1964) Ramseier (1960, 1961) studied the effect of nisin on observed that nisin permitted germination in 56 strains Clostridium butyricum and concluded that (a) nisin was of various species of Bacillus, but prevented post­ bactericidal against both young and old vegetative germination swelling, rupture of the spore coat and forms, but that the degree of sensitivity was dependent outgrowth of the vegetative forms. l"Nisaplin" is obtainable from the Biologicals Division, Aplin & Barrett Ltd., Yeovil, Somerset.

40 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

The way in which nisin acts is unexplained. Hall Cross-resistance with medically important anti­ (1966) suggested that since nisin is surface-active it biotics does not occur and no problems arise from the might be adsorbed onto the spore-coat (cf. Thorpe, development of nisin-resistant flora in the gut since 1960) to form a complex with the spore wall (Hawley, nisin is destroyed by digestive enzymes (Heinemann 1961 b). Since nisin can be removed from spores by and Williams, 1966; Jarvis and Mahoney, 1969). incubation with trypsin (which does not inactivate Claypool, Heinemann, Voris and Stumbo (1966) nisin) the possibility of nisin being bound through ester reported that the resistance to nisin of oral microflora or amide linkages is possible and provides circum­ did not increase following consumpition of chocolate stantial evidence for the correctness of this hypothesis. milk containing nisin and no changes in the microflora However, Tramer (1964) reported that nisin is not were observed. firmly bound to spores of B. stearothermophilus.

Alppications of nisin Resistance to nisin Cheese. Since strains of Str.lactis are used in cheese The opening of the bacterial spore-coat on out­ starter cultures nisin occurs naturally in many cheeses, growth is usually characteristic ohhe species (Laman­ and its effect in cheese has been extensively investi­ na, 1940) and is not radically affected by changes in the gated. Hirsch, Grinstead, Chapman and Mattick (1951) environment (Gould, 1962). Spores (type M) of small­ suggested the use of nisin for the control of gas­ celled species of Bacillus generally open by rupture of producing anaerobes during ripening of Swiss-type the spore"wall, whilst spores (type-L) of large-celled cheeses (e.g. Emmenthaler, Gruyere, Danish-Swiss, species open by lysis of the wall. Gould and Hurst etc.). Although Hirsch and co-workers (1951) and (1962) and Gould (1964) showed that type L spores McClintock, Serres, Marzolf, Hirsch and Mocquot were resistant to more than 100 units nisin/ml and that (1952) reported that nisin did not affect the desirable type M spores were generally sensitive to 2-10 units/mi. microfiora of Swiss-type cheese (i.e. thermophilic The presence of a nisin-inactivator in type L spores was streptococci and propionic bacteria), Ritter (1957) and reported by Gould and Hurst (1962), but little of this Winkler and Frohlich (1957, 1958) reported abnormal inactivator could be detected in type M spores. Jarvis ripening of cheeses due to the effect of nisin on the (1966 a, 1967 a) confirmed these observations and lactobacilli. Pulay (1956) found that the use of nisin­ demonstrated that the inactivator was an enzyme which producing starter cultures only retarded the develop­ was active against both nisin and subtilin, but not ment of butyric acid bacteria until the nisin was against other polypeptide antibiotics tested. The reduced to below the minimum inhibitory level and enzyme also occurs in sporulating vegetative forms of further suggested that use of nisin might result in B. cereus and other species of Bacillus. Evidence to spoilage by coliforms. Lilley (1958) suggested the show that the 'nisinase' is probably an esterase or deliberate addition of nisin preparations at a later amidase has been obtained (Jarvis, 1967 b, 1968). stage of manufacture in order to overcome any adverse effect on the starter organisms and to provide an ade­ Moycho and Gromska (1956) observed that B.subtilis quate level of nisin during the ripening period. was sensitive to nisin, although Carlson and Bauer(l957) were able to increase the resistance for one strain by Although many reports confirm that nisin is able serial cultivation through increasing concentrations of to control butyric acid production in Edam and Gouda nisin. Shahani (1962) and Halasz and Juhasz (1963) cheese (see for instance, Lipinska 1956, 1961; Lilley, reported that spores of B.subti/is were resistant to 1958; Lipinska, Strzalkowska, Goettlich and Soltys, nisin, but this was not confirmed by Gould (1964) or 1962) other workers have reported poor eye formation by Jarvis (1967 a). in Edam cheese made with pasteurized milk and nisin­ I Induced resistance to nisin has been reported for producing starters (Galesloot and Pette, 1957) and li' many organisms and is frequently dependent upon the retarded ripening of the cheese (Galesloot, Raadsveld ,, and Pette, 1957). Bylund, Norrgren and Sjostrom ' production of nisin-inactivating enzymes. Kooy (1952) and Pette and Kooy (1953) reported the production of (1954) used nisin-producing starter cultures to control an extracellular nisinase by strains of Lact.plantarum, the growth of butyric acid bacteria in Herregard and Gibbs and Hurst (1964) reported the isolation of cheese. nisin-resistant lactobacilli and micrococci from nisin­ Although over 90% of English-type cheeses (i.e. treated bacon. Induced resistance in Staph.aureus Cheddar and Cheshire) contain viable spores of gas­ (Carlson and Bauer, 1957) and in lactic streptococci producing clostridia, spoilage is not normally caused (Galesloot, 1956; Lipinska and Strzalkowska, 1959) by these organisms, probably due to the high acid was reported to be dependent on the production of and salt levels (Berridge, 1953). Consequently the use nisinase. The inducible nisinase of Str.thermophilus of nisin in English cheeses is of little value, although was isolated by Alifax and Chevalier (1962) and was it will frequently be found in these cheeses. shown to be specific for nisin as substrate and to bave optimal activity at pH 7.0. Hirsch (1951) reported that Processed Cheese. The spoilage of processed cheese nisin-resistant strains of E.co/i and Str.faecalis were is caused largely by gas-producing clostridia (Berridge, unable to inactivate nisin and similar results with 1953). The germination of spores present in the cheese, E.coli and Kl.aerogenes were obtained by Ramseier or other ingredients, may be stimulated by the heat­ (1959). shock given during processing and growth may ensue 41 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti. in the anaerobic environment of foil-packed cheese. Tramer (1966) reported an improvement in the Spoilage, as indicated by gas production, may be colour and flavour of sandwich pastes as a result of controlled by the incorporation of 100-200 units incorporating nisin and reducing the processing nisin/g cheese (Hawley, 1957 a; Lipinska and Strzal­ conditions. Wheaton and Hays (1964) investigated kowska, 1959; Tramer, 1966). the use of nisin in foods intended for sale from "hot­ vending machines". They found that 40 units nisin/g. The use of cheese containing nisin as raw material prevented flat-sour spoilage in inoculated packs of for processing may overcome legal difficulties in cream-style corn and chow mein, but they were certain countries (e.g. Holland) but it does not permit unable to control spoilage caused by putrefactive ready control ofthe nisin concentration and is neither anaerobes even with 100 units nisin/g. convenient nor economic (Hall, 1966; Tramer, 1966). Since nisin is ineffective against yeasts, moulds and gram-negative bacteria (Mattick and Hirsch, 1947) its Detection and Assay of Nisin in Foods. use in cheese processing will not mask the use of Many microbiological methods for the assay of poor quality raw materials nor poor manufacturing nisin have been reported (for review see Marth, 1966), conditions. but these methods are generally unsuitable for assay of nisin in food extracts. Two different agar diffusion Canned Foods. Commercially sterilized foods norm­ methods were proposed by Tramer and Fowler (1964) ally contain viable spores of extremely heat resistant, for estimation of nisin in foods. The first method thermophilic species (e.g. B. stearothermophilus, C./ which is suitable for routine use is based on the thermosaccharolyticum, C/. sporogenes, etc.) which can reverse phase assay procedure of Kosikowski and develop and produce spoilage if suitable environmental Ledford (1960) and uses heat-shocked spores of conditions arise. Since nisin inhibits development of B.stearothermophilus in a non-nutrient saline agar. bacterial spores it was logical that attempts should be The method is semi-quantitative up to 10 units made to use it in canning, since its use might permit nisin/ml food extract. lower thermal processes without any increase in spoilage or health risk and with consequent im­ The other method uses M.jlavus in a nutrient provements in the product and economies in produc­ medium containing the surfactant Tween 20, to tion. enhance diffusion of the nisin. An involved procedure for extraction of nisin and preparation of diluents Large-scale investigations into the use of nisin in makes the method unsuitable for routine assay in canned foods given a "minimum botulinum cook" unskilled hands. However, this method forms the were instigated in 1955 and the control of thermo­ basis of the British Standard Method for estimation of philic and putrefactive anaerobes and thermophilic Nisin in Cheese (B.S.I. Report, 1964), and is suitable flat sour bacteria has been reported by many workers for estimation of nisin using either petri dishes or (see Hawley, 1957, 1962). Reduction in post-processing large-plate techniques (Jarvis, 1966 b). spoilage, with nisin levels up to 200 units/g, has been reported for canned beans in tomato sauce and peas A sensitive micro-method for the assay of nisin was (Gillespy, 1957), evaporated milk (Gregory, Henry proposed by Stumbo, Voris, Skaggs and Heinemann and Kon, 1964), sterilized milk and tomatoes (Tramer, (1964). The technique depends upon the nisin-sensi­ 1966), chocolate-flavoured milk (Heinemann, Stumbo tivity of heat-damaged spores of B.stearothermophi/us and Scurlock, 1964; Tramer, 1966), carrot puree and and is not suited to routine applications, but has the mushrooms (Vas, 1964), soups (Bardsley, 1962; advantage that results are not affected by the presence Hawley, 1962) and for other foods. Some reports in the food of emulsifying salts. have shown, however, that under conditions of prolonged storage, delayed thermophilic spoilage may No methods are available for the absolute identifica­ arise (Hawley, 1962); such delayed spoilage probably tion of nisin in food extracts. Hawley (1957) identified reflects the chemical inactivation of nisin at neutral nisin on the basis of its heat stability and anitmicrobial spectrum, and this procedure has been improved by pH (see above). Gibbs & Hurst (1964) were unable to control spoilage in heavily contaminated soups Fowler (personal communication) but the procedure even at levels up to 500 units nisin f g., and this is too involved for routine application. The possibility confirms that nisin cannot be used as a means of of using the "nisinase" of B.cereus (Jarvis, 1966 a, masking poor quality raw materials. 1967 a, b) for the identification of nisin is being current­ ly investigated and preliminary results suggest that Other Food Applications. Hawley (1961 a) reported this method may be feasible (Jarvis and Berridge, 1968). that nisin reduced spoilage of meats such as tongue, luncheon meat and crustaceans which receive a Legislation & Public Health Aspects pasteurization process and Gola (1962) used nisin to prevent spoilage of non-refrigerated pasteurized The use of nisin in cheese, processed cheese and hams. Earlier Eastoe and Long (1958) had suggested certain other food products is permitted in many the use of nisin to prevent clostridial spoilage of countries (see Appendix), usually without limitation gelatin solutions used in food products. Gibbs and on amount and often without the necessity to declare Hurst (1964) found nisin unable to prevent spoilage its use on the label. Since nisin is a natural constituent of pre-packed bacon and reported the rapid develop­ of cheese, countries such as Norway which prohibit ment of nisin-resistant lactobacilli and micrococci. the generalized use of antibiotics in foods, permit its 42 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti. use in cheeses. An interesting contrast occurs in the If the use of nisin offers a potential reduction in the case of West Germany where nisin is permitted 1n spoilage rate of a food, without a change in the process canned foods but not in processed cheese, milk or conditions, or if a reduction in processing conditions milk products. In Australia, nisin is permitted in can be achieved without any increase in the spoilage cheeses and in canned foods where the pH is below rate, then the use of nisin may he justified. Nisin might 4.5-this regulation is presumably intended to prevent also be considered for use in new products where the any possible hazard from the reduction of processing minimum heating conditions without nisin are too conditions below the level of the "minimum botulinum severe for a satisfactory product to he obtained. cook" in low acid and neutral pH foods. However, every application of nisin should be con­ sidered only on the basis of results obtained in a In the U.K., the use of nisin is permitted without suitable controlled series of expermiental productions. limitation on amount and without the need to declare its use on the label; its use is restricted to canned foods, NYSTATIN and PIMARICIN which are foods in hermetically sealed containers and Nystatin (syn. Mycostatin) and pimaricin (syn. which have been sufficiently heat processed to destroy Tennecetin) are polyene (tetraene) macrolide anti­ Cl.botulinum in food or container, or which have a pH biotics produced by Streptomyces noursei and Strept. ofless than 4.5, cheese and clotted cream. (Preservatives natalensis respectively (Hazen & Brown, 1950, 1951; in Foods Regulations, 1962). Struyk et a/., 1957). Details of their structures and properties have been published (Patrick et a/., 1958 Since nisin is not used clinically, is atoxic (Fraser, a, b; Berry, 1963; Ceder eta/., 1963; Golding, eta/., Sherratt & Hickman, 1962) and is only used in con­ 1966; Korzybski, Kowszyk-Cindifer and Kurylowicz, junction with some other method of preservation, no 1967). Both antibiotics are fungistatic at low con­ public health problems arise from its use and the centrations of use and have little or no effect on emergence of nisin-resistant organisms is unlikely to bacteria, viruses or actinomycetes. occur. Further, nisin does not induce the production of nisin-resistant strains in the consumer since it is Mode of Action inactivated by intestinal enzymes. (Heinemann and Williams, 1966; Jarvis and Mahoney, 1969). Treatment of Saccharomyces cerevisiae and Candida albicans with nystatin was reported to cause a rapid However, the FDA have rejected applications for increase in the permeability of cell membranes to small the use of nisin in heat-treated canned foods, on the ions, such asK+ (Marini, Arnow and Lampen, 1961; basis that inadequate data is available (Anon., 1966). Zygmont, 1966). The increase in permeability exerted The report called for data on {I) Variations in the an indirect influence on glycolysis. Lampen and co­ nisin sensitivity of the vegetative cells of a number of workers (1962) reported the binding of nystatin to the cultures of spoilage organisms of processed foods as cell wall and cell membrane, and suggested that the compared with variations of spore forms; (2) Existence binding site on the cell wall contained a sterol. This and emergence of nisin-resistant organisms and on hypothesis is consistent with the ability of certain cross-resistance to· therapeutically important antibio­ sterols to complex polyenes, thereby preventing the tics; (3) whether nisin might otherwise interfere with inhibition of growth of normally susceptible fungi The action of therapeutic antibiotics; and (4) commercial only sterol which has been shown to effectively an­ use. tagonize the antifungal activity of five polyene anti­ biotics including both pimaricin and nystatin was After other data shows that the use of nisin is ergosterol (Zygmont and Tavormina, 1966). A correla­ potentially safe, the FDA have been asked to approve tion between the molecular size of a polyene antibiotic an application for investigational use and commercial and the degree of damage has been reported; the distribution of the products. Before final approval will smaller the polyene molecule the greater the damage to be given, the pilot studies are required to provide the membrane, but both large and small polyenes are information on {I) spoilage in each batch of product bound to the sterol area of the cell membrane (Cirillo, (2) residual nisin and the change in nisin-susceptibility Harsch and Lampen, 1964). of microbial flora; (3) oral and pharyngeal flora of The use of nystatin and other polyene antibiotics individuals consuming the product; (4) nisin activity, against Neurospora crassa resulted in a decrease in dry if any, in the mouth; and (5) any untoward reactions weight relative to the untreated controls, and loss of or sensitivity in a selected sample of consumers. "The cytoplasmic constituents into the medium. These committee recommends that such a commercial trial results were consistent with the antibiotic increasing should be limited at first to one or two processed foods the cell-membrane permeability (Kinsky, 1961, 1962). on which considerable data are already available, primarily heat-treated chocolate milk, or secondarily Microbiological Activity tomato juice". Klis, Witter and Ordal (1959) determined the con­ In this country we already accept that the use of centrations of antibiotic required to inhibit the growth nisin introduces no public health hazard. The only of food spoilage yeasts and moulds for 96 hr. Organ­ question is whether the use of nisin offers any real isms tested included species of Absidia, Mucor, Rhizo­ advantage to the consumer, the manufacturer or to pus, Aspergillus, Penicillium, Rhodotorula, Saccharo­ both parties. myces and Geotrichum. Nystatin, at 5 to 10 ppm, both 43 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti. in broth and agar cultures inhibited growth of all woe and room temperature. At 4.4°C both concentra­ organisms tested except for Absidia spp. which re­ tions of pimaricin were equally effective, but at the quired 25 ppm. Pimaricin inhibited the same organisms higher storage temperatures the higher concentration at the same or slighly lower concentrations. Similar of antibiotic was the more effective. At 4.4°C, fungal results have been reported for other yeast and mould growth in pimaricin-treated inoculated samples was strains (Ayres eta/., 1964 a; Stanley and English, 1965; controlled over a period of 77 days storage, whereas Korzybski et a/., 1967). Rangaswami and Damodoran in the inoculated control samples yeast growth occur­ (1957) reported on the in vitro inhibition by nystatin red after 14 days. of various fungi pathogenic to bananas. Growth of Helminthosporium torulosum and G/eosporium musa­ Cheese. Neither pimaricin (50 ppm) nor nystatin rum was found to be inhibited at an antibiotic con­ (20 ppm) were effective in extending the storage life of centration 2.5 ppm and Fusarium oxysporium at!Oppm. cottage cheese stored at W°C and 15.5°C, and bacterial Spore germination was inhibited by 10, 12.5 and 25 spoilage occurred at the higher temperature. At 4.4°C, ppm nystatin respectively. pimaricin (50 ppm) extended the storage life of the cheese by I 0 days to 27 days and nystatin (20 ppm) The growth of Staph. aureus, Bacillus cereus var by approximately 8 days to 21 days (Shahani, Nilson mycoides, B.subtilis, Salmonella typhi, Klebseiella spp. and Downs, 1959). The cheese was treated by washing and Mycobacterium tuberculosis was not inhibited by the curd with water containing the antibiotic and by nystatin at 100 ppm (Korzybski et a/., 1967). The holding the curd in antibiotic solutions. Hard cheeses lack of activity of polyene antibiotics against bacteria may be protected from mould growth by dip treatment may be associated with the absence of sterols from in a suspension of pimaricin (WO ppm), or by coating bacterial cell membranes. the cheese with polyvinyl acetate containing 500 ppm pimaricin (Anon, 1963 b), Applications in foods Meat and Poultry. Nystatin has been used at levels of 5 and 10 ppm to retard yeast growth on CTCtreated Commercially, neither pimaricin nor nystatin, have chicken meat (Yacowitz eta/., 1957). It was found that had great use but their effect in del.aying spoilage of a both mould and yeast growth was effectively con­ number of foods has been investigated. trolled. The nystatin did not penetrate the chicken Fruits. The effect of a number of polyene antibiotics skin into the muscle and was inactivated after boiling on the storage life of strawberries, raspberries, peaches the chicken. Volkova (1961) reported the inhibition of and tomatoes has been investigaed (DiMarco and mould growth on refrigerated CTC-treated meat by the Davis, 1957 a, b; Ayres and Dennison, 1958; Ayres concomitant use of 250 ppm nystatin. et a/., 1964 a, b), using two methods of application. Little improvement in the storage life or quality was Other foods. The use of pimaricin in other foods in­ reported for strawberries and raspberries which were cluding grape juice, lemons, apples and pickled products sprayed with antibiotic before harvesting, but some has been reported (Anon., 1963 b). York (1966) report­ improvement resulted from post-harvest dipping into ed that the heat-resistance of Saccharomyces cerevisiae antibiotic solutions. Pimaricin was slightly effective at was lowered by the presence of pimaricin and suggested a concentration of 10 ppm and nystatin at 20 ppm, that spoilage of acid foods by this organism might be j but 50 to 100 ppm were required for maximal effect. retarded by a combination of mild heat treatment and Fruits treated with 100 ppm were no more spoiled the addition of pimaricin. after 9 days storage then were untreated fruits stored j Detection and Assay for 5 days. Tomatoes, treated by spraying the paint with I a 100 ppm solution, or by post-harvest dipping into a Many methods for the chemical and microbiological 1 50 to 100 ppm solution had only a slighlty longer assays of pharmaceutical preparations of both nystatin storage life at l2°C than had water-treated controls. and pimaricin have been reported, but these are Fruit juice and concentrates. Shirk and Clark (1963) generally unsuitable for use with food extracts. An reported the use of pimaricin in retarding the spoilage agar diffusion assay for pimaricin in orange juice, of fresh orange juice. The presence of 20 ppm pimari­ using Saccharomyces cerevisiae (ATCC 9763) as the cin in orange juice stored at 2.5°C to 4.0°C reduced the test organisms and nystatin assay agar, has been described by Shirk, Whitehill & Clark (1962). The mould and yeast count both in non-inoculated and range of the assay is from I to 8 pg/ml (ppm). The inoculated (Saccharomyces cerevisiae) samples. Anti­ biotic treated samples remained unspoiled after 8 assay may be used for other foods provided a suitable weeks' storage, whereas untreated inoculated controls extraction procedure is adopted. A similar assay may were spoiled after storage for one week. After 12 be used for nystatin in foods (Ayres et a/., 1964 a) weeks, 70% of the added pimaricin was active, and, whilst a method developed for assay of nystatin in at the concentration used, did not impart any off­ animal feeds may also be applicable (Pagano, 1963). flavour to the juice. Tube dilution assays (see for example Gold, Stander & Pansy, 1953; Pagano and Stander, 1955) and a respiro­ Shirk, Kline ,and Clark (1966) added pimaricin at metric assay (Gerke, Gentile, Stander and Pagano, 10 and 20ppm to orange juice concentrates (42° and 65° 1959) may also be applicable to food extracts. Specific Brix), both uninoculated and inoculated with an methods for the identification of nystatin and pimari­ osmophilic yeast and subsequently stored at 4.4°C, cin in food extracts are not available. 44 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

Toxicity TETRACYCLINES Both nystatin and pimaricin are of low toxicity to Introduction humans when taken orally (Hazen and Brown, 1951; Seneca, 1955; Lynch et a/., 1960). In clinical trials, The tetracyclines comprise a group of related anti­ pimaricin showed side effects including diarrhoea, biotics. Chlortetracycline (CTC; Aureomycin) is when given orally at dose levels of 125,150 mg/day produced by Streptomyces aureofaciens, oxytetracy­ (Lynch et al., 1960), but these levels are considerably cline (OTC; Terramycin) by Strept. rimosusand tetracy­ greater than those at which pimaricin would be used cline (TC; Achromycin) is produced synthetically from CTC or from the fermentation broth in the production in food preservation. The acute oral LD50 of pimaricin for male and female rats was found to be 2.73 and of CTC. Details of their chemical and physical pro­ 4.67 gfkg respectively and 1.42 gfkg for male rabbits. perties have been described (Regna, 1959; Umezawa Rats survived a three month feeding trial of 8,000 ppm H, 1964; Korzybski et a/., 1967. They are bacterio­ in their daily diet without adverse effects, as also did static in action, and have similar antimicrobial spectra. rats fed at 1,000 ppm daily for 2 years. In the latter Mode of Action experiment, the reproduction of the rats over two The tetracyclines may act as inhibitors of various generations was not affected, although pups from enzyme systems, as chelating agents and as inhibitors pimaricin-fed dogs had lower mean body weights on of protein synthesis (Newton, 1965; Laskin, 1967). weaning than had pups from control animals (Levin­ They inhibit protein biosynthesis by inhibiting the skas, Ribellin and Shaffer, 1966). At oral dose levels of specific binding of amino-acyl sRNA to the ribosomal between 70,000 and 1.4 million units nystatin/day for messenger RNA {Franklin, 1963, 1964; Suarez and 32 days, no adverse effects were noted in humans and Nathan, 1965). blood concentration of antibiotic rose only to 7.5 to The effectiveness of tetracyclines in inhibiting the 9 unitsfml (Strade, H. cited by Seneca, 1955). The low growth of selected microorganisms in beef was found toxicity when taken orally probably reflects the poor to be much greater at low concentrations of use than absorption of these antibiotics from the gut, most had been expected from in vitro sensitivity experiments. being voided with the faeces (Seneca, 1955; Barr, Strains of Proteus vulgaris and of a Pseudomonas spp. 1959). were both found to be more sensitive to CTC in fresh Although the fungal flora of the gut is not normally beef, the strain of Pr.vulgaris being inhibited by 100 a siguificant fraction of the total, it was shown that ppm of antibiotic in broth .but by only 3 ppm in beef. nystatin inhibited the gut fungi of humans and other This effect was not noted in aged nor in heated beef animals, but no toxic effects were noted (Sternberg (Jay, Weiser and Deatherage, 1957 a, b, 1958). It was eta/., 1953; Anon, 1968). Pimaricin at 50 ppm inhibited suggested that CTC, because of its chelating properties, yeasts in the rat gut when tetracycline was also in­ competed with the organisms for essential ions in the corporated in their feed at a concentration of 200 ppm meat (Jay eta/., 1957 a, b; Weinberg, 1957). Snell and (Manteo and Hoogerheide, 1958). Cheng (1960) reviewed the inhibiton of specific enzyme systems by tetracyclines. The listed effects were diverse and they concluded that it was not possible to be Public Health Aspects and Legislation precise about the mode of action of the antibiotics in At the present time the use of nystatin and pimaricin a given situation. to control fungal growth in food appears to be limited to the use of pimaricin in cheese rind covering in Microbiological activity Belgium and of nystatin on the skin (but not in the The tetracyclines are broad spectrum antibiotics, flesh) of bananas in the United Kingdom (see Appen­ active against a range of gram-positive and gram­ dix). In neither of these cases is a siguificant ingestion negative bacteria, acid-fast actinomycetes, spirochaetes of the antibiotic by humans likely to occur. In the and rickettsia but without effect on fungi. They are U.S.S.R. the use of nystatin to control fungal growth primarily bacteriostatic in action (Otto, Alford, Grundy on CTG·treated meat has been permitted for industrial and Silvester, 1961). Resistance to tetracyclines, when trials (Shevtchenko, 1966). In the U.S.A., a proposal to used therapeutically, develops slowly but cross­ use pimaricin to reduce fungal spoilage in canned resistance occurs between the different tetracyclines. orange juice and in orange juice concentrate has not Generally salmonellae and Proteus spp. are less suscept­ been accepted by the F.D.A., who have asked for ible than are gram-positive bacteria such as Staphy­ further information on the toxicity and on the effects lococcus aureus (Korzybski eta/., 1967). of its use in fruit juice (Anon, 1966). Varying reports have been made of the sensitivity The use of pimaricin and nystatin as external pre­ of bacteria responsible for the spoilage of flesh foods. servatives cannot reasonably be criticized. However, Goldberg, Weiser and Deatherage (1953) showed that when incorporated in a foodstuff they do not satisfy of three hundred strains of microorganisms isolated all the criteria for use of antibiotics in foods. Although from beef, none were resistant to CTC. Brown and they appear to be satisfactory on the basis of their low Weidemann (1958) reported that 80% of 81 bacterial oral toxicities, little is known about the development isolates (probably predominantly pseudomonads) from of resistance, cross-resistance io other anti-fungal beef were inhibited in vitro by 10 ppm CTC and 73% antibiotics, changes in spoilage patterns of given were inhibited by 10 ppm OTC. Similar results were foods, etc. reported by Velankar (1958). All bacterial isolates 45 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti. from commercially processed chickens, untreated with Taylor and Davis, 1960, 1961). Dubrova (!963) ere, which had been allowed to spoil at 7.2°C were reported an increase in storage life of about 15 days found to be sensitive to ere, but 24-44% of isolates for CTC-treated (25 ppm) poultry. The effectiveness from ere-treated chickens were resistant to 15 ppm of the antibiotic in retarding spoilage was shown to ere (Ng, Vaughn and Stewart, 1957). Thatcher and be very much reduced when the storage temperature Loit (1961) reported that ere treatment resnlted in was increased to !5°C (Shrimpton, 1957; Hobbs eta/., an increased level of resistance amongst organisms 1960). Poultry is treated normally by immersion in isolated from poultry. Antiobitic-resistant strains 'slush ice' or in refrigerated water containing the were isolated both from treated and untreated birds antibiotic at 10 ppm (Barnes and Shrimpton, 1958). and included strains of species of Pseudomonas, Achromobacter and Flavobacterium. Both CTC-resi­ Meat. Several methods are available for the treat­ stant and CTC-sensitive strains of species of Pseu­ ment of meat with antibiotic. The antibiotic (50--- domonas, Achromobacter, Flavobacterium, Bacillus 100 ppm) may be applied to the surface of the meat, and Lactobacillus were isolated from perch (Sebastodes e.g. by spraying, which may then be followed by alutus), but more ere-resistant strains accumulated wrapping in antibiotic-impregnated film (Firman, with increasing CTC concentration and with increas­ Bachmann, Heyrich and Hopper, 1959; Ayres, 1959). ing storage time in the presence of ere (Lee and An alternative method is by injection of the antibiotic Sinnhuber, 1967). Dubrova (1963) found that the prior to or immediately after slaughter (Weiser et a/., resistance of Achromobacter liquefaciens and of three 1953; Ingram, Barnes and Shewan, 1956; Firman strains of Pseudomonas fiuorescens developed more et a/., 1959). Possibly the most satisfactory method slowly at oo than at 20°C in in vitro experiments. of treatment for meat is by intravenous injection of the live animals immediately before or as a spray immediately after slaughter (Firman et a/., 1959). Applications The storage life of ground beef was found to be The storage life of fish, poultry and meat may be extended for 5-9 days at !0°C when treated with extended by the use of tetracyclines, usually in com­ either ere or OTC (Goldberg, Weiser and Deatherage bination with low temperatures. Both ere and OTC 1955). Wrapping fresh beef in pliofilm impregnated are used, CTC being generally the more effective in with ere was reported to increase the storage life preserving flesh foods. Their application inhibits the by two days at and by one day at !5°C (Ayres, growth of spoilage bacteria of the surface epithelia soc 1959). and so delays the penetration of the flesh and its subsequent spoilage. Changes in the flora of flesh foods resulting from Fish. In an investigation of the efficacy of a number treatment with tetracyclines. of antibiotics in the preservation of raw fish it was shown that both ere and OTC were effective in The flesh of newly caught fish is assumed to be retarding spoilage at concentrations of 10 ppm (Tarr, sterile but on the skin and in the gastro-intestinal Southcott and Bissett, 1952). In a laboratory scale tract large numbers of microorganisms are usually trial an extension in the storage life of approximately present. Fish from "colder" waters usually have ten days was obtained for codling and haddock by psychrophilic bacteria of the Pseudomonas, Achromo­ keeping the fish in ice containing 5 ppm ere (Shewan bacter, Flavobacterium and Micrococcus genera present and Stewart, 1958). In commercial trials detectable on the skin. Over a period of storage in ice, spoilage differences in quality between fish stored in untreated is caused by growth of these bacteria, in particular ice and in antibiotic ice became evident after ten to by strains of Achromobacter and Pseudomonas, non­ twelve days of storage (Shewan and Stewart, 1958). pigmented members of the latter becoming dominant In commercial practice two ot three days extension after storage for 15-20 days. Fish from "warmer" in storage life may be obtained (Woods, 1966). waters have fewer psychrophiles persent and greater Treatment of fish with antibiotic may be carried out numbers of mesophiles, e.g. Bacillus spp., micrococci by storage in ice containing 5 ppm antibiotic or, for and coryneforms (Shewan and Stewart, 1958; Shewan, fillets, by dipping in an antibiotic solution (10 ppm) Hobbs and Hodgkiss, 1960; Shewan, 1961; De Silva, and storing at 0---1 oc (Tarr, 1955). 1964). The changes in the bacterial skin flora of the herring, resulting from the addition of either OTC Poultry. Ayres and co-workers (1956) investigated or ere to the storage ice, were studied by De Silva the effect of dipping eviscerated chicken into antibiotic (1964). OTC resulted in an earlier selection of the solutions for two hours at 1·7°C. Of several antibiotics dominant pseudomonads as compared to controls tested, ere was found to be most effective at con­ immersed in ordinary ice; CTC also resulted in the centrations of 3-10 ppm, but OTC was as effective selection of pseudomonads but strains of Achromo­ when used at 30 ppm. The storage life of eviscerated bacter spp. formed an important part of the flora poultry was extended from thirteen to eighteen days up to and after about 20 days of storage, due pre­ by using ere in the dip ice at a concentration of sumably to the selection of resistant strains. Fluorescent 10 ppm and storing at I oc (Barnes and Shrimpton, pseudomonads, which normally do not form an 1958). The development of off-odours in commercially important part of the flora, were favoured by the processed chickens and turkeys was shown to be antibiotics, particulady by OTC. Antibiotics can do delayed for 5-7 days and 10 days respectively, by little to assist in the preservation of herring because the use of 10 ppm CTC in the dip tanks. (Walker, other more rapid changes, resulting from chemical 46 The use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti. spoilage, occur (Anon, 1964); however, qualitative (1955) reported that five minutes heating at 90°C changes in the bacterial flora are similar to those that reduced the level of CTC in the flesh of fish from occur with other fish. 5 ppm to less than 0·3 ppm. A progressive re9uction in the initial amount of CTC (21 ppm) in "bonito" The flora of poultry and meat stored at low tempera­ muscle was demonstrated on heating and after 60 tures is similar to that of fish, the important factor min at 76°C no residue was detectable (Tomiyama, in determining the spoilage flora being temperature. Yone and Mikajiri, 1957). CTC (3 ppm) present in Thatcher and Loit (1961) isolated strains of species of the muscle of chicken was destroyed by boiling for Pseudomonas, Alcaligenes, Achromobacter and Flavo­ 15 min or by roasting at 230°C for 30 min (Kohler, bacterium from untreated and CTC•treated commer­ Miller and Broquist, 1955). These results are not all cial poultry. The resistance to CTC of Achromobacter in agreement. Dubrova (1963) cast doubts on claims spp. from treated poultry was significantly higher that the boiling of poultry containing 7 ppm CTC (112 ppm) than that of strains from untreated poultry resulted in complete inactivation of the antibiotic, (28 ppm). Barnes and Shrimpton (1958) reported but supposed that such residues as were present that the spoilage of untreated and OTC-treated would not be harmful to humans. poultry was caused by pseudomonads, wliilst spoilage of CTC-treated poultry was caused by strains of Achromobacter spp. and yeasts. Yeast spoilage of Microbiological Assay CTC-treated poultry was reported also by Dubrova (1963) and by Ayres et a/., (1953). Assay of tetracyclines in foods may be carried out by the agar diffusion method. Tomiyama et al. (1957) Strains of Saccharomyces cerevisiae, Sacch. dariensis, described a method using spores of Bacillus cereus Rhodotorula minuta, Geotrichum candidum, Torulopsis (strain No. 5) as the test organism. Extraction of the globosum, Candida spp., Cladosporium spp., Mucor antibiotic from tissue was carried out in a Waring spp., Penicillium spp. and Rhizopus nigrificans were blender with an acetone-water-HC! solvent, followed isolated from CTC-treated poultry which had spoiled by centrifugation and adjustment of the supernatant (Njoku-Obi, Spencer, Santer and Eklund, 1957; to pH 4·2. In the preparation of the standard, to Walker and Ayres, 1959). As they had isolated no allow for the binding effect of the antibiotic with pathogenic species, Walker and Ayres (1959) concluded protein, untreated tissue was ground with the standard that there was no public health danger in the use of CTC solution. Successive I :I dilutions were prepared CTC. However, Njoku-Obi et a!., (1957) isolated a from this to obtain a final CTC concentration of strain of Candida parapsilosis, a human pathogen, 0·062 ppm. Paper discs were used for application from antibiotic-treated poultry. Incorporation of of the antibiotic to the assay plate. Dubrova (1963) nystatin (at 5 and 10 ppm) into the dip ice was effective described an agar diffusion assay using vegetative in preventing the growth of fungi and development of cells of Bacillus subtilis (strain L,) which were spread fungal odours in CTC-treated chickens stored for as a "lawn" over the surface of the agar; the antibiotic 15 days (Yacowitz eta/., 1957). solutions were applied in wells cut in the agar. Goldberg, Weiser and Deatherage (1953) reported the isolation from beef of strains of species of Pseu­ Toxicity and Hypersensitivity domonas, Alcaligenes, Flavobacterium, Micrococcus, Streptococcus, Bacillus, Clostridium, Corynebacterium, The effects on both humans and other animals of Escherichia and intermediate coliforms. A similar long term feeding of tetracyclines, at concentrations group of bacteria was isolated from beef by Jay, similar to and greater than those used in food preserva­ Weiser and Deatherage (1957) who found that the tion, have been investigated. Trials on rats showed presence of CTC reduced the numbers of coliforms, that the toxicity of CTC taken orally at high dose enterococci and Bacillus spp. isolated. levels was very low even over a two year period (Dessan and Sullivan, 1961). Chicken meat, treated Persistence of tetracyclines in food with CTC at a concentration of 1000 ppm (i.e. more than 100 times greater than the level normally used Cod stored in ice containing 5 ppm CTC showed a in the preservation of food) was fed to rats with no gradual but unequal build-up to antibiotic. After adverse effects (Bruggeman, Karg and Barth, 1961). twenty four days storage, the upper skin surface contained 12-14 ppm antibiotic but the lower skin In a study of the effect on adult humans of feeding contained only 2-3 ppm. In the flesh immediately 10 mg OTC per day (equivalent to 5-10 ppm anti­ below the upper skin there were 4 ppm and in that biotic in the total diet) for six months, Goldberg below the lower skin 0·8-1·0 ppm. In the deep flesh (1962) demonstrated that slight changes occurred in no antibiotic was detected during the first 10 days the gut flora including the development of tetracycline storage and thereafter the concentration never exceeded resistant coliforms (resistant to 10 ppm or more). 2 ppm. Cooking by boiling for 30 min, steaming for However, OTC-resistant coliforms also occurred prior 60 min or frying in fat for 3-5 min reduced the to antibiotic administration and OTC resistance was antibiotic residue in the flesh to a level at which it shown to be transitory. Hypersensitivity (skin tests) could not be detected and that in the skin by about did not develop and at this level of feeding no anti­ 60 %- Baking for 20 min at 350°C had little effect biotic was detectable in the blood serum (sensitivity on the antibiotic (Shewan and Stewart, 1958). Tarr of the assay method was 0·025 ppm). 47 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

Legislation and public health nspects from fowl in Great Britain during 1957 to 1959 were resistant to CTC. McWhorter, Murrell and Edwards Legislation permitting the use of tetracyclines in (1963) reported that Salm. typhimurium displayed a foods has concerned the preservation of raw fish and, much higher incidence of resistance to CTC than did to a lesser extent, dressed poultry; their use in meat other serotypes and that resistance amongst cultures preservation has been limited (see Appendix). The from animals other than fowl was due largely to the foods in which they are, and have been, used are flesh high incidence of resistance among strains from foods subject to rapid microhiological deterioration; bovine sources. They concluded that there had been the use of antibiotics has been to control the surface a considerable increase in the frequency of eTC­ bacterial growth so that the consumer benefits from a resistant salmonellae concomitant with the use of "fresher" product. It is possible} however, to use these antibiotics as feed supplements. antibiotics not to provide a "fresher" product but to use the extension in storage life to obtain commercial The risk of food poisoning organisms being present economies (e.g. with fish, by extending the time the in foods which may be treated with antibiotics varies trawler is at the fishing ground), or to try to use them with the food. Fish do not suffer normally from to cover insanitary processing conditions. salmonella infection but they may act as carriers for short periods if they have been in polluted waters, the The decision of the American F.D.A. (Anon, 1966) risk being much greater for fresh water fish. Marine to rescind permission for use of CTC and OTC in fish caught in the open sea have been found to be food preservation was based on a number of factors, free of salmonellae (Shewan and Liston, 1955; chief amongst these being that the use of tetracyclines Shewan, 1962). However, after fish have been caught had resulted in the selection of naturally resistant contamination may occur because of poor hygiene in strains of microorganisms and induced the emergence the handling of the fish (Buttiaux, 1962). The latter of resistant strains. The selection of resistant strains author stated that it would be a mistake to rely on of microorganisms is most likely to occur when an antibiotics to avoid hazards caused by unhygienic antibiotic is used at, or just about, the minimum handling of the fish. If fish treated with antibiotic inhibitory concentration (MIC). Since tetracyclines are is held at temperatures of 3°C or below there should bacteriostatic, and are used in food preservation at be no hazard; however, if this temperature is exceeded levels close to the MIC, resistant strains of micro­ significantly no such guarantee can be given. Poultry organisms have emerged. Tetracyclines are also used and meat animals may harbour salmonellae in the in animal feedstuffs and in some countries the use of alimentary canal and meat derived from these animals tetracyclines both in animal feed and as a preservative has been shown to be frequently contaminated with of food derived from animals fed on such feed is species of Salmonella (Hobbs, 1961, 1965). permitted. The danger that this practice may increase Transfer of drug resistance may occur amongst the possibility of inducing the emergence of resistant members of the Enterobacteriaceae (Watanabe and strains has been stressed elsewhere (Anon, 1963). The Fukasawa, 1961; Watanabe, 1963; Anderson, 1968). danger when the resistant strain is a food poisoning Organisms which have not been in contact with the organism was illustrated by Hobbs and co-workers drug may become resistant to it by transfer of the (1961). They investigated whether the use of CTC to drug resistance factor from resistant organisms. increase the storage life of dressed poultry (in con­ Transfer is not limited to related strains but may occur ditions simulating commercial practice) would affect between strains of different species or genera of the the control of salmonellae on the carcass and whether Enterobacteriaceae. The spread of drug resistance may resistant strains behaved differently from sensitive occur rapidly and may reach high levels. Anderson strains. They deliberately introduced known strains of and Lewis (1965) reported that of 450 cultures of salmonellae into the alimentary canal of chickens and Salmonella typhimurium examined, 61 % were resistant found that sensitive strains of Salmonella typhimurium to one or more drugs including the tetracyclines, and were inhibited by CTC treatment in a manner similar that in Salm. typhimurium type 29 drug resistance to other spoilage organisms, but that a resistant strain appeared to have reached 100%. This strain is found of the same species grew on the skin of CTCtreated not only in calves but also in human infections. chicken held at l5°C at a rate similar to that on control In Great Britain a joint committee was set up in birds. At 22°C the antibiotic resistant strain grew more rapidly on treated birds than it did in competition July 1968 to examine the use of antibiotics in animal husbandry and in veterinary medicine with particular with spoilage organisms on untreated birds. For these reference to the phenomenon of infectious drug reasons they concluded that the treatment of poultry resistance. Whether or not the use of antibiotics, with CTC was open to objection, particularly if the including the tetracyclines, as feed supplements has chickens were not held at refrgeration temperatures been a principal cause of the development of resistant after, as well as before, retail sale. strains of food poisoning organisms, resistant strains Huey and Edwards (1958) showed that resistant have emerged and their existence should give reason strains of Salmonella typhimurium appeared after for further consideration to be given to the permitted tetracyclines were introduced into use and that the use of tetracyclines as food preservatives. Further, percentage of such cultures increased with continued other evidence given here suggests that the tetra­ use of antibiotics both as therapeutic agents and as cyclines do not satisfy the criteria for use of anti­ feed additives. Garside, Gordon and Tucker (1960) biotics in food which were listed in the introduction found that 13·5% of 362 strains of Salmonella isolated to this paper. 48 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

TYSLOYIN 0 II Tylosin, which is a macrolide antibiotic produced H C by strains of Streptomyces fradiae (Hamill, Haney, \I\ Stamper and Wiley, 1961; McGuire et a/., 1961), is c active against gram-positive bacteria, mycobacteria II CH3 C and pleuropneumonia-like organisms. It is a weak \I\ base, sparingly soluble in water but readily forms C H II water-soluble salts and esters. A water•soluble deriva­ Mycinose - CH3 - CH tive, tylosin lactate', is available commercially. I CH I ; Mild hydrolysis of tylosin (C 45H 71N017 M.W. 903) yields the sugar Mycarose (I) and a new antibiotic C•H• -1H lC.,HuO desmycosin, the properties of which are similar to 0 CHO those of tylosin. Refluxing of tylosin at pH 2 yields 0-b- Mycaminose-Mycarose the sugar Mycinose (II), whilst vigorous hydrolysis of both desmycosin and tylosin at pH I yields Mycami­ (IV) Partial structure of Tylosin nose (III) and degradation products (Hamill et a/., 1961). A tentative partial structure (IV) has been proposed by Gorman (Malin, personal communica­ tion, 1966).

'I Mode of Action and Bacterial Resistance

OH The activity of tylosin against spore-forming bacteria was first reported by Malin (1958) and has since been confirmed by many workers. Denny, Reed and Bohrer (1961) and Denny, Sharpe and Bohrer -(1961) showed that tylosin did not affect the heat 'I HO resistance of spores of B.stearothermophi/us or Cl. I botulinum and suggested that it prevented outgrowth 0 0 of the germinated spore. Hawley (1961 c) and Green­ berg & Silliker (1962 a) observed that tylosin inhibited (I) Mycarose (II) Mycinose growth of vegetative forms but did not prevent spore germination. Gould (1964) and Poole and Malin (1964) showed that tylosin inhibited growth of the newly-emergent vegetative forms and did not affect germination or post-germination development of the spore (cf. nisin.).

Jarvis and Cheetham (unpublished data, 1968) have compared the effect of tylosin, nisin and subtilin on both vegetative forms and heat-shocked spores of several species of Bacillus. The results (Table I) suggest that tylosin is less active than nisin on a molar 0 basis but this may merely reflect the different stages (III) Mycaminose at which nisin and tylosin act.

'Tylosin lactate is manufactured by Eli Lilly & Co., Indianapolis 6, U.S.A. 49 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

TABLE 1-Comparison of the minimum inhibitory concentrations of tylosin, nisin and snhtilin wbicb prevent colony formation on agar by several species of Bacillus (Jarvis & Cheetham, unpublished data, 1968).

Nisin A • (Jimole) Subtilin t (Jimole) Tylosin (JJmole)

Organism Vegetative t Spore§ Vegetative Spore Vegetative t Spore§ Inoculum Inoculum Inoculum Inoculum Inoculum Inoculum

B.cereus > 0·70 > 0·70 5.60 5-60 3-33 3-33

B.polymyxa > 0·70 0·70 5·60 0·56 2·77 > 5·54 B.megaterium 0·53 0·13 - - HI 0·55 B.subtilis > 0·70 0·04 2-80 1·40 3-33 1-67

B.licheniformis > 0·70 0·08 2-80 1-40 3-33 1-67

B.stearothermophilus 0·04 <0·01 - - 0·55 0·55

• Jarvis (1967); t Gould (1964) t Vegetative inoculum: 400 organisms/plaque (for method see Jarvis, 1967) § Spore inoculum: 400 heat-shocked spores/plaque (Jarvis, 1967) I I

Vegetative forms of a putrefactive anaerobe develop­ the use of tylosin lactate in semi-preserved fish products ed resistance to tylosin when subcultured through stored at 20°C and showed that gas production in 2 ' increasing concentrations of the antibiotic (Poole and batches of "titbits" did not occur until after 10 and I Malin, 1964). Jarvis and Cheetham (unpublished data, 17 weeks when 20 ppm tylosin were used, whereas ! 1968) obtained a 300-fold increase in the resistance of in controls without tylosin gas-production was detect­ 'i a strain of B.cereus (NCIB 8579) in four sequential able after 3 and 4 weeks respectively, and no adverse transfers through increasing concentrations of tylosin, organoleptic effects were noted. In anchovy, tylosin ,j and the level of resistance was maintained for several did not delay gas formation significantly but it gave transfers through antibiotic-free medium. A two"fold a significant increase in the storage life of caviar. Both I increase in resistance was obtained in B.licheniformis Erichsen (1966) and Yoseki, Nemotot and Amana !I (NCIB 8061) but none was obtained in B.polymyxa (1966) observed a significant rapid decrease in the (NCIB 8094). Development of resistance to tylosin in activity of tylosin in fish products. I Staph.aureus was reported to be of the same type as .i' that against erythromycin, and resistant organisms Several reports have shown that Cl.botulinum is !' exhibited cross resistance to tylosin, desmycosin, inhibited by tylosin (Malin, 1963; Malin, Dobbs and erythromycin and oleanomycin but not to penicillin Martin, 1964; Greenberg and Silliker, 1964; Malin or other antibiotics tested (Greenberg, 1958; McGuire and Greenberg, 1964). Even with gross under-process­ eta/., 1961). ing of inoculated canned foods, no toxin formation occurred in cans containing 10 ppm tylosin but toxin occurred in tylosin-free controls. Sheneman (1964) Applications of tylosin in food preservation reported the inhibition of botulin formation in white­ Wheaton and Hays (1964) used 2·5 ppm tylosin in fish chubs processed at low temperature in brine conjunction with a reduced thermal process for the containing 100 ppm tylosin lactate. control of flat-sour spoilage in low-acid food stuffs such as chow mein, cream of chicken soup, tuna and Staphylococci are very sensitive to tylosin in vitro noodles and cream-style corn. The use of nisin and (Greenberg, 1958; McGuire et a/., 1961) although tylosin for the control of flat-souring was compared resistant strains develop readily. Greenberg and by Segmiller, Xerones and Hutchings (1965), who Silliker (1962 b) observed that the growth of coagulase­ concluded that tylosin was the more effective in low­ positive staphylococci in processed cheese, ham, acid foods whilst nisin was the more effective in acid sausages and ice.cream mix was inhibited by tylosin. foods. Wheaton, Burroughs and Hays (1957), Camp­ Malin (1964) observed that lactic acid bacteria were bell, Sniff and O'Brien (1959) and Denny et a/., (1961 frequently resistant to tylosin and suggested its use a, b) used tylosin to control thermophilic spoilage in for the control of sporeforming bacteria in lactic a range of canned foods. Erichsen (1966) investigated fermentations (e.g. cheese and yoghurt). 50 I

The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

Detection and Assay foods have been rejected on the basis of cross­ resistance to therapeutically important antibiotics such Tylosin may be extracted and identified by chroma­ as erythromycin and oleandomycin (Anon, 1966). tography and spectroscopy. However, these methods are of little use in the routine identification of tylosin in food products. Several methods for the assay of Conclusions tylosin have been proposed but an agar diffusion method, using Sarcina lutea as the test organism, is the In this review consideration has been given only to most suitable method for assay of tylosin and desmyco­ those antibiotics which are currently permitted in sin in food extracts (Kavanagh & Dennin, 1963). foods, or for which applications have been filed. The Malin (personal communication) reports that the use of other antibiotics has been investigated, frequent­ National Canners Association uses B.stearothermo­ ly without success, although useful preservative effects philus 1518 in a method which is essentially the same have been obtained in some instances. In the latter as that proposed for the assay of nisin (Stumbo et al., cases, their use has not been advocated because they 1964). The "Testab D"l method (Ryder, 1965) is did not satisfy the criteria for use in the food industry useful for rapid assay of tylosin and other antimicro­ (e.g. because of a high toxicity rating or because of bials but the tablets are not available commercially clinical use, etc. . . . .). Details of many of these in the U.K. investigations have been given by Marth (1966). Yoseki et a/ (1966) used the S.lutea assay to investi­ The object of this paper has been to consider whether gate the loss of tylosin activity in heated fish sausage. or not the "permitted antibiotics" satisfy the ideal Low recoveries of activity were obtained when the criteria for use in foods and to consider their use both fish was extracted at neutral pH levels, 50% recovery in relation to ··the scientific data and the current with extraction in dilute acid (pH 4·0) -and 100% legislation governing their use. On the basis of this recovery after proteolytic digestion of the fish protein. ·survey our conclusions are that tylosin and the tetra­ They concluded that mild heat (85° for 30 min.) did cyclines do not really satisfy the criteria for use and not destroy the tylosin but that its activity was lost that, in the case of both nystatin and pimaricin, due to adsorption on the fish protein; "bound" insufficient data on factors affecting their use are tylosin had no antibacterial activity. By contrast, presently available. Although nisin satisfies the criteria almost 100% recovery was obtained with tylosin in on most aspects doubt remains as to whether its use is starchy materials, even after heating, and good re­ justified on the basis of effectiveness, except in certain covery was obtained from the raw flesh of fish. Jarvis specific applications such as in processed cheese and and Erridge (unpublished data, 1965) obtained quan­ in canned products such as chocolate milk. titative recovery of activity from tylosin.Jactate treated If antibiotics are to be used in foods without comminuted raw beef using the methods of .Ryder introducing hazards to public health, further research (1965) and of Kavanagh and Dennin (1963). must be made into the application of some of the many thousands of antibiotics which have been discarded by the pharmaceutical industry as being Legislation and Public Health unsuitable for clinical use. Amongst these discards some may exist which would satisfy the criteria for That tylosin is effective in preventing post-processing use in foods and which are not related closely to spoilage in canned foods is not in doubt and the antibiotics in clinical use. One would hope that such inhibition of toxin formation by Cl.botulinum suggests investigations might lead to the establishment of a that use of tylosin might provide a health safeguard special group of antibiotics, the use of which would be in the event of foods being grossly under-processed. restricted by international legislation to that of food However, the possibility of gross under processing of preservation. foods is undesirable and the presence of low levels of tylosin might cover up spoilage whilst permitting some toxin formation. The use of any antibiotic in canned Acknowledgements foods without the safeguard of a minmum botulinum cook could not be countenanced with due regard for We are indebted to Mr. S. Green, College Librarian, public safety. At the present time the use of tylosin to Miss S. P. Foster, Librarian at Glaxo Research in foods is prohibited in all countries, although Laboratories, Stoke Poges, Bucks., and to the sta!I of residues might arise in meats from the use of tylosin the Information Department at the British Industrial in animal feeds. However, Anderson, Worth, Small Biological Research Association, Carshalton, Surrey, and Harris (1966) have shown that tylosin is atoxic for their assistance in obtaining material used in this when administered to experimental animals. review. We are also indebted to the many overseas governments for supplying details of their food The ready development of resistance to tylosin in legislation, to Mr. G. G. Fowler of Aplin & Barrett vitro suggests that health hazards might arise from the Ltd., Yeovil, Som., and to Dr. B. Malin of Eli Lilley in vivo development of resistant organisms. Recent and Co. Ltd., Indianapolis 6, U.S.A., for providing petitions to the FDA for the use of tylosin in certain unpublished information.

lAvailable from Eli Lilly & Co. Ltd., Indianapolis 6, U.S.A. 51 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

References Barnes, E. M. and Shrimpton, D. H. (1958). The Alifax, R. and Chevalier, R. (1962). Study of the effect of the tetracycline compounds on the storage nisinase produced by Streptococcus thermophilus. life and microbiology of chilled eviscerated poultry. J. Dairy Res., 29, 233. J. appl. Bact., 21, 313. Barr, F. S. (1959). Tennecetin: A new antifungal Anderson, A. A. and Michener, H. D. (1950 a). Effect of subtilin on bacterial spores. Bact. Proc., 1950, 28. antibiotic-Toxicological studies. Antibiotics Chem­ other., 9, 406. Anderson, A. A. and Michener, H. D. (1950 b). Berridge, N. J. (1949). Preparation of the antibiotic Preservation of foods with antibiotics. I. The nisin. Biochem. J., 45, 486. complementary action of subtilin and mild heat. Fd Techno/., Champaign, 4, 188. Berridge, N. J. (1953). The antibiotic nisin and its use in the making and processing of cheese. Cherny. Ind., Anderson, E. S. (1968). The ecology of transferable 1953, 1158. drug resistance in the Enterobacteria. A. Rev. Microbial., 22, 131. Berridge, N.J., Newton, G. G. F. and Abraham, E. P. (1952). Purification and nature of the antibiotic Anderson, E. S. and Lewis, M. J. (1965). Drug resist­ nisin. Biochem. J., 52, 529. ance and its transfer in Salmonella typhimurium. Nature, Lond., 206, 579. Berry, M. (1963). The macrolide antibiotics. Q.Rev. chem.Soc., 17, 343. Anderson, R. C., Worth, H. M., Small, R. M. and Harris, P. N. (1966). Toxicological studies on tylosin: British Standards Institute, B.S. 4020 (1966). Estima­ tion of nisin in cheese. its safety as a food additive. Fd Cosmet. Toxico/., 4, I. Brown, A. D. and Weidemann, J. C. (1958). The Anon. (1963 a). The public health aspects of the use of taxonomy of the psychrophilic meat spoilage antibiotics in food and feedstuffs. Wid Hlth Org. bacteria. A reassessment. J.appl.Bact., 21, II. techn. Rep. Ser., No. 260. Briiggemann, J., Karg, H. and Barth, K. (1961). Anon, (1963 b). Pimaricin for food preservation. Reaction of albino rats to chronic feeding with Techn. Rep., American Cyanamid Co. chicken meat pretreated with chlortetracycline. Studies on generations of rats. Arzneimittel-Forsch., Anon. (1964). Antibiotics and the preservation of wet 11, 1022. fish. Torry Advisory Note No. 19; Torry Research Station, Aberdeen. Buttiaux, R. (1962). Salmonella problems in the sea. in Fish as Food, Vol II, p 503-519 (ed. by G. Borg­ Anon. (1966). Revocation of antibiotic clearances on strom); Academic Press, London. raw poultry, fish proposed. Fd chem. News, 8 (Aug. 22nd 1966), 25. Bylund, G., Norrgren, 0. and Sjostrom, G. (1954). The use of antibiotic producing strains of Strepto­ Anon. (1968). Comparison of side effects of tetra­ coccus lactis to combat butyric acid fermentation cycline and tetracycline-plus nystatin. Brit.Med.J., in cheese. An orientating experiment. Svenska 4 (5628), 411. Mejeritidn., 46, 433. Ayres, J. C. (1959). Use of coating materials or film Campbell, L. L. and Sniff, E. E. (1959). Effect of impregnated with chlortetracycline to enhance subtilin and nisin on the spores of B.coagulans. colour and storage life of fresh beef. Fd Techno!., J.Bact., 77, 766. Champaign, 13, 512. Campbell, L. L., Sniff, E. E. and O'Brien, R. T. Ayres, J. C. and Denisen, E. L. (1958). Maintaining (1959). Subtilin and nisin as additives that lower the freshness of berries using selected packaging mater­ heat-process requirements of canned foods. Fd ials and antifungal agents. Fd Techno/., Champaign., Techno!., Champaign, 13, 462. 12, 562. Carlson S. and Bauer, H. M. (1957). A study of Ayres, J. C., Kraft, A. A., Denisen, E. L. and Pierce, probl~ms associated with resistance to nisin, an L. C. (1964 a). The use of macrolide antifungal antibiotic produced by Streptococcus lactis. Arch. antibiotics in delaying spoilage of fresh small fruits Hyg. Bakt., 141, 445. and tomatoes. in Microbial Inhibitors in Foods, Ceder 0. Waisvisz, J. M., Van der Hoeven, M. G. p 185-193. (ed. by N. Molin); Almqvist & Wiksell, ' and' Ryhage, R. (1963). On the structure ofptmancm. . . Stockholm. Chimia, 17, 352. Ayres, J. C., Kraft, A. A. and Pierce, L. C. (1964 b). Cheeseman, G. C. and Berridge, N.J. (1959). Observa­ Delaying spoilage of tomatoes. Fd Techno!., Cham­ tions on the molecular weight and chemical compo­ paign, 18, 1210. sition of nisin A. Biochem.J., 71, 185. ' Ayres, J. C., Walker, H. W., Fanelli, M. J., King, Cirillo, V. P., Harsch, M. and Lampen, J. 0. (1964). A. W. and Thomas, F. (1956). Use of antibiotics in Action of the polyene antibiotics filipin, nystatin prolonging storage life of dressed chicken. Fd and N-acetyl candidin on the yeast cell membrane. Techno/., Champaign, 10, 563. J.gen.Microbiol., 35, 249. Bacharach, (1957). Antibiotics: the legal position. Claypool, L., Heinemann, B., Voris, ~·. a~d Stumbo, Cherny. Ind., 1957 (36), 1208. C. R. (1966). Residence time of msm m the o~al Bardsley, A. (1962). Antibiotics in food canning. cavity following consumption of chocolate m!lk Fd Techno/., Aust., 14, 532 & 606. containing nisin. J.Dairy.Sci., 49, 314. 52 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

Deatherage, F. E. (1955). The use of antibiotics in the Galesloot, Th.E. and Pette, J. W. (1957). The forma­ preservation of foods other than fish. Proc. 1st. tion of normal eyes in Edam cheese made by nisin­ inter. conference on the use of antibiotics in agri­ producing starters. Ned. Malk-en Zuiveltijdschr., 11, culture. p 211. 144. Denny, C. B., Reed, J. M. and Bohrer, C. W. (1961). Galesloot, Th.E., Raadsveld, G. W. and Petie, J. W. Effect of tylosin and heat on spoilage bacteria in (1957). The influence of nisin-producing starters canned com and canned mushrooms. Fd Techno/., upon the ripening of Edam cheese. Ned. Me/k-en Champaign, 15, 338. Zuiveltijdschr., 11, 152. Garside, J. S., Gordon, R. F. and Tucker, J. F. (1960). Denny, C. B., Sharpe, L. E. and Bohrer, C. W. (1961). The emergence of resistant strains of Salmonella Effects of tylosin and nisin on canned-food spoilage typhimurium in the tissues and alimentary tracts of bacteria. App/.Microbiol., 9, 108. chickens following the feeding of an antibiotic. DeSilva, N. N. (1964). Changes in the spoilage flora Res. vet. Sci., 1, 184. during preservation of fish with antibiotics. in Gerke, J. R., Gentile, J., Stander, H. and Pagano, Microbial Inhibitors in Foods, p 245-259 (ed. by J. F. (1959). Respirometric assay of nystatin in N. Molin); Almqvist & Wiksell, Stockholm. animal tissues. Antibiotics A., (1959-60), 563. Dessan, F. I. and Sullivan, W. J. (1961). A two-year Gibbs, B. M. and Hurst, A. (1964). Limitations of study of toxicity of chlortetracycline-hydrochloride nisin as a preservative in non-dairy foods. in in rats. Toxic. appl. Pharmac., 3, 645. Microbial Inhibitors in Foods, p 151 (ed. by N. Molin); Almqvist & Wiksell, Stockholm. DiMarco, G. R. and Davis, B. H. (1957 a). Prevention Gillespy, T. G. (1957). Nisin Trials. Fruit & Veg. Cann. of decay of strawberries with post-harvest treat­ Quick Freez. Res. Assoc. Leaflet No 3, Chipping ments. PI.Dis.Reptr., 41, 460. Campden, Glos. DiMarco, G. R. and Davis, B. H. (1957 b). Prevention Gola, J. (1962). Science and Research in the Food of decay of peaches with post-harvest treatments. Industry. Collected reports of the Research Institute Phytopathology, 47, 244. for Meat, Brno., 10,239. Gold, W., Stander, H. and Pansy, F. E. (1953). Dubrova, G. B. (1963). The use of antibiotics for the Assay methods for nystatin. Antibiotics. A. (1953- preservation of food products. Translation from the 54), 195. Russian, pub. by Nat/. Lending Library for Science Goldberg, H. S. (1962). Evaluation of some potential and Techno/., Boston Spa., Yorks. public health hazards from non-medical uses of Duggar, B. M. (1948). Aureomycin, a product of the antibiotics. in Antibiotics in Agriculture, p 389-404 continuing search for new antibiotics. Ann. N.Y. (ed. by M. Woodbine); Butterworths, London. Acad. Sci., 51, 177. Goldberg, H. S., Weiser, H. H. and Deatherage, F. E. Eastoe, J. E. and Long, J. E. (1958). The effect of (1953). Studies on meat. IV. The use of antibiotics nisin on the growth of cells and spores of Clostridium in preservation of fresh beef. Fd Techno!., Cham­ welchii in gelatin. J. Appl. Bact., 22, I. paign, 7, 165. Erichsen, I. (1967). The effect of tylosin lactate on the Golding, B. T., Rickards, R. W., Meyer, W. E., shelf-life of some fish products. 2nd. Inter. Congr. Patrick, J. B. and Barker,-M. (1966). Structure of Fd Sci. Techno/., Warsaw, p 351, Abstract E.1.15. the macrolide antibiotic pimaricin. Tetrahedron Lett., (30), 3551. Farber, L. (1959). Antibiotics in food preservation. Gould, G .. W. (1962). Microscopic observations on A. Rev. Microbial., 13, 125. the emergence of cells of Bacillus spp. from spores Firman, M. C., Bachmann, H. J., Heydrich, F. J. under different cultural conditions. J. Appl. Bact., and Hopper, P. F. (1959). A comparison of methods 25, 35. for applying acronise chlortetracycline to beef. Gould, G. W. (1964). Effect of food preservatives on Fd Techno/., Champaign, 13, 529. the growth of bacteria from spores. in Microbial Inhibitors in Foods, p 17 (ed. by N. Molin); Almqvist Franklin, T. J. (1963). The inhibition of incorporation & Wiksell, Stockholm. of leucine into protein of cell-free systems from rat liver and Escherichia coli by chlortetracycline. Gould, G. W. and Hurst, A. (1962). Inhibition of Bacillus spore development by nisin and subtilin. Biochem. J., 87, 449. 8th inter. Congr. Microbiol., Abstract A2-11. Franklin, T. J. (1964). The effect of chlortetracycline Gowans, J. L., Smith, N. and Florey, H. W. (1952). on the transfer of leucine and transfer ribonucleic Some properties of nisin. Brit. J. Pharmacal., 7, 438. acid in rat liver ribosomes in vitro. Biochem. J., Greenberg, R. A. (1958) unpublished data, Swift & 90, 629. Co., Chicago, U.S.A. Frazer, A. C., Sherratt, M. and Hickman, J. R. Greenberg, R. A. Silliker, J. H. (1962 a). The aciion (1962). The biological effects of food additives (I) of tylosin on spore-forming bacteria. J. Fd Sci., Nisin. J. Sci. Fd Agric., 13, 32. 27, 64. Galesloot, Th. E. (1956). Lactic acid bacteria which Greenberg, R. A. and Silliker, J. H. (1962 b). The destroy the antibiotic nisin. Ned. Me/k-en Zuivel­ effect of tylosin on coagulase-positive staphylococci tifdschr., 10, 143. in food products. J. Fd. Sc., 27, 60. 53 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

Greenberg, R. A. and Silliker, J. H. (1964). Spoilage Hirsch, A. and Grinstead, E. (1954). Methods for the patterns Clostridium botulinum inoculated canned growth and enumeration of anaerobic spore-formers foods treated with tylosin. in Microbial Inhibitors in of cheese, with observations on the effect of nisin. Foods, p 97-103 (ed. by N. Molin); Almqvist & J. Dairy Res., 21, 10 I. Wiksell, Stockholm. Hirsch, A., Grinstead, E., Chapman, H. R. and Gregory, M. E., Henry, K. M. and Kon, S. K. (1964). Mattick, A. T. R. (1951). A note on the inhibition Nutritive properties of freshly prepared and stored of an anaerobic sporeformer in Swiss-type cheese evaporated milks manufactured by a normal com­ by a nisin-producing streptococcus. J. Dairy Res., mercial procedure, or by reduced thermal process 18, 205. in the presence of nisin. J. Dairy Res., 31, 113. Hirsch, A. and Mattick, A. T. R. (1949). Some recent Gross, E. and Morrell, J. L. (1967). The presence of applications of nisin. Lancet, ii, 190. dehydroalanine in the antibiotic nisin and its Hitchins, A. D., Gould, G. W. and Hurst, A. (1963). relationship to activity. J. Amer. chem. Soc., 89, The swelling of bacterial spores during germination 2791. and outgrowth, J. qen. Microbiol., 30, 445. Halasz, A. and Juhasz, E. (1963). Activity changes of Hobbs, B. C. (1961). The public health significance of nisin due to heat and X-ray irradiation effects. Salmonella carriers in livestock and birds. J. appl. Elelm. !par., 17, 160. Bact., 24, 340. Hall, R. H. (1966). Nisin and food preservation. Hobbs, B. C. (1965). The contamination of meat Process Biochem., 1, 461. supplies. Mon. Bull. Minist. Hlth., 24, 123. Hamill, R. L., Haney, M. E., Stamper, M. and Wiley, Hobbs, B. C., Reeves, J. C., Garside, J. S., Gordon, P. F. (1961). Tylosin, a new antibiotic: II. Isolation, R. F., Barnes, E. H., Shrimpton, D. H. and Andersen properties and preparation of desmycosin, a micro­ E. S. (1960). Antibiotic treatment of poultry in biologically active degradation product. Antibiot. relation to Salmonella typhimurium. Mon. Bull. Chemother., 11, 328. Minist. Hlth., 19, 178. Hawley, H. B. (1957). Nisin in food technology. Fd Huey, C. R. and Edwards, P. R. (1958). The resistance Mnfr., 32, 370 & 430. of Salmonella typhimurium to tetracyclines. Proc. Soc. exp. Bioi. Med., 97, 550. Hawley, H. B. (1961 a). The use of the thermal adjunct Ingram, M., Barnes, E. M. and Shewan, J. M. (1956). nisin in the processing of meat products packed in Problems in the use of antibiotics for preserving plastic films. CIIA-BIPCA, 7th Symposium sur les meat and fish. Fd Sci. Abs., 28, 121. Matieres Etrangeres dans les Aliments, 11-17th October, Belgrade. Ingram, M., Buttiaux, R. and Mossell, D. A. A. (1964). General microbiological considerations in the choice Hawley, H. B. (1961 b). Nisin as a thermal adjunct of anti"microbial food preservatives. in Microbial for processing meats packaged in plastic film. Inhibitors in Foods, p 381 (ed. by N. Molin); Packaging, 32, 93. Almqvist & Wiksell, Stockholm. Hawley, H. B. (1961 c). The use of nisin in food pro­ Jarvis, B. (1966 a). Observations on the inactivation of cessing. Discourse, Institute Food Technology, Uni­ nisin by enzymes from Bacillus cereus and B. versity of Moscow. polymyxa. Cherny. Ind. 1967, (21), 882. Hawley, H. B. (1962). The uses of antibiotics in Jarvis, B. (1966 b). Technique for measurement of canning. in Antibiotics in Agriculture, p 272 (ed. by antibiotic inhibition zones. J. Dairy Sci., 49, 318. M. Woodbine); Butterworths, London. Jarvis, B. (1967 a). Resistance to nisin and production Hazen, E. L. and Brown, R. B. (1950). Two antifungal nisin-inactivating enzymes by several species of agents produced by a soil actinomycete. Science, Bacillus. J. Gen. Microbiol., 47, 33. N. Y., 112, 423. Jarvis, B. (1967 b). Inactivation of nisin by enzymes Hazen, E. L. and Brown, R. B. (1951). Fungicidin, an from Bacillus cereus and B.polymyxa. 4th meeting antibiotic produced by a soil actinomycete. Proc. Fed. Europ. biochem. Soc., Oslo, Abstract 502, p 126. Soc. exp. Bioi. Med., 76, 93. Jarvis, B. (1969). Mechanism of enzymic inactivation Heinemann, B., Stumbo, C. R. and Schurlock, A. of nisin by an enzyme from Bacillus cereus (in (1964). Use of nisin in preparing beverage-quality preparation). sterile chocolate-flavoured milk. J. Dairy Sci., 47, 8. Jarvis, B. and Berridge, N. J. (1968)c The application of enzymes which inactivate antibiotics. Paper Heinemann, B. and Williams, R. (1966). Inactivation presented at a Symposium on Microbial Enzymes, of nisin by pancreatin. J. Dairy Sci., 49, 312. Society of Chemical Industry, Sept. 1968. Heinemann, B., Voris, L. and Stumbo, C. R. (1965). Jarvis, B., Jeffcoat, J. and Cheeseman, G. C. (1968). Use of nisin in processing food products. Fd Molecular weight distribution of nisin. Biochim. Techno/., Champaign, 19, 592. biophys. Acta, 168, 153. Hirsch, A. (1951). Growth and nisin-production of a Jarvis, B. and Mahoney, R. R. (1969). Inactivation of strain of Streptococcus lac/is. J. gen. Microbiol., 5, nisin by «-chymotrypsin. J. Dairy Sci., submitted 208. for publication. 54 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

Jay, H. M., Weiser, H. H. and Deatherage, F. G. Lewis, J. C., Michener, H. D., Stumbo, C. R. and (1957 a). Studies on the mode of action of chlorte­ Titus, D. R. (1954). Antibiotics in food processing: tracycline on the preservation of beef. Appl. Micro­ Additives accelerating the death of spores by moist bioi., 5, 400. heat. J. agric. Fd Chern., 2, 298. Jay, J. M., Weiser, H. H. and Deatherage, F. E. Lilley, M. D. (1958). Food preservation with the (1957 b). Further studies on the preservation of antibiotic nisin. Research, 11, 472. beef with chlortetracycline. Fd Techno/., Champaign, 11, 563. Lipinska, E. (1956). Microbiological methods of control of butyric acid fermentation in cheese. Jay, J. M., Weiser, H. H. and Deatherage, F. E. Acta. Microbial. Polon., 5, 271. (1958). Inhibition in beef of chlortetracycline resistant bacteria by sub-bacteriostatic concentra­ Lipinska, E. (1961). Sugar fermentation in Edam tions of the antibiotic. App/. Microbial., 6, 343. cheese made with nisin-producing and nisin-resistant Kavanagh, F. and Dennin, L. J. (1963). Tylosin. in starters. Prac. Ins/. Przemylsu Mleczarskiego, 8, I. Analytical Microbiology, p 369 (ed. by F. Kavanagh), Lipinska, E. and Strzalkowska, M. (1959). Experiments Academic Press, New York. in training pure cheese cultures to resist antibiotics Kinsky, S.C. (1961). Alterations in the permeability of produced by Streptococcus lactis. Proc. 15th int. Neurospora crassa due to polyene antibiotics. J. Dairy Congr., 2, 572. Bact., 82, 889. Lipinska, E., Strzalkowska, M., Goettlich, W. and Kinsky, S. C. (1962). Effect of polyene antibiotics on Soltys, W. (1962). Application of nisin-resistant the protoplasts of Neurospora crassa. J. Bact., 83, starters to the production of Edam cheese. Proc .• 351. 16th int. Dairy Congr., B 849. Klis, J. B., Witter, L. D., and Ordal, Z. J. (1959). Lynch, H. J., Furcolow, M. L., Yates, J. L., Tosh, F. E. The effect of several antifungal antibiotics on the and Larsh, H. W. (1960). Toxicity and absoroption growth of common food spoilage fungi. Fd Techno/., studies in humans of newer antifungal agents. Champaign, 13, 124. Antimicrobial Agents A., (1960). 551. Kohler, A. R., Miller, W. H. and Broquist, H. P. McClintock, M., Serres, L., Marzolf, J. J., Hirsch, A. (1955). Aureomycin chlortetracycline and the con­ and Mocquot, G. (1952). The inhibitory action of trol of poultry spoilage. Fd Techno/., Champaign, 9, nisin-producing streptococci on the development of 151. anaerobic spore•bearers in processed Gruyere Kooy, J. S. (1952). Strains of Lactobaccil/us p/antarum cheese. J. Dairy Res., 19, 187. which inhibit the activity of the antibiotics produced McGuire, J. M., Boniece, W. S., Higgens, C. E., by Streptococcus lac tis. Ned. Me/k-en Zuiveltijdschr., Hoehn, M. M., Stark, W. M., Westhead, J. and 6, 323. Wolfe, R. N. (1961). Tylosin, a new antibiotic: Korzybski, T., Kowszyk- Cindifer, Z. and Kurylowicz, I. Microbiological studies. Antibiot. Chemother., 11, W. (1967). Antibiotics-Origin, Nature and Properties 320. Vol. I ; Pergamon Press, London. McWhorter, A. C., Murre!, M. C. and Edwards, P. R. Kosikowski, F. W. and Ledford, R. A. (1960). A (1963). Resistance of salmonellae isolated in 1962 reverse-phase assay test for antibiotics in milk. to chlortetracycline. Appl. Microbial., 11, 368. J. Amer. vet. Med. Assocn., 136, 297. Malin, B. (1958) Unpublished data. Eli Lilley & Co., Lamanna, C. (1940). The taxonomy of the genus Indianapolis 6, U.S.A. Bacillus. I. Modes of spore germination. J. Bact., 40, 347. Malin, B. (1963). The control of fermentation with Lampen, J. 0., Arnow, P., Borowski, Z. and Larkin, tylosin. Paper presented at the Global Impacts of A. (1962). Location and role of sterol at nystatin Applied Microbiology Conference, Stockholm, Swe­ binding sites. J. Bact., 84, ll52. den, July 1963. Laskin, A. I. (1967). Tetracyclines. in Antibiotics, Vol. Malin, B. (1964). Potential concomitant utilization of I Mechanism of Action, p 331-359 (ed. by D. Gott­ tylosin and reduced heat treatment. Dissertation at leib and P. D. Shaw); Springer-Verlag, Berlin. the University of Arizona, U.S.A., June, 1964. Lee, J. S. and Sinnhuber, R. 0. (1967). Selection of Malin, B., Dobbs, R. and Martin, L. (1964). The microbial population in chlortetracycline-treated complementary action of tylosin and sodium Ocean Perch (Sebastodes alutus). Appl. Microbial., chloride on Clostridium botulinum type E. Paper 15, 543. presented at the 24th meeting Inst. of Fd Techno/., Levinskas, G. J., Ribelin, E. and Shaffer, C. B. (1966). Washington D.C., U.S.A., May 1964. Acute and chronic toxicity of pimaricin. Toxicol. Malin, B. and Greenberg, R. A. (1964). The effect of appl. Plzarmacol., 8, 97. tylosin lactate on spoilage patterns of inoculated Lewis, J. C. and Snell, N. S. (1951). The amino acid and non-inoculated cream style corn. in Microbial composition of subtilin. J. Amer. chem. Soc., 73, Inhibitors in Foods, p 87 (ed. by N. Molin); Almqvist 4812. & Wiksell, Stockholm. 55 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

Manten, A. and Hoogerheide, J. C. (1958). The Patrick, J. B., Williams, R. P., Wolf, C. F. and Webb, influence of a new antifungal antibiotic Pimaricin J. S. (1958 b). Pimaricin I. Oxidation and hydrolysis on the yeast flora of the gastrointestinal tract of products. J. Arner. chem. Soc., 80, 6688. rats and mice during tetracycline administaration. Pette, J. W. and Kooy, J. S. (1953). The inhibition of Antibot. Chernother., 8, 381. butyric acid fermentation in cheese by antibiotic­ Marini, F., Arnow, P. and Lampen, J. 0. (1961). The producing starter. Proc. 13th int. Dairy Congr., 3, effect of monovalent cations on the inhibition of 1172. yeast metabolism by nystatin. J. gen. Microbiol., Poole, G. and Malin, B. (1964). Some aspects of the 24, 51. action of tylosin on Clostridium species PA 3679. Marth, E. H. (1966). Antibiotics in foods-naturally J. Fd Sci., 29, 475. occurring, developed and added. Residue Rev., 12, 65. Preservatives in Foods Regulations (1962). Statutory Instrument No. I 532; H.M.S.O., London. Mattick, A. T. R. and Hirsch, A. (1944). A powerful inhibitory substance produced by group N strepto­ Pulay, G. (1956). Research on Streptococcus lactis cocci. Nature, Lond., 154, 551. strains producing antibiotics against clostridia. Proc. !4th int. Dairy Congr., 2 (2), 432. Mattick, A. T. R. and Hirsch, A. (1947). Further observations on an inhibitory substance (nisin) Ramseier, H. R. (1959). The action of coliform bacteria from l.actic streptococci. Lancet, ii, 5. on the stability of nisin with special regard to the influence of the test medium. Proc. !5th int. Dairy Moycho, W. and Gromska, W. (1956). Antaguonistic Congr., 2, 566. action of Streptococcus lactis against Bacillus subtilis and Pseudomonas fluorescens observed in Ramseier, H. R. (1960). The action of nisin on Clos­ milk. Acta Microbial. Polon., 5, 267. tridium butyricurn Prazm. Archiv. Mikrobiol., 37, 57. National Canners Association (1951). Annual Report Ramseier, H. R. (1961). The application of nisin in for 1950, Washington, D.C., U.S.A. the dairy industry. Schweiz. Milchztg. Wissenschaftl. Beilage Nr. 74, I. Newton, G. G. F. (1965). Mechanism of antibiotic action. A. Rev. Microbial,. 19, 209. Rangaswami, G. and Damodaran, K.A.P.S. (1957). In vitro action of mycostatin on fungi pathogenic Newton, G. G. F., Abraham, E. P. and Berridge, N.J. to banana. Indian Phytopath., 10, 126. (1953). Sulphur-containing amino acids of nisin. Nature, Land., 171, 606. Regna, P. P. (1959). The chemistry of antibiotics. in Antibiotics, their Chemistry and Non,medical Uses, Ng, H., Vaughn, R. H. and Stewart, G. F. (1957) p 58 (ed. by H. S. Goldberg); Van Nostrand & Co., Antibiotics in poultry meat preservation: Develop­ Inc., Princeton, U.S.A. ment of resistance among spoilage organisms. Appl. Microbial., 5, 331. Ritter, W. (1957). On the inhibition of desirable organisms from cultured cream. Schweiz. Milchztg., Njoku-Obi, A. N., Spencer, V. J., Sauter, E. A. and 71, 255. Eklund, M. W. (1957). A study of the fungal flora of spoiled chlortetracycline-treated chicken meat. Appl. Ryder, R. (1965). A novel approach to the micro­ Microbial., 5, 319. biological analysis of antimicrobials. Paper presented to the Microbiology Group, Society of ChemiCal O'Brien, R. T., Titus, D. S., Devlin, K. A., Stumbo, Industry, Jan. 1965. C. R. and Lewis, J. C. (1956). Antibiotics in food preservation. II. The influence of subtilin and nisin Sarkisov, A. Ch. {1966). The problem of non-medical on the thermal resistance of food spoilage bacteria. uses of antibiotics. in Antlbwtics: Advances in Fd Techno/., Champaign, 10, 352. Research, Production and Clinical Use, p 616 (ed. by M. Herold & Z. Gabriel); Butterworths, London Otto, H., Alford, E. F., Grundy, W. E. and Sylvester, · and Czechoslovak Medical Press, Prague. J. C. (1961). Antibiotic bactericidal studies: bacteri­ cidal and bacteriostatic tests with various antibiotics. Segmiller, J. L., Xerones, H. and Hutchings, I. J. Antimicrobial Agents A., (1961), 104. (1965). The efficacy of nisin and tylosin lactate in selected heat-sterilized food products. J. Fd Sci., Pagano, J. F. (1963). Microbiological method for 30, 166. assaying nystatin in animal feeds. J. Ass. off. agric. Chern., 46, 438. Seneca, H. (1955). In vivo and in vitro effect of nystatin on Endamoeba histolytica. Antibiotics A., 1955-56, Pagano, J. F. and Stander, H. (1955). Bioassay of 697. Nystatin (Mycostatin) in body fluids. in Therapy of Fungus Diseases p 186-194 (ed. by T. H. Sternberg Shahani, K. M. (1962). Inhibitory effect of nisin on & V. D. Newcomer); Little, Brown & Co., Boston, various organisms. J. Dairy Sci., 45, 827. U.S.A. Shahani, K. M., Nilson, K. M. and Downs, P. A. Patrick, J. B., Williams, R. P. and Webb, J. S. (1958 a). (1959). Effects of antifungal antibiotics upon the Pimaricin II. The structure of pimaricin. J. Arner. keeping quality of cottage cheese. Proc. 12th int. chern. Soc., 80, 6689. Dairy Congr., 2, 926. 56 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

Sheneman, J. M. (1964). Prevention of .type E Clos­ Suarez, G. and Nathan, D. (1965). Inhibition of amino­ tridium botulinum toxin formation in smoked white­ acyl sRNA binding to ribosomes by tetracyclines. fish chubs with tylosin lactate. Paper presented at Biochem. biophys. Res. Commun., 18, 743. the 24th A. Meeting Inst. Fd Techno!., Washington Tarr, H. L. A. (1955). Control of bacterial spoilage D.C., U.S.A., May, 1964. of fish with antibiotics. Proc. !st. int. Congr. on the Shevtchenko, M. G. (1966). Hygienic problems of Use of Antibiotics in Agriculture, p 199-209; Nat. foreign substances in food products in the U.S.S.R. Acad. Sci., Nat. Res. Council Pub!. No. 397. 2nd int. Congr. Fd 'Sci. Techno/., Warsaw, Abstract Tarr, H. L. A., Southcott, B. A. and Bissett, H. M. H.3.2., p 527. (1952). Experimental preservation of flesh foods Shewan, J. M. (1961). The microbiology of sea water with antibiotics. Fd Techno/., Champaign, 6, 363. fish. in Fish as Food, Vol. l, p 487-560 (ed. by G. Borgstrom); Academic Press, London. Taylor, J. I., Hirsch, A. and Mattick, A. T. R. (1949). The treatment of bovine streptococcal and staphy­ Shewan, J. M. (1962). Sanitation. Part 1: Food lococcal mastitis with nisin. Vet. Record., 61, 197. poisoning caused by fish and fishery products. in Fish as Food, Vol. 2, p 443-446 (ed. by G. Borgs­ Thatcher, F. S. and Loit, A. (1961). Comparative trom); Academic Press, London. microflora of clortetracycline treated and non­ treated poultry with special reference to public Shewan, J. M., Hobbs, G. and Hodgkiss, W. (1960)· health aspects. Appl. Microbial., 9, 39. The Pseudomonas and Achromobacter groups of bacteria in the spoilage of marine white fish. J. appl. Thorpe, R. H. (1960). The action of nisin on spoilage Bact., 23, 463. bacteria. The effect of nisin on the heat resistance of Bacillus stearothermophi/us spores. J. appl. Bact., 23, Shewan, J. M. and Liston, J. (1955). A review of food 136. poisoning caused by fish and fishery products. J. appl. Bact., 18, 522. Tomiyama, T., Yone, Y. and Mikajiri, K. (1957). Uptake of aureomycin chlortetracycline by fish and Shewan, J. M. and Stewart, J. (1958). Experiments in its heat inactivation. Fd Techno/., Champaign, 11, the use of antibiotics in fish preservation. Bull. 290. Inst. Int. Froid., 2, 549. Tramer, J. (1964). The inhibitory action of nisin on Shirk, R. J. and Clark, W. J. (1963). The effect of B. stearothermophilus. in Microbial Inhibitors in pimaricin in retarding the spoilage of fresh orange Foods, p 25 (ed. by N. Molin). Almqvist & Wiksell, juice. Fd. Techno/., Champaign, 17, 1062. Stockholm. Shirk, R. J., Kline, E. F. and Clark, W. L. (1966). Tramer, J. (1966). Nisin in food preservation. Cherny. The effect of pimaricin in the control of yeast Ind., (1966) 446. growth in organge juice concentrate. Fd Techno/., Tramer, J. and Fowler, G. G. (1964). Estimation of Champaign, 20, 91. nisin in foods. J. Sci. Fd Agric., 15, 522. Shirk, R. J. Whitehill, A. R. and Clark, W. L. (1962). Umezawa, H. (1964). Recent advances in chemistry and The bioassay of pimaricin and its binding effect in biochemistry of antibiotics, p 148; Nissin Tosho orange juice. J. Fd Sci., 27, 605. Insatu Co. Ltd., Tokyo. Shrimpton, D. H. (1957). The use of chlortetracycline Vas, K. (1964). Nisin in the food industry. Dt. Lebens­ to retard the spoilage of poultry carcases. J. Sci. mitt. Rdsch., 60, 63. Fd Agric., 8, 485. Velankar, N. E. (1958). Inhibition of bacteria from Snell, J. F. and Cheng, L. (1960). Studies on the mode marine sources by aureomycin. Proc. Indian Acad. of action of tetracyclines (II). in Developments in Sci., 47, 87. Industrial Microbiology, Vol. 2, p 107-131 (ed. by Volkova, L. P. (1961). The use of nystatin for com­ S. Rich); Plenum Press Inc., New York. bating meat mould. Mikrobiologiya, 30, 158. Stanley, C. and English, M. P. (1965). Some effects of Walker, E. A., Taylor, J. H. and Davis, J. G. (1960). nystatin on growth of four Aspergillus spp. J. gen. The effect of chlortetracycline on eviscerated chilled Microbial., 40, 107. chickens commercially processed in Great Britain. Sternberg, T. H., Tarbet, J. E., Newcomer, V. D., J. appl. Bact., 23, 145. Huddleson, H. G., Weir, R. H., Wright, E. T. and Walker, E. A., Taylor, J. H. and Davis, J. G. (1961). Egbert, R. 0. (1953). Antifungal effects of nystatin The effect of chlortetracycline on eviscerated on the faecal flora of animals and man.Antibiot. A., turkeys commercially processed in Great Britain. (1953-54), 199. J. appl. Bact., 24, 104. Struyk, A. P., Huett, J., Drost, G., Waisivozz, J. w.. Walker, H. W. and Ayres, J. C. (1959). Characteristics Van Eck, T., and Hoogerherde, J. C. (1957). of yeasts isolated from processed poultry and the Pimaricin-a new fungal antibiotic Antibiot. A., influence of tetracycline on their growth. Appl. (1957-58), 879. Microbial., 7, 251. Stumbo, C. R., Voris, L., Skaggs, B. G. and Heine­ Weinberg, E. D. (1957). The mutual effects of anti· mann, B. (1964). A procedure for assaying residual microbial compounds and metallic cations. Bact. nisin in food products. J. Fd Sci., 29, 859. Rev., 21, 46. 57 The Use of Antibiotics in Food Preservation. B. Jarvis and M. D. Morisetti.

Weiser, H. H., Goldberg, H. S., Cahill, V. R., Kunkle, Woodbine, M. (1962). Antibiotics in Agriculture; L. E. and Deatherage, F. E. (1953). Observations Butterworths, London. on fresh meat processed by infusion of antibiotics. Woods, B. M. {1966). Use of tetracyclines in food Fd Techno/., Champaign, 7, 495. preservation. Cherny. Ind., (1966), 615. Watanabe, T. (1963). Infective heredity of multiple Yacowitz, H., Pansy, F., Wind., S., Stander, H., drug resistance in bacteria. Bact. Rev., 27, 87. Sassaman, H. L., Pagano, J. F. and Trejo, W. H. Watanabe, T. and Fukasawa, T. (1961). Episome­ (1957). Use of nystatin (mycostatin) to retard mediated transfer of drug resistance in Entero­ yeast growth on chlortetracycline-treated chicken bacteriaceae. I. Transfer of resistance factors by meat. Poult. Sci., 36, 843. conjugation. J. Bact., 81, 669. York, G. K. (1966). Effect of pimaricin on the resist­ Wheaton, E., Burroughs, J. D. and Hays, G. L. (1957). ance of Saccharomyces cerevisiae to heat, freezing Flat-sour spoilage of tomato juice and its control and ultraviolet irradiation. Appl. Microbial., 14, 451. with subtilin. Fd Techno/., Champaign, 11, 286. Wheaton, E. and Hays, G. L. (1964). Antibiotics and Yoseki, M., Nemoto, Ch. and Amano, K. (1967). the control of spoilage in canned foods. Fd Techno/., Behaviour of tylosin in fish products on the basis Chmnpaign, 18, 549. of antibacterial action. 2nd int. Congr. Fd Sci. Techno/., Warsaw, Abstract £.1.16., p 352. Winkler, S. and Frohlich, M. (1957). The effect of nisin on the microorganisms most important to the Zygmont, W. A. (1966). Intracellular loss of potassium ripening of Emmental cheese. Milchwissensch. in Cm1dida a/bicans after exposure to polyene anti­ Berichte, 7, 125. fungal antibiotics. Appl. Microbial., 14, 953. Winkler, S. and Frohlich, M. (1958). Investigations of Zygmont, W. A. and Tavormina, P. A. (1966). Steroid Emmental cheese with nisin. Milchwissensch. Ber­ interference with antifungal activity of polyene ichte, 8, 87. antibiotics. Appl. Microbial., 14, 865.

58 --~~--~~--~~~------~------..~~-- ...... ~ "

APPENDIX: ANTIBIOTICS USED IN FOOD PRESERVATION IN DIFFERENT COUNTRIFS

Country Antibiotic Permitted in Residue Notes Source Tolerance I. Argentina! ere i. Poultry 5-10 ppm Use of these antibiotics is not permitted in beef, veal, Government decree no. 4238, OTe ii. Fish mutton or lamb chapter XVIII items 3.15 and 3.16 2. Australia Nisin I. Cheese No limit Food standards are elaborated by the Food Standards Approved food additives. a) Federal ii. Canned fruit products committee of the National Health and Medical Research The National Health and (including tomatoes) with Council, and if approved by Council, recommended to Medical Research Council pH<4.5 States and Territories for adoption and subsequent May 1966 and amendment iii. Canned soups in hermetic- inclusion in legislation Nov 1967 ally sealed containers which have been sufficiently heat treated to destroy any Clostridium botulinum present 2. Nisin I. Cheese No limit Label must show that preservative is added Amendment to Pure Food Act b) New South il. Canned tomato paste 1908-1961, 27.10.61 (Published Wales iii. Canned tomato juice N.S.W. Gazette No. 82) iv. Canned fruit with pH<4.S 2. Nisin i. Cheese No limit Queensland Food an c) Queensland ii. Canned fruit products Drug Regulations ~ (including tomatoes) with pH<4.5 2. Nisin I. Cheese No limit Food and Drugs Act 1908-1954 d) South if. Canned tomatoes Amendment Food additive Australia iii. Canned fruit with pH<4.5 legislation No. 49, 30.11.61 2. Nisin Cheese No limit Food and Drug standard e) Victoria regulations 1966 3. Brazil' ere I. Animals (live) by intra- Governmental decree OTe muscular or intraperitoneal no. 691, 13.3.62 inoculation ii. Birds-immersion in aqueous solution 4. Belgiuml Nisin Cheese 0.0005% Nisin was not included in the main list of permitted Nisin - Ministerial food additives dated 18.9.67, but has been re- decree 28.10.64 introduced on a provisional list Pimaricin In cheese rind covering 0.05%of Pimaricin - Ministerial rind decree 18.9.67 material 5. Canada ere i. Poultry (raw) ?ppm It is not intended that these antibiotics be used WHO Technical report OTe ii. Fish preservation in ice 5ppm specifically as food preservatives or permitted in no. 260 and Personal iii. Fresh fillets of fish in 10ppm packaged poultry or fish which might be consumed Communication dipping tanks without preparation and cooking --- .. ------·- APPENDIX: ANTIBIOTICS USED IN FOOD PRESERVATION IN DIFFERENT COUNTRIES (continued)

Country Antibiotic P~rmitted in Residue Notes Source Tolerance 6. Chile Tetracyclines Fish preservation in ice Containers of treated fish must comply with food Chilean Ministry of Health regulations (Decree 770) 26.9.39, and must have decree no. 1207 (Diario Official indicated the name of the antibiotic, date of of 9.12.1958) application to ice and length of duration of activity with the statement "treated with ice containing antibiotic to retard alteration" 7. Czechoslovakia Nisin Cheese 200 Anon. (1964). units/g BIBRA Bull, 3, 27 B. Francet Nisin Processed cheese No limit Ministry of Agriculture 24.11.62 9. Great Britain ere Raw fish 5 ppm A. Because the 'Therapeutic Substances Act' permits Preservatives in food OTe the sale of antibiotics to medical practitioners regulations 1962 Nystatin Skin, not flesh of bananas No limit and certain other authorised persons only, it was not Nisin i. Cheese possible nntil11.7.64 (By statutory instrument no. 883) ii. Clotted cream No limit for the fishing industry to use tetracyclines for the iii. Canned foods preservation of raw fish. B. No label declaration is required 10. Greece Nisin An application for permission to use the antibiotic has been submitted for governmental approval 11. Italy' Nisin Cheese 500,000 Ministerial decree of 31.3.65 g) Ru/kg )I Ru= 0.025Jlg) 12. Israel ere i. Poultry 7 ppm A. The manufacture or sale of food containing Public Health OTe ii. Meat antibiotics is forbidden except by special permit (Preservative in food) iii. Fish granted by the Director-General of the Ministry of Regs. 5725

Nisin Fruit and vegetables packed in None Health. 1965 and 1967 -~. hermetically sealed containers B. The label must show that preservative is added and preserved by heat treatment Cheese (not including soft No limit white cheese) 13. Japan ere Fish preservation in ice <0.1 ppm ere may be added to ice only for use in 'distant' Personal conununication e.g. Fish for fish paste trawlers. Further, CfC is not regarded as a food additive in this context. Generally no antibiotics are permitted as food additives

14. Mexico Nisin All foods No limit 15. New Zealand Nisin An application for permission to use the antibiotic has been submitted 16. Norway Nisin Cheese No limit Nisin is not considered to be a medicament and is not Ministry of Social Affairs governed, therefore, by the provisions of the Act of - Directorate of Health, Oslo 20.6.64 on Medicaments and poisons. Further nisin is considered as a raw material in the production of cheese and is not governed by the provisions dealing with food additives ' ~•. ~~-~~~~~~~···~-~-~~~----~-

APPENDIX: ANTIBIOTICS USED IN FOOD PRESERVATION IN DIFFERENT COUNTRJES (continued) Country Antibiotic Permitted in Residue Notes Source Tolerance 17. Peru2 Wide spectrum i. Raw food to be consumed 7ppm A. The use of an antibiotic not authorised in code Peruvian Food Code 1963 antibiotics cooked is absolutely prohibited, except where expressly e.g. authorised. ere ii. Ice for preservation of fish 7ppm B. No antibiotic may be added to milk, directly or OTC indirectly 18. South Africa The use of antibiotics as preservatives in food is not Food drug and disinfectants permitted Act, no. 19 of 1929 Antibiotics in foods

19. Spain It is prohibited to sell or use for human consumption Spanish food code part IV foodstuffs .... containing antibiotics, .... Chapter XXXV Published under decree no. 2484 (1967) of 21.9.67 20. Sweden Nisin i. Cheese No limit Nisin was permitted to be used in fish products Pure food law The Swedish ii. Sterile condensed milk and icdluding canned crustaceans in the 1965 list of Code of statutes 1951. cream .. permitted food additives, but this was rescinded in List of permitted additives of the 1968 list for 1968 21. U.S.A. ere i. in/on uncooked poultry 7ppm A. On 6.9.67 the residue tolerance for these Food & Drug Administration ii. Uncooked fish shrimp or 5ppm antibiotics was revoked and the FDA withdrew Federal Register Vol32 no. 172 scallop approval for the use of chlortetracycline and oxytetracycline in food preservation. j: OTC iii. in/on uncooked poultry 7ppm B. The withdrawal of approval as in (A) above did ::l not affect the residue tolerances of chlortetracycline .\i\~ .. '·:~ and oxytetracycline. (Pesticide law; sections 121/1014, ·~ 121/1046) resulting from their use in animal feeding 22. U.S.S.R. ere Raw fish in ice 0.25 ppm A. The use of biocin (erC) ice is permitted only for (a) Shevtchenko, M.G. (1966) use in long distance transportation of fish and residues Abs. 2nd Inter. Congr. Fd Sci. must be absent from culinary-treated fish. Techno/., Warsaw, 527. B. Industrial trials of meat treated with biomycin and nystatin are permitted; residue tolerances are biomycin

j

'l Hueck, H. J. Int. Biodetn Bull. 5, (2), 63-66, (1969). Experiences with biological tests in the field of the biodeterioration of materials. 3. Some principles of the testing of material-protectants.

EXPERIENCES WITH BIOLOGICAL TESTS IN THE FIELD OF THE BIODETERIORATION OF MATERIALS. 3. SOME PRINCIPLES OF THE TESTING OF MATERIAL-PROTECTANTS. H. J. Hueck1

Summary A discussion is given of properties of material­ Erfahrungen mit biologischen Prilfverfnhren auf dem Gebiet der protectants which should be considered in biological testing of Zerstorung von Werkstoffen durch Organismcn. 3. Einige Grund~ materials protected against biodeterioration. It is pointed out Sitze fOr die PrUCung von Schitzmitteln fiir Werkstoffe. Die that the biotic test then only has indicator value for the proper Eigenschaften von Schutzmitteln, die in biologischen Prlifver~ fahren fUr geschlltzte Werkstoffe beriiksichtigt werden sollen, functioning of the material. Evaluation of material-protectants werden besprochen. Es wird dargelegt, daB die biotische Prilfung should in principle be effected through an ageing procedure that our Aussagewert hat fUr die ordentliche Benutzbarkeit des includes physical, chemical and biotic (environmental) influ­ Materials. Die Prilfung von Schutzmitteln furWerkstoffe sollte ences. The biological test of protected materials should be applied grundsatzlich cine Alterungsbeanspruchung einschlieBen, die at different periods of the ageing process, and thus indicate the physikalische, chemische und biotische (umweltbedingte) presence or absence of the protection under investigation. Ein.flusse berucksichtigt. Biologische Prlifungen von geschiltzten Werkstoffen soli ten zu bestimmten Zeiten des Alterungsprozcsscs durchgeflihrt werden, urn die Wirksamkeit oder Unwirksamkeit des untersuchten Schutzmittels zu beweisen. Experiences avec des essais biologiques dans lc domainc de Ia dCtCrioration biologique des matCriaux. 3. Quelques principcs Expericncias con pruebas biol6gicas en el campo del In Bio­ des essais des produits de protection des matCriaux. Les proprietes deterioraci6n de los :Matcrialcs. 3. Cicrtos princlpios de In des produits de protection des matCriaux qui devraient Ctre prueba de los protectores de los materialcs. Se discuten las prise.~ en consideration Iars des essais biologique des matCriaux propiedades de los protectores de los materiales que hay que proteges contre la dE:tE:rioration biologique sont discutees. On considerar en Ia prueba biol6gica de los matcriales protejidos attire l'attention sur le fait que le test biotique n'a alors une contra la biodet~rioraci6n. Se subraya el hecho que en tales valeur indicatrice que pour le fonctionnement correct du circunstancias la prueba bi6tica tiene valor s61o como indicador material. L'evaluation des produits de protection des matCriaux para el debido funcionamiento del material. SegU:n los principios la evaluaci6n de los protectores deberfa hacerse por medio devrait, en principe, se baser sur un protedC de vieillissement qui de un procedimiendo de deteriorar por envejecimiento que comporte des influences physiques, chimiques, et biotiques comprenda tambiCn los inftujos ffsicos, qufmicos y bi6ticos del (milieu ambiant). Les essais biologiques des matCriaux proteges media ambiente. Se deberia hacer la prueba biol6gica de los devraient Ctre effectuE:s a diffCrentes Ctapes au cours du processus materiales protejidos a varias etapas del procedimiento de de vieillissement, indiquant ainsi Ia presence ou \'absence de Ia envejecimiento para que asi se revele Ia presencia o Ia ausencia protection Ctudic!e. de la protecci6n indagada.

I. Definitions material to which it is applied. Hence it is the proper functioning of the material which should be the The older literature on the biological testing of decisive parameter for evaluating the success of a proofness of materials, as reviewed in the books of certain chemical compound in protecting a material Siu (1951) and Greathouse and Wessel (1954), dis­ against biotic attack. Only as far as biocidal activity tinguishes roughly two groups of test: pure culture in this situation is directly correlated with the protec­ and similar laboratory tests of short duration, and tion provided to the material, does it make sense to exposure tests simulating conditions of practical use measure this activity as the main parameter. Hueck such as weathering, tropical chamber, soil burial and Adema (1967) have experimentally shown for tests, etc. A more detailed analysis of test types is antifouling paints that erroneous results will ensue, if given by Hueck-Van der Plas (1965). biocidal value is taken as the only parameter. It is Pure culture tests on rotproofed materials are indeed already common practice to use e.g. tensile clearly related to in vitro screening tests for biocides, strength in rotproofed materials, increase in stiffness which in general allow a rapid and relatively precise in plasticized PVC, electrical resistance in insulating evaluation of the toxic properties of chemical com­ materials etc. as parameterers in biological tests of pounds. With the introduction of materials in the test, materials. This principle not only holds good for some complications arise. First of all, we are dealing fungicides, but also for bacteria, insects and other with a heterogeneous system, the biologically active biodeteriogens including rodents and barnacles. The agent being distributed over a matrix with properties chemical compounds used in such systems, therefore, usually completely different from its own. The dis­ should be properly called material-protectants, as a tribution and the physicochemical state of the active generic name for those compounds that are applied agent thus become important (Hueck et al., 1968). for the prevention of biodeterioration of materials, Furthermore it is no longer the biocidal quality as as defined by the author earlier (Hueck, 1965). As such that matters, but its protective value for the protection may also be provided against other in-

tCentraal Laboratorium TNO, Postbox 217, Delft, The Netherlands. (Copy received 8th May, 1969). 63 Experiences with biological tests in the field of the biodeterioration of materials. 3. Some principles of the testing of material-protectants. H. J. Hueck. fluences than those of biotic origin, it might be useful enzymatic degradation of the protectant. In the to specify "bioprotectants" if only protection against indicator phase, which is reached after the protective biodeterioration is meant, but usually it will be clear threshold has been passed, the only thing that matters from the context at what type of agent the protection is that biodeteriogens are able to develop, thus is aiming. indicating the endpoint of the reaction which was under way in the ageing phase. It is, therefore, quite un­ 2. Ageing phase and indicator tests necessary to carry out protracted biotic tests, because The uncoupling of the protection of materials from any indication of the occurrence of biodeteriogens notions of fungicides, fungicidal protection and fungi­ suffices for the diagnosis that through the ageing the cidal tests (resp. bacteria, rodents, algae, insects, protection has reached the protective threshold. fouling organisms) is desirable for other reasons, too. Ageing is caused by a complex of physical, chemical Although biocidal treatments have been used exten­ and biotic processes which we may describe as sively to protect materials against biodeterioration, it environmental stress on the material protectant. The is by no means the only technique for this purpose. preferance of technologists and engineers for such A well-known case in point is the "Arigal" treatment practical tests as weathering, raft tests and soil burial, of cotton (cf. Ruperti eta/., 1964) that basically renders is due to the fact that in these tests they have a broad the cotton inert to biotic attack by incorporation of a variety of environmental stress factors at their com­ resin without imparting biocidal qualities. Acetylation mand, together with a natural supply of indicator and cyanoethylation of cotton are other instances of organisms. The obvious drawback of such arrange­ making a material inert to biotic attack without ments is that the dosage of stress is extremely difficult involving biocidal activity. It is perhaps interesting to to define, both quantitatively and qualitatively. A point out the parallel which exists in chemical corro­ perhaps less conspicuous drawback may be that sion, where, on the one hand, biocidal protection may indicator organisms are not necesarily always present; be compared with cathodic protection and, on the they may be absent at the very moment that the other hand, protection by making materials inert to protective threshold is crossed. In the raft testing of biodeterioration may be compared with passivation antifouling paints, this latter factor has been found of metals. to be of great importance. In the marine testing station The presence of organisms, with regard to biological of our laboratory at Den Helder, the occurrence of an material tests, has another meaning than that in appreciable fall of barnacles may be relied on virtually biocidal testing. In biocidal testing, organisms react only during the three summer months. During the directly with the biocidal agent; in the testing of material other nine months of the year, leaching of the test protectants, organisms play the role of an indicator. panels goes on, but without a reliable presence of Their ability to grow gives an indication of the failure indicator organisms. The same holds true for weathering of the protection of the material that should be experiments in climates with marked seasonal variation afforded by the protective treatment under investiga­ or the occurrence of long dry spells in which micro­ tion. organisms fail to develop. Kempton, Maisel and Kaplan (1963) have shown in The obvious remedy for this situation is to separate an analysis of the soil burial process, that breakdown the ageing phase from the indicator phase in the of fungicidally protected cotton by biotic attack evaluation of material-protectants. Rather intuitively occurs only after a preceding presumably non-biotic this procedure has already been practiced in the field process which lowers the concentration of the active of biodeterioration, when laboratory tests for testing compound until a level is reached which allows the "the permanency" of the protection were developed. development of cellulolytic organisms and the ensuing In the author's experience, which is of course not breakdown of the cotton. Van Londen and De Wolf, exhaustive, the following ageing procedures are used: in a series of publications pertaining to the testing of antifouling paints (1963, 1964, 1966, 1968), have washing -mothproofness of wool reached the same conclusion in that two phases in dry cleaning -mothproofness of wool the breakdown of a protective paint must be distin­ leaching in -rotproofness of textiles, paints, guished: continuous flow wood (I) the leaching out of the active agent, rotor"leaching -antifouling paints (2) the actual settling of fouling organisms. light exposure to -mildew proofness of paints, textiles These two phases require different experimental tools. xenon tube For a proper discussion of the phenomena, in the heat treatment -unusual, but occurs in tropical present context, it is convenient to distinguish these proofing of electrical apparatus two phases as and in storage experiments of (a) the ageing phase, paints. (b) the indicator phase. However, much confusion arises from the fact that In the ageing phase, physical, chemical and biotic hardly any biological test of material"protectants is influences may lower the protective potential of the designed on the basis of providing a minimal environ­ protectant, e.g. by leaching, oxidation or actinic or mental stress, if used as an indicator phase test only. 64 Experiences with biological tests in the field of the biodeterioration of materials. 3. Some principles of the testing of material-protectants. H. J. Hueck.

On the contrary, biological tests are valued as being in an environment (aqueous culture media) which more or less aggresive; this means that they differ in provides some "environmental stress". However, this "stress". Some people even compare performance of latter influence should be minimised. This can be material protectants on the basis of the breakdown achieved by using tests of short duration, under achieved in such a test, e.g. the residual strength of optimal conditions for the biodeterioration of the cotton in experiments of a specified duration whereas, blank ( = unprotected) material. There are also in the last analysis, an ali-or-none answer is the practical reasons for advocating biomaterial tests of only one required in the indicator phase. A better short duration only. Walchli (1967) has shown that approach appears to be the half value period (Hueck, culture conditions change during the test, which 1960) in tests where environmental stress prevails, leads to deviating results in tests of longer duration such as the soil burial test, and where the presence of (> 16 days). indicator organisms is no problem. This parameter (half value period) has a drawback, however. It is 4. Functional tests presumed that the period of exposure is related linearly to the ageing of the material protectant and, further" It is not sufficient to rely on the growth or non­ more, that the ageing in a soil burial bed is represen­ growth of biodeteriogens only. The decisive factor in tative of ageing in practical situations. On this latter material protection is the proper functioning of the point however, we know next to nothing. Yet is is material in question. With plasticised PVC, for exam­ of paramount importance to investigate the ageing ple, slight growth of fungi may occur on impurities procedure, as a superficial acceptance of ageing present in or on the material, this growth not being conditions may easily lead to erroneous results. Van related to the plasticiser as such. Only the loss of Louden (1963) has studied ageing of antifouling paint plasticiser will impair the functioning of the material films by taking microscopical cross-sections. He com­ and, hence, it is this loss that matters, be it determined pared ageing of paint films on ships' hulls, on test as weight loss, as stiffness or as change of the elasticity panels in the (usual) raft tests and on rotor-leached modulus. test strips. He found that the ageing pattern in raft The determination of such properties is subject to tests was not comparable with that on ships, whereas statistical variations. In order to decide whether a that of rotor leaching showed much more agreement. change in such a property has indeed occurred in a For antifouling paints there is a rather simple situation, biological indicator test, we must rely on a quantitative in that the fiow of water is apparently the predominant response to the presence of the biodeteriogen. It follows ageing factor. With other materials, however, the from this requirement that we must possess a quan­ situation may not be so simple. titative description of the breakdown process of the material under influence of the biodeteriogen. Quane 3. Requirements of a test system for material­ tification only makes sense if we are able to reproduce . protectants results at will, which calls for a definition of deteriorat­ A test system for the evaluation of material"protect­ ing capacities of the deteriogen and its environment. ants which avoids the difficulties outlined above If all these requirements are fulfilled, we may define should consist of the following: limits of functional properties of materials which,under the circumstances of the test, indicate a positive (a) The protected material should be exposed to biodeteriogenic influence. In many military specifica­ representative environmental stress; tions this sytem is already used in a crude form. The (b) The environmental stress should be applied in U.S. Federal Specification CCCT, 191b, no. 5750 measureable and reproducible quantities; indicates as insufficiently rotproofed all materials (c) During the application of environmental stress, which subjected to a soil burial test show more than samples should be taken which are destined for 10% loss in strength. This margin of 10% must be biological indicator testing; allowed, because tensile strength of the unchanged sample cannot be determined with much greater (d) A biological indicator test should demonstrate accuracy, unless special precautions are taken. Any the possibility of the development of a representa­ change over this limit is attributed to the influence of tive biodeteriogen and thus the impairment of biodeteriogens (c.q. rotting). It is clear from this function of the material; example that, in such a test, proof must be given that (e) The result of the evaluation should be expressed the organisms present can, in the time afforded, in terms of quantities of environmental stress indeed bring about a change of a certain percentage that can be endured by the protected system of the mean strength of the material, the amount of (material + material - protectant), before the the percentage being determined by a statistical threshold of protection is crossed. analysis of the accuracy of the method. A definition of breakdown capacity of the biodeteriogen, therefore, The ideal biological test, in this respect, would be is necessary in all tests. one that gave only an indication of the biodegrad­ ability of the material under investigation without In earlier publications (Hueck, 1969; Hueck and offering "extra environmental stress". Obviously, this Van der Toorn, 1965) the present author has shown is difficult to achieve as the organisms themselves are some preference for the determination of the half part of the environmental stress and can only thrive value period as a parameter in biomaterial tests. This 65 Experiences with biological tests in the field of the biodeterioration of materials. 3. Some principles of the testing of material-protectants. H. J. Hueck. requires a somewhat tedious procedure, which, how­ Hueck, H. J. (1960). An analysis of the soil burial test. ever, in the same laboratory yields quite reproducible Berichte des 4. Int. Kongresses ftir Pflanzenschutz results. In this laboratory the common practice for Hamburg 1957, Vol. 2. Braunschweig, pp 1773- evaluating material,protectants, which is in principle 1776. the same as the ones used elsewhere (cf. Kaplan, Hueck, H. J. and van der Toorn, J. (1965). An inter­ (1967), is the following: laboratory experiment with the soil burial test. (a) leaching for different periods; Int. Biodetn Bull. 1, 31-40. Hueck, H. J. (1965). The biodeterioration of materials (b) exposure to xenon tube light for different periods; as a part of hylobiology. Material und Organismen, (c) determination of half life period in soil burial 1, 5-34. tests after each period of (a) and/or tb). Hueck, H. J. and Adema, D. M. M. (1967). Some problems in the testing of materials with algae. The determination of the half life period thus serves Material und Organismen 2 (2), 141-152. only as an indicator test. Mter protracted leaching, Hueck, H. J., La Brijn, J., Copper, J. A. and van Ham, the half life period should be equal to that determined J. (1968). Investigations on the biological activity for blank ( = untreated) cotton. In practical tests of pentachlorophenyl esters. In "Biodeterioration of (acceptance tests) a soil burial test of 14 days duration materials" edit. A. H. Walters & J. J. Elphick. is used as an indicator test. The requirement is that Proc. 1st Int. Biodetn Symposium. Elsevier Pub!. the results of such tests, before and after the specified Co., Amsterdam. ageing period (usually one day leaching), should not differ significantly from each other and, moreover, Kaplan, A. M. (1967). Prevention of microbiological should not lead to a significant change of one or other deterioration of materials in the United States­ functional property of the material, if compared with Present state of the art. TNO-Nieuws 22, 227-236. material not subjected to the test. The determination Kempton, A. G., Maisel, H. and Kaplan, A.M. (1963) of the half life period tends to be more accurate, A study of the deterioration of fungicide-treated providing at the same time some insight in the actual fabrics in soil burial test. Text. Res.J. 33 (2), 87-93. aggressivity of the test. The amount of work involved, however, prohibits its use for routine purposes. It will Van Landen, A. M. (1963). A study of ship bottom be appreciated that ageing period and aggressivity of paints in particular pertaining to the behaviour the indicator test are, generally speaking, arbitrarily and action of antifouling paints. Netherlands chosen quantities. Much research will be needed Research Centre TNO for Shipbuilding and before more appropriate values will be generally Navigation, Report Nr. 54C. accepted in routine testing. Van Landen, A. M. (1964). The mode of action of antifouling paints: Interaction between antifouling It could be argued that a simple pure culture test paints and seawater. Netherlands Research Centre might be a substitute for the soil burial test. The TNO for Shipbuilding and Navigation, Report reason that we still prefer soil burial is the mixed Nr. 62C. inoculum it offers, and the possibility of working with Ruperti, A., Bryler, N., and Aenishanslein, R., (1964). non-sterilised cotton. Even an indicator test must Baumwollkonservierung mit Arigal S. V.F.Fachorgan deal with a situation representative of practical 19, 348-356. circumstances. As long as we do not know to what extent pure cultures under specified conditions are Siu, R. G. H. (1951). Microbial decomposition of representative of such situations, we try to be as cellulose. Reinhold Pub!. Corp., New York. accurate as possible, and use soil burial tests which iii Walchli, 0. (1967). Der Einflus der Beliiftung auf die our mind have some resemblance to actual practice, zerstOrende Wirkung der Bodenmikroorganismen auf though our preferance is based on intuition rather zellulosehaltigen Textilien beim Erdvergrabungsver­ than accurate knowledge. fahren. Material und Organismen 2 (2), 85-96. De Wolf, P. and van Louden, A. M. (1966). Anti­ References fouling compositions. Nature 209, 272-274. Greathouse, G. A. and Wessel, C. J. (1954), Deteriora­ De Wolf, P. (1968). The problem of quality control tion of materials. Reinhold Pub!. Corp., New York. in antifouling. J. Oil. Col. Chern. Assoc. 51, 944-960.

66 Forse, W. J. Int. Biodetn Bull., 5, {2), 67-75, (1969). The incidence and effects of bird impacts witb aircraft.

THE INCIDENCE AND EFFECTS OF BIRD IMPACTS WITH AIRCRAFT W. J. Forse1

Summary. The nature of bird hazards to aircraft is outlined Hiiufigkeit und EinfUissc von VogeJaufschUigen auf Flugzeuge. with details of the parts of an aircraft which are particularly Die Art der Gefiihrdung von Flugzeugen durch VOgel wird susceptible to bird strikes: windscreens; engines; radomes and erHiutert mit Angaben von den Flugzeugteilen, die durch tailplanes. The various test methods used to evaluate the effects Vogelaufschl3ge besonders gef3hrdet sind: Windschutzscheiben, Triebwerke,Antennenschutz, HOhenleitwerke. Die verschiedenen of bird impact on aircraft components are described and the Prlifverfahren, die angewandt werden, um die Auswirkungen possible effects on the seriousness of the bird hazard problem des Vogclaufschlags auf Flugzeugteile zu bestimmen, werden of current trends towards increased speed and size of aircraft beschrieben und die mOglichen Konsequenzen werden in are considered. Betracht gezogen, die sich aus dcr Emsthaftigkeit dieses Prob­ lems hinsichtlich der Tendenz schncllerc und grODere Flugzeuge L'Incldence et les effets du choc causC aux avions par les oiscaux. zu bauen. ergeben. La nature des ·dangers par les oiseaux auxqucls sont exposes les avions est dCmontree, indiquant les details des parties des avions Frecuencia y efectos del choque de los p:'ijaros contra los aviones. qui sont les plus vulnCrables aux attaques par les oiseaux: Se esboza Ia indole de los peligros que constituycn los p:'ijaros para los avioncs y se suministran detalles de las partes del avi6n pare-brises; rnoteurs; radomes et empennage. On donne une mas susceptibles de choque-parabrisas, motores, radomos y description des nombrcuscs mCthodcs d'essai utilisCes pour alas traseras. Se describen los diversos mCtodos empleados para Cvaluer Ies effets causes par le choc des oiseaux sur les diffCrcntes evaluar el efecto del choque de los p:ijaros contra los componen­ parties composant un avion ainsi que les effets possibles de Ia tes del avi6n, adem:is de los efectos probables sabre tal problema relation entre l'accroissement de Ia vitesse et de Ia taille des ocasionados por las tendencias actuales bacia cl aumento de la avions et le choc par les oiseaux. velocidad y el tamano de los aviones.

In the autumn of 1962 an aircraft on a routine Most of the reported collisions occur with small service flight in the Southern United States was flying birds, with the exception of local hazards like the at 6,000 ft. when it collided with a group of migrating Indian Vultures. Another such local problem is the birds. These were later identified as Whistling Swans North American Water Fowl population, extending of 12 to 14 lbs. in weight. The impact occurred on the from the arctic and down the major rivers to the tailplane of the aircraft and the resultant structural Southern United States. This becomes even more damage led to complete tailplane failure, the loss of dangerous during the migration periods. Figure 2 the aircraft and the lives of all those on board. Another shows the distribution by weight of birds involved aircraft at Boston, Mass. flew into a dense flock of in incidents in Western Europe over the three year starlings which were ingested into the engines. In this period 1961-1963. It should be noted that there were case the weight of the individual birds was only a probably a larger number of collisions than shown few ounces, but the number of birds involved over• with birds under t lb. in weight which were not whelmed the engines, and the aircraft crashed with a reported sfuce the impact was unnoticed and the loss of over sixty lives. damage was negligible. From this plot it can be seen Fortunately such disastrous encounters are rare, that about 75% of reported incidents occnr with bnt the incidence of bird strike is an ever present birds under 3 lb. in weight. menace to all aircraft. Reported collisions in airline The figures quoted above are an indication of the service on a world wide basis occur at an approximate order of frequency of bird impact incidents. It must rate of once in each 1,000 honrs flying time or some be emphasised that considerable variations occur, 2 or 3 incidents per aircraft per year. Most of these dependant on the flight path of the aircraft, local bird produce little or no damage. The damage to the populations along that flight path and the time of the structure is a function of the energy of impact. Thus year. the damage is proportional to the weight of the bird Since about 70% of all incidents occur at heights of L, but to the square of the velocity of impact. The less than 2,000 ft. above local ground zero it means frequency of incidents also depends upon local that the main hazard in normal airline service is on I conditions and bird popnlation. For instance in the take off and landing. The take off is additionally Indian Sub-Continent the presence of large numbers hazardous since birds tend to congregate at the ends I of vultures of 8 to 10 lbs. in weight leads to a par­ of runways probably due to the slightly higher local ticularly serious hazard. temperatures of the tarmac. The landing case involves I The majority of incidents occur at or near ground a long slow approach with a slow rate of descent. level and over 70% of all accidents occur within Fortunately in both cases the damage is minimised 2,000 ft. of the local gronnd level. The variation of by the comparatively low forward speed of the aircraft, reported incidents relative to altitude is shown in which reduces the energy of impact. The normal Fig. I, and refers to accidents in Europe over a three airline operational conditions are such that the fnll year period. The inevitable freak accidents do occur cruising speed of the aircraft is not reached until the and one aircraft is reported to have hit a bird at an altitnde is greater than that of the densest bird popula­ altitude of 21,000 ft. tion. This is not the case with the latest types of

1Structural Test Laboratories, British Aircraft Corporation, Weybridge Division, Weybridge, Surrey, England. (Copy received 9th December, 1968). 67 The incidence and effects of bird impacts with aircraft. W. J. Forse. military aircraft, operating at heights of a few hundred and deflect it into the engine. Here the main feet at supersonic speeds on low level sorties. danger is in the break-up of the engine compressor Difficulties of exact identification of the birds blades which rotate at very high speeds. If damaged, involved in these accidents exist, since the evidence the out of balance forces are then likely to cause after impact is rarely identifiable unless the structure break-up of the compressor. The engine manu­ has been penetrated by the remains of the carcase. facturers are, of course, fully aware of this problem The pilot has occasionally been able to identify the and rigorous testing is carried out to ensure that bird as it approaches, but closing speeds of 300 the above mentioned sequence of failure is un­ miles/hour or more are not favourable conditions for likely. bird watching. However, a note of excessive caution It is now a requirement in many countries that was expressed in one report of an American accident the engine should continue to function after investigation which stated that 'Feathers were found ingesting starlings at a rate of I starling for every in the wreckage, believed to be of avian origin.' The 50 square inches of air intake frontal area. This imagination boggles at an alternative explanation. means that some 15 to 20 starlings are injected The majority of reports cite only a limited range of into the engine simultaneously, for test purposes. species and prominent among them are Seagulls, Pigeons, Water Fowl and Vultures. The gregarious 3. Radomes. birds represent a special hazard, particularly to Radomes are constructed from thin dielectric engines, which may be stopped by the number of materials to ensure transparency to the radar birds ingested. In this context seagulls, starlings and pulses. Like the windscreen the radar installation plovers are in evidence. One R.A.F. aircraft after is designed to look forward in the direction of take off from an airfield on the Scottish coast was flight. This unfavourable position from the aspect forced to land after such an encounter with a group of of bird impact is mitigated by the streamlined seagulls, and on inspection was found to have over shape of the radome. The double curvature gives 100 separate identifiable impact positions. improved resistance to impact and the shape tends It has been suggested that there is a tendency for to deflect the bird and prevent direct penetration. birds to fly towards an approaching aircraft, although little reliable evidence is put forward to prove the 4. Tailplanes. point. There did occur one special case which might Although the tailplane has a small frontal area be used as justification of this hypothesis. A small and the risk of impact is not very high it does sailplane in the alps was attacked by au eagle and a represent a very vulnerable area. In the previous classic type of air battle ensued until the sailplane instances cited any primary failure, although left the eagle's territory. serious, is not catastrophic. Even in the unlikely The damage almost invariably occurs on the forward event of a complete engine failure due to bird facing components of the aircraft due to the high ingestion the aircraft will continue to fly on the speed of the aircraft relative to the bird. Most of the remaining engines. A window failure is not in structure so exposed is sufficiently strong in construc­ itself catastrophic, although secondary damage to tion to withstand the majority of bird impacts without the pilot and cockpit may have dire results. But special modification, but some areas present special the tailplane represents an area which can and difficulties. These are as follows:- has led to disaster due to primary damage. The structure of the tailplane based on usual design I. Windscreen Glazing. criteria is lighter than most of the other areas at risk and impact can penetrate causing loss of In construction the pilot's windscreen must be flat to prevent optical distortion, and be as large strength and subsequent failure. Impact with larger birds can penetrate both the leading edge as possible to ensure the maximum field of vision. It should also be fitted as near as aerodynamically and trailing edge and can damage or jam the permissible to a plane normal to the line of flight. elevator with consequent loss of control. These criteria are, as in all matters of aircraft Test Methods. design, subject to compromise due to other considerations but remain the prime design re­ In order to determine the effects of bird impact on quirements. Since all known transparent materials aircraft components it has been necessary to develop are less than ideal impact resistant materials and ground testing methods to simulate the damage. The if failure does occur the debris is in close proximity current system employed at the British Aircraft to the head of the pilot, then it can be readily seen Corporation (Weybridge Division) test laboratory that this area is of particular importance. consists of a large air gun capable of firing projectiles of up to 10 lbs. in weight at energies up to 50,000 ft. 2. Engines lbs. Typically this means that we can simulate the Most parts of an aircraft are at risk in direct impact of a 4 lb. bird at speeds up to 450 m.p.lf. proportion to their frontal area. At low forward The gun consists of an air reservoir of 60 cu. ft. speeds the large air flows down the intakes of the capacity which will work at pressures up to 150 pounds engines do produce a 'vacuum cleaner' effect which per square inch. The outlet from this chamber is tends to attract any foreign matter in their vicinity sealed by a replaceable diaphragm housed in the 68 The incidence and effects of' bird impacts with aircraft. W. J. Forse. firing mechanism. The diaphragm is a thin fabric is shown in Figure 4 which shows the damage to the impregnated with rubber and is 6 inches in diameter leading edge. The gun is fired by means of a rapid acting peripheral One experimental modification to reduce the cutter which removes the diaphragm instantaneously. damage to this tailplane was to replace the 20 gauge Downstream of the firing mechanism is a 6 inch light alloy skin of the leading edge with one of 18 diameter barrel which is I 5 feet in length. Prior to gauge stainless steel. The results of impact with a firing, the projectile is set up in this barrel and rests 7·75 lb. bird at 24,000 ft. lbs. energy is shown in in a lightweight foamed plastic cup. The latter runs Figure 5. This clearly illustrates the problem of free in the barrel but acts as a seal during the time of preventing bird impact damage. The tougher skin acceleration dowu the barrel. On cutting the diaphragm has succeeded in resisting penetration but the damage the missile is accelerated down the barrel with the is more widespread since all the energy is now dissi­ plastic cup and the cut diaphragm, and hits the test pated on the leading edge. target after a typical distance of 3 feet from the muzzle. Tests have been carried out to ensure that Tests on Cockpit Glazing. the broken piece of cup, diaphragm and the air blast Most of the work carried out in the B.A.C. test do not cause damage to the specimens. facility has been on laminated glass windscreens with The velocity of impact is controlled from calibra­ vinyl interlayers. With such a typical installation it tion data by the initial pressure in the air reservoir. has been found that with energies of 12,000 ft. lb. The velocity is checked at each firing by either high or above then the impact leads to a severe fracture of speed photography against a marker board or by the toughened glass. A typical result at this energy is means of a photo,electric cell and timer system. shown in Figure 6. Whilst the inner laminate is intact and the window is structurally sound it is no longer In early tests the projectile used was a freshly possible to see through it. killed bird (usually a chicken) of the required weight. For many reasons this was unsatisfactory. The most At higher velocities the damage becomes more important of these was that after firing, the test site pronounced until typically at 26,000 ft. lbs. severe was littered with fragments of the disintegrated bird, delamination occurs, although complete penetration which proved not only unpleasant but also unhygenic. is prevented. The results of such an impact are shown From a purely technical aspect it proved almost in Figure 7, viewed from inside the cockpit. impossible to procure a bird of the correct weight for Above this energy complete penetration is likely a particular test. with the results shown in Figure 8. In this instance It has been shown (and since confirmed by our own the test cockpit had been enclosed with fibre insulation tests) that satisfactory results can be obtained by to enable the cockpit temperature to be maintained using a 'synthetic' bird made from a gelatine-water at service temperature. A four pound bird was fired mixture. This system was developed by the National with an energy of 28,000 ft. lbs. at the centre window Gas Turbine Research Establishment, for similar with the results shown. The window has been com­ tests on engines. In this way a cylindrical dummy can pletely penetrated and the broken glazing and remains be made of the correct diameter to suit the gun and of the carcase have demolished the insulation. The of such a length as to produce the exact weight larger pieces of glass are arrowed. Before the test required. commenced a model of the pilots head was placed in the appropriate position in the cockpit. The head Bird Impact Tests on a Typical Tailplane Structure. consisted of a wooden block covered with modelling clay and was wearing goggles. The damage to this A series of tests have been carried out at various model is shown in Figure 9. impact energies on a typical aircraft tailplane. The gun was set up as previously described with the muzzle The above tests were carried out at normal ground three feet from the target. The complete assembly is level temperatures outside the cabin. If the air tempera· shown in Fig. 3. The tailplane used had not been ture is reduced outside the cabin the damage caused desigued with specific reference to bird impact by bird impact is increased. This is due to the in­ criteria and later tests were carried out to provide creased stiffness of the vinyl at low temperature which methods of reducing the damage caused. The tests produces greater deceleration forces on impact. For were carried out using a four pound bird at a number instance the case shown above showed that penetration of velocities and at various positions along the leading occurred at energies above 26,000 ft. lb. at 20°C. edge. With the outer temperature at -50° this value reduces to 9,000 ft. lb. This is not a likely flight The tests showed that the structure at the tip of condition since the windscreens are normally heated the tailplane was penetrated at a lower velocity than to prevent misting, and this keeps the vinyl interlayer at the root. This was probably due to the greater at a high temperature. In the event of a heating failure, deceleration of the bird by the stiffer nosing at the however, the situation could be dangerous. tip of the tailplane. The minimum velocity required to penetrate the leading edge with a four pound Future Developments. bird was 350 ft./sec. The damage caused reduced the Since new aircraft are flying at successively higher torsional stiffness of the tail plane by 76%. speeds it would at first sight appear that the prol::lem The result of a further test using a 7 lb. bird at of bird impact is increasin5. Fortunately several 480 ft./sec. yielding an impact energy of 25,000 ft. lbs. factors help to prevent this. 69 The incidence and effects of bird impacts with aircraft. W. J. Forse.

Firstly the very high speeds which are now proposed The one area which could show a marked increase will not occur at altitudes where any bird can exist. in trouble is in the field of very high speed military The aircraft of the supersonic era will be flying at aircraft. The requirement here is for an aircraft to sub-sonic speeds until well above the most unlikely travel at high supersonic speeds at very low altitude bird. Secondly, although the proposed aircraft have in order to make a sortie below the opposition radar very high cruising speeds they must have comparable defences. This puts the aircraft into the dense bird runway characteristics with existing types. This means population at maximum energies of impact. An aircraft that although the landing and take off speeds may at twice the speed of sound will collide with a 4 lb. increase slightly the change will be small when com­ bird at an energy of 250,000 ft. lbs. or ten times as pared with the dramatic increase in cruising speed. high as the worst impact described above. For com­ parison purposes this energy is of the same order as In addition the aerodynamic requirements for high that of a large car travelling at 60 m.p.h., but concen­ speed demand longer slimmer aircraft with small trated into the small area of contact. Even if no frontal area. This reduces the risk proportionally. structural damage results from such a collision there is a major problem in stability, and loss of control One other type of development tendency is towards may well result. This type of operation is essentially larger aircraft of the air bus type. Here the move is military and the risks involved may well be less than towards very large aircraft of sub-sonic performance. those encountered at higher altitudes where the Projected air bus designs have some 3 or 4 times the aircraft is exposed to radar controlled missiles. frontal area of existing types and consequently are Within the limits of present technology it seems highly exposed to a similar increase in risk, although the unlikely that civil aircraft at high speed will ever energies of impact remain similar. Some of the vul­ operate under similar conditions. nerable areas of these aircraft, however, do not These are the problems which must be met when increase in size pro rata. The cockpit glazing for flying in an environment already occupied by birds instance will be of similar dimensions to those already which cannot be regulated by ground control proce­ in service, as will the radomes. Other parts of their dures. Since such control is an unlikely development structures will be of heavier construction for reasons we must expect our aircraft to collide with birds and other than bird impact. The larger diameter fuselage design them to withstand the effects of such collisions. for instance will require heavier gauge skin to satisfy Within the foreseeable future it seems unlikely that the pressurisation requirements and the bigger span the risks will increase substantially and although the of the tail plane will require more substantial members problem must be treated as a serious hazard we have to support it. These factors will all help towards accumulated sufficient experience to be able to reduce reducing the dangers from bird impact. the risks to an acceptable level.

70 The incidence and effects of bird impacts with aircraft. W. J. Forse.

50 occu RRENCE OF BIRD IMPAC T. DISTRIBUTION BY ALTITUDE ABOVE LOCAL GROUND ZERO. - REPORTED INCIDENTS IN EUROPE OVER J YEAR PERIOD.

40

30 o[o OF TOTAL INCIDENTS. ---'-

20

10

I 0 500 1000 2000 3000 FIG. I. ALTITUDE (FEET).

50 PCCURRENCE OF BIRD IMPACT • DISTRIBUTION BY WEIGHT. REPORTED INCIDENTS IN EUROPE OVER 3 YEAR PERIOD.

40

30 0 /o OF TOTAL INCIDENTS.

20

10-

0~-----L----~L-----~----~----~ 2 3 4 WEIGHT OF BIRD (POUNDS l. 71 The incidence and effects of bird impacts with aircraft. W. J. Forse.

FIG 3. TEST SET- UP OF GUN AND TAILPLANE .

FIG 4. DAMAGE TO TAILPLANE LEADING EDGE BY 71b BIRD AT 480ft /sec ( 25.000 ft. lb)

72 The incidence a nd effects of bird impacts with aircraft. W. J. Forse.

FIG S. DAHAG E TO STAI NLES S STEEL LEADI NG EDGE BY 7 • 751b BIRD AT ~~5 ft/ sec ( Z~ 1 0 0 0 ft lb ).

FIG 6. DAMAGE TO CENTRE WINDOW AFTER IMPACT OF 12.000 ft lb ENERGY .

73 The incidence and effects of bird impacts with aircraft. W. J. Forse.

FIG 7. DAMAGE TO WINDOW AFTER IMPACT AT 26.000 ft lb ENERGY.

74 The incidence and effects of bird impacts with aircraft. W. J. Forse.

FIG 8. DAMAGE TO CABIN AFTfR IMPACT AT 28.000 ft lb ENERGY.

FIG 9. DAMAGE TO PILOT AFTER FAILURE OF WINDOW.

75 ·I I Grant, C. and Savory, J. G. Int. Biodetn Bull., 5 (2): 77-94, (1969). Methods for isolation and identification of fungi on wood. • METHODS FOR ISOLATION AND IDENTIFICATION OF FUNGI ON WOOD' C. Grant' and J. G. Savory'

Summary. Practical details of isolation techniques for and Verfahren zur lsoliernng und Bestimmnng von Pilzcn anf Holz. identification of wood·desawins fungi are listed. Appendix I Praktische Einzelheiten von Verfahren zur Isolierung und contains infonnation on med.a and techniques for the study of Bestimmung von holzzerstorenden Pilzcn werden aufgefllhrt. the fruiting of Basidiomycetes in culture and Appendix II is a Anhang I gibt Auskunft ilber Kultunnedien und Verfahren zur bibliography of culture descriptions with an index of fungal Untersuchung von FruchtkHrpem von Basidiomyceten in species. Kultur, und J\nhang II bestehti aus einer Bibliographie von Kulturbeschreibungen mit einem Index fUr Pilzarten. Methodes pour I'Isolement et I'Identilitation des Cbampignons sur Ie Bois. Les dCtails pratiques des techniques d'isolement M~todos para aislar e identifkar los bongos en Ia madera. Se et !'identification des champignons xylophages soot enum6res. registran los detalles prActices de Ia tCcnica de aislar e identificar L'Appendice I contient des infonnations sur les milieux et los bongos que destrozan Ia madera. El primer ap6ndice contiene les techniques concernant t•etude deJa fructification des Basidio­ informes sobre los medics y Ia tknica para el estudio de Ia mycetes en culture et l'Appendice II est une bibliographie fro.ctificaci6n de los Basidiomycetes en cultura. El segundo relative aux. descriptions de cultures, comportant un index des ap~ndice es una bibliografia de descripciones de culturas con un especes fongiques. fndice de las especies fungosas.

Introduction 7. Examine surface fungus growths by the cellophane tape-<:over glass technique {Endo, R. M. 1966) or This is a compilation of notes, instructions and ref­ by direct mounts in water or lactophenol. erences originally prepared for staff use and now published for the information of other Timber Mycolo­ 8. Note stains, e.g. sapstain or stains associated with gists. The techniques quoted have been developed at the rot. this and other Laboratories; some have been in use 9. Examine sections under the microscope (FPRL for many years and have already been described in Leaflet No. 40). Choose samples from the advancing other publications. Professor C. G. Chesters of edge of the decay zone; avoid severely decayed wood. Nottingham University first brought the Twig Culture 10. Damp chamber suitably moistened material. (See technique to the notice of J. G. Savory. Appendix I.)

The Material Source of Inocula 1. If possible, select material embracing the full 1. Note the source of individual inocula or groups · range of decay from rotten to sound wood. Obtain of inocula and also the date of inoculation. ample material; a whole log or complete structural 2. Make isolations from less decayed wood and from unit whenever this is possible. zones of discoloration. Very decayed wood is less 2. Ascertain the previous history of the material. liable to contain viable mycelium and may be infected 3. Give each sample an accession number and by secondary invaders. record all observations. Isolation Techniques Examination Adopt isolation techniques suitable for the sample and the project. Discussion of some of the methods is 1. Confirm the identity of the timber. ('Things given in Greaves and Savory (1965), and in the Ph.D. b'ain't always what they seem', Gilbert and Sullivan.) thesis by H. Greaves dated October 1966. 2. Note the distribution of rot in the sample, for 1. Use of the Pressler Increment Borer. (This tool is example in relation to heartwood and sapwood; in usually employed by foresters to extract a solid core sawn timber look for clues as to its presence or absence of wood from growing trees.) After sterilisation of the at the time of conversion. borer itself and the associated plug extractor by 3. Note location in tree, e.g. butt rot, stem rot. fiaroing in alcohol, the borer is again dipped in alcohol Adhering alcohol is then ignited and the flame issuing 4. Note relation to previous history, e.g. pruning cuts, from the end of the borer is used to sterilise the surface logging injury, flood damage in building, etc. of the area of timber to be sampled. The borer is 5. Determine type of rot-white, brown, pocket, etc. driven into the wood; the plug is removed by the plug (See Appendix I.) extractor and transferred aseptically either to an 6. Note presence of visible fungus growth; sheets of empty sterile petri-disb, where it can be cut aseptically mycelium on the surface or in shakes in the wood; into a number of inocula, or the plug inoculum is fruit bodies or traces thereof on wood surfaces. directly transferred to a slope of medium. The plug

'Ministry of Technology, Forest Products Research Laboratory, Princes Risborough, Aylesbury, Bucks, England. •A revised version of the paper published by the Forest Products Research Laboratory in May, 1968. (Copy received, May, 1969). 77 Methods for Isolation and Identification of Fungi on Wood. C. Grant and J. G. Savory. is loosened from the extractor by forcing it against the Isolations may be made direct from fruit bodies of inner surface of the test tube with a rolling movement. wood-destroying Basidiomycetes by taking portions 2. The Two-Chisel Technique. A sterilised t-inch aseptically from beneath the surface of the fleshier (13 mm) chisel is used to remove the superficial layer parts and planting them on agar. Bacterial contamina­ of decayed wood and underlying wood is then sampled tion is often a problem so it is desirable to include with the aid of a t-inch (6 mm) sterilised chisel. The inoculations on to malt agar acidified by the addition resultant shaving is placed on agar with a pair of of t per cent malic acid and to plant some inocula on sterilised forceps. Best results are obtained if the the dried agar at the extreme toe of the slope. shaving is stuck on edge so that half of its cross­ section is embedded in the agar; complete saturation Aids to Successful Isolation of the chip is then avoided. I. Choice of Culture Vessel. Petri-dishes are very 3. The Cut Block System. Cut a slice about I em convenient for ease of access for observation or thick across the cross-section of the wood sample. subculturing but they dry out too rapidly to allow Split blocks of convenient size for planting in the successful isolation of many Basidiomycetes. For culture vessel, be it petri-dish or test tube, from this these it is often desirable to use test tube slopes. material with a chisel. Mount the blocks on the point Inocula from which nothing grows should be retained of a stiff wire inoculating needle and lightly flame for twelve weeks because the Basditiomycetes often sterilise before planting them aseptically on the remain quiescent in the inocula for six weeks or more. medium; if the blocks are very small it is easy to kill 2. PrecDrying of Wood Prior to Isolation. If bacteria the fungus by overheating. prove troublesome the best method of control is to 4. The Split-Billet Technique (Shigo, A. L., 1965). dry the wood from which the inocula are taken for Inocula are taken from a freshly split surface of a two or three days beforehand in the Laboratory. sample billet by cutting samples of matchstick cross­ 3. The Choice of Media. Standard media used are section longitudinally from the freshly exposed 2 or 5 per cent malt and 5 per cent malt acidified by surface with the aid of a sterilised U-gouge or scribe. the incorporation of t per cent malic acid. Use of Sterilised forceps are used to transfer the inoculum Czapek's medium may encourage thinner growth and to medium. Shigo plants a dozen or more small hence greater ease of selecting individual fungi in samples around the edge of a petri-dish. mixed culture. An infinite variety of selective media is available, for example, media containing antibiotics 5. The Saw-Cut Technique (Greaves and Savory, for the avoidance of bacteria: Russell's orthophenyl 1965). This method provides much smaller inocula phenol medium (Russell, P. 1956) for the selection of than the others. Small particles of sawdust are obtained by cutting the material being samples with a sterilised fungi able to cause white rot: Eggins and Pugh's cellulose medium (Eggins, H. 0. W. and Pugh, G. J. F. hack-saw blade. The particles are allowed to fall over 1962), modified after Bravery (1968), for selecting the surface of petri,dishes of nutrient media. Planting cellulose attacking fungi. Various media low in of too may sawdust particles on any one dish of nutrient value, such as vegetable, soil or sawdust nutrient medium should be avoided. By restricting the decoction agars may prove useful on occasion. number of particles~, and by choice of suitable media, it is often possible to grow out single microfungi from The Products of Isolation individual particles of soft rotted material. Basidio, mycetes have only been isolated from mixtures with It is important to maintain continued observations soft rot fungi when methods employing small particle on culture vessels containing inocula. Slow growing size have been employed. organisms can often be recovered from mixed culture if subcultures are made from them immediately they 6. The Drill Technique (Levy, J. F., 1967). The drill become noticeable and before they can be overgrown is inserted in a standard carpenter's brace. A !-inch by faster growing organisms on the same dish. If it (13 mm) twist drill or smaller can be used. The drill is the intention to culture wood-rotting Basidiomycetes and the wood surface are sterilised by flaming; the these can often be recognised by their silky appearance drill is introduced into the sample and particles in culture. Inocula which give rise to fungi imperfecti obtained from the hole are planted out in the same (e.g. Penicillium), easily recognisable by their appear­ manner as in the sawdust technique. Particles can ance, can be rejected if Basidiomycetes are being often be conveniently taken from the drill by tapping sought, but if soft rot is in question, all of the fungi the drill on the base of a sterilised petri-dish and isolated must be considered. Staining fungi can usually subsequently pouring agar at a temperature of 45'C. be recognised in culture because their hyphae darken 7. Isolation from Fruit Bodies. If fruit bodies are in time and very often they darken the malt agar available, a number of different techniques can be medium itself. employed; spore suspensions can be made up from fruit bodies of bark inhabiting Ascomycetes in bark; Book-Keeping spore projection on to agar can be employed for some I. Accession Number. Each piece of wood from which Basidiomycetes, the fruit body is stuck to one side of isolation is made should be identified by its own the petri•dish lid and allowed to project spores down accession number, e.g. M.456. If several pieces have on to the medium beneath. The dish lid is rotated been given the same accession number, then each at shortening intervals so that there will be variation piece should be identified by the accession number in the number of spores deposited. plus a suffix letter, e.g. M.456b. 78 Methods for Isolation and Identification of Fungi on Wood. C. Grant and J. G. Savory.

2. Date of Culturing. This should he affixed to each Forest Products Research Laboratory. Leaflet No. 40. tube or group of tubes. It is often convenient to hold The preparation of wood for microscopic examina­ a group of tubes in a beaker or similar container tion. HMSO, Revised November 1956. with a single label on stiff card in the early stages. Greaves, H. (1966). New concepts of the decay of 3. Tnbe Number. When fungal growth is seen a tube timber by micro-organisms with reference to the number should he affixed, e.g. M.456b/12. long-term durability of wood from archaeological sites. Ph.D. Thesis, University of London. 4. Serial Number. If several fungi are isolated from one tuhe they should in tum be identified by numbers, Greaves, H. and Savory, J. G. (1965). Studies of the e.g. M.456b/12/3. In every case the date of sub­ microfungi attacking preservative-treated timber, culturing should be noted on each tube; with particular reference to methods for their isolation. J. Inst. Wood Sci., No. 15: 45-50. 5. Culture Collection. If it is intended to retain cultures temporarily for further work or permanently Levy, J. F. (1967). Necessity for developing reliable because they have been identified, they should be techniques for the isolation and identification of transferred to a permanent culture collection, being fungi from wood. Wood, 32 (6): 37-39. given a new number in the process. The new number Nobles, M. K. (1948). Studies in forest pathology. VI. should be noted on the accession list and the original Identification of cultures of wood-rotting fungi. accession number should be noted in the culture list. Canad. J. Res., C, 26: 281-431. Nobles, M. K. (1965). Identification of cultures of Identification of Fungi in Culture wood-inhabitingHymenomycetes. Canad.J. Bot., 43: 1097-1139. Non-Basidiomycetes may be identifiable from obser­ vations on the character of the spores produced and Russell, P. (1956). A selective medium for the isolation the manner in which they are borne. In the case of the of Basidiomycetes. Nature, Lond., 177(4518): 1038-9. Fungi Imperfecti it is particularly important to note Shiga, A. L. (1965). Organism interactions in decay from the colonies whether the spores are borne dry and discoloration in beech, birch, and maple. U.S. or wet and by making microscopical observations on Forest Serv. Res. Paper Ne-43. dry-mounted material to form opinions on the manner in which the spores are attached, i.e. in chains, in APPENDIX 1: FRUITING OF BASIDIOMYCETES clusters or singly. IN CULTURE Distinguishing between Basidiomycete and non­ Basidiomycete cultures is not always easy; the absence Cultures destined for fruit body production should .. of clamp connections in microscope preparations is he exposed to the light but shielded from 'direct sun­ not a positive indication that the culture is a non­ light. In general it is advisable to disturb the cultures Basidiomycete. Identification ofBasidiomycete cultures as little as possible; should they be moved they must is usually difficult, but will be facilitated if fruit bodies be replaced with their original orientation. can be produced (see Appendix 1). Unknown cultures should be compared with any named Basidiomycete Media Employed cultures which are available and microscopic characters 1. Agar. As only a few fungi fruit readily on 2 or 5 should be observed as required by the FPRL card key per cent malt agar media its use for this purpose is (Findlay, W. P. K., 1947) and Miss Nobles' keys limited. It may be important to use large tubes so that (Nobles, M. K., 1948 and 1965). Regrowing the fungus a bulk of medium can be provided. Fruiting may on petri"dishes, malt agar boiling tubes, sawdust sometimes by initiated by transferring a culture to a boiling tubes should be undertaken, as appropriate. low temperature for a few hours. Literature should be studied as there are many scattered descriptions of Basidiomycetes in culture 2. Sawdust Medium. This medium provides a useful (see Appendix II). means of determining the type of rot produced by a fungus, i.e. white or brown rot. Subsequently fruiting may take place in the culture vessel and although References fruit bodies produced in this way are not always Bravery, A. F. (1968). Microbiological breakdown of typical they may provide clues as to the identity of cellulose in the presence of alternative carbon the fungus. sources. J. Sci. Fd Agric., 19: 133-135. Preparation of the Sawdust Medium. The medium Eggins, H. 0. W. and Pugh, G. J. F. (1962). Isolation used at the FPRL is a modification of that proposed of cellulose-decomposing fungi from the soil. Nature by Badcock (1941). Hardwood fungi are grown on Lond., 193 (4810): 94-5. heech sawdust, and softwood fungi on either Norway or Siberian spruce sawdust. If supplies of these are Endo, R. M. (1966). A cellophane tape-cover glass not readily available then sawdust of any easily technique for preparing microscopic slide mounts of decayed wood may be substituted. The culture vessels fungi. Myco/ogia, 58: 655-9. used are boiling tuhes but any suitably sized glass Findlay, W. P. K. (1947). The use of perforated cards containers can be employed providing they are for the preliminary identification of fungi. Trans. capable of withstanding the temperature and pressure British Mycol. Soc., 31: 106-111. of autoclave sterilisation. 79 Methods for Isolation and Identification of Fungi on Wood. C. Grant and J. G. Savory.

To fill one boiling tube the following quantities of readily if the tube is laid almost flat with the open end ingredients are required. raised slightly and always orientated in the same way. Badcock (1943) described a modification of the saw­ Sawdust 20 g (10-20 mesh (0·6•1·2 mm) particle dust/tube technique whereby he obtained fruit bodies Maize meal 0·6 g}A t size) Bone meal 0·4 g cce1 era or of 81 species of wood-rotting fungi. Water 40-60 mi (200-300% moisture content) Techniques It has been found necessary to incorporate the 1. Wood Block Technique (Plate 2(a)). A method for 'accelerator' as many fungi do not make vigorous the artificial production of Basidiomycete fruit bodies growth on sawdust alone. Water should be added until on wood blocks was developed by Tamblyn and the mixture has the right 'feel'. This is when the Da Costa (1958). It has been used at the FPRL with sawdust particles will stick together readily to form a few modifications. quite large clumps. Experience has shown this to be within a moisture content range of between 200 and Materials. Badcock's sawdust medium as previously 300 per cent. described. Wood blocks of the same timber as the sawdust The ingredients should be thoroughly mixed and used. The block measures 7·5 X 7·5 X 4·5 em. It has lightly packed into boiling tubes. To obtain uniform a well 6 em in diameter and 0·5 em in depth in the distribution throughout the tube it is necessary to upper surface as a water reservoir and a hole 6 em gently tap the base of the tube on a bench. Filling diameter X 2 em deep in the lower face to fit over should be to within about 2:!; inches (6 em) from the the mouth of the jar. This hole is further recessed by top. After plugging with cottonwool the tubes are a 4 em diameter x 1 em deep cavity to take a wooden • autoclave sterilised. Beech sawdust medium may plug measuring 4 em in diameter X 6·5 em in length. require several periods of autoclaving as the wood The direction of the grain is an important feature; itself contains a natural microflora. Softwood sawdust in the wood block it should run parallel to one of usually requires only a single period of 30 minutes the long axes whereas in the plug it should run at 15 p.s.i. (1·05 Kgfcm 2). After sterilising the tubes vertically. The former ensures that water from the may be stored for several months in a refrigerator reservoir is not able to soak through into the jar and used as required. It should be borne in mind, easily, whilst the latter enables the fungus to make however, that some desiccation takes place and re• rapid growth up the grain through the plug into the wetting of the medium may be necessary. This can wood block. be achieved either by adding sterile water to the tubes under sterile conditions (e.g. within a hood) or Method. The sawdust medium is packed into a by adding tap water and then reautoclaving. 16 oz glass jar fitted with a ventilated screw cap and autoclave sterilised for one hour at 15 p.s.i. The fungus Use for determining White and Brown Rot reactions. is inoculated into the sawdust and incubated in the The fungus to be tested should first be grown on malt dark at 2rc until a vigorous mycelium develops agar until well established and then a piece of inoculum throughout the medium. The wood block assembly removed and buried against the glass to a depth of is then soaked in water for one or two hours and approximately 1 inch (2·5 em) in the sawdust. autoclave sterilised for one hour at 15 p.s.i. (on each Incubation should initially be in the dark at room of three successive days if the wood is beech). When temperature unless it is suspected that the fungus cool it is placed aseptically in the mouth of the jar has special temperature requirements. If possible so that the plug is pushed into the sawdust and the several replicates should be prepared and incubated block rests securely over the neck. A clean polythene under different conditions. bag is put over the block and the top of the jar and The white/brown rot reaction usually occurs within secured with an elastic band to exclude contamination a fortnight; some species may take longer. Brown rot and maintain a high humidity. fungi will darken the sawdust gradually. This is more Incubate at room temperature (although a brief easily detectable in light-coloured sawdusts although exposure to a somewhat lower temperature may be beech lightens somewhat at first with subsequent beneficial) in a cabinet with a glass lid and glass side darkening. White rot fungi initially form a rich panels receiving diffused light. Direct exposure to brown colouration followed by bleaching of the sunlight should be avoided. Water reservoirs in the sawdust. cabinet maintain a high ambient humidity and as soon as the fungus reaches the surface of the wood Plate 1 shows Polystictus versicolor (a white rot block the polythene bag is carefully removed. Occa­ fungus) growing on beech sawdust. sional watering ensures that drying-out does not take Inducing Fruiting. When the fungus has made good place. Although some contamination is inevitable this growth throughout the medium it is desirable that it does not usually present a serious problem except on should be exposed to the light. This should not be beech blocks where Chaetomium and Trichoderma direct sunlight and strong artificial light should be species have proved troublesome. avoided. As an aid to fruit body formation the cotton­ 2. Moisture Gradient Technique (Plate 2(c) Upper). wool plug may be removed and a moistened pad of Where it is thought that a fungus requires a moisture absorbent cottd'nwool placed in the neck of the tube gradient in order to fruit the following technique may to prevent drying•out. In general, fruiting occurs more prove useful. 80 Methods for Isolation and Identification of Fungi on Wood. C. Grant and J. G. Savory.

B

Polyst ictus versicolor growing on Badcock's sawdust medium (beech) React ion A. Natural colour of the sawdust after B. Zone of initially darkened medium one week C. Area where bleaching is beginning to take place

PLATE 1 81 (a) Modified Tamblyn/ Da Costa wood block assembly for obtaining fruit bodies of wood-destroying Basidiomycetes .

culture mtdiu .. ·

wood block--#--

~~~~~~~-----=----~~-=~~~~-wottr crystallising dish/

(b) Nectria cinnabaTina fruiting (c) Upper - Moisture gradient technique in twig culture vessel . Lower - 'Damp chambering' apparatus . PLATE 2 82 Methods lor lsolallon and Identification of Fungi on Wood. C. Grant and J. G. Savory.

Materials. A 'feeder' strip of wood provides a (iv) Vegetative growths of fungus should be visible a substrate in which a moisture gradient may be within a week or two. If no growth is visible after obtained. A stick of suitable dimensions is placed in about 12 weeks the sample can be rejected. Fruit a I litre conical flask (or container of similar capacity) body formation may take much longer than this. It which has wood chips or sawdust medium of the same may be necessary to replenish the water in the con­ timber to a depth of approximately I inch (2·5 em) tainer from time to time. in the bottom. The wood chips and the feeder strip 4. Twig Culture Technique {Plate 2(b)). This method should be thoroughly moistened. The flask is plugged is most useful for obtaining fruiting bodies of bark with cottonwool and the assembly sterilised for a inhabiting fungi. suitable period dependent on the timber species employed (15 p.s.i. for 30 minutes is adequate for Materials most timbers with the exception of beech). (i) Culture vessels are prepared containing at least Method. The fungus is inoculated on to the chips ! inch (1·3 em) depth of 2 per cent malt agar medium or sawdust and incubated in the dark at room tem­ in their base. Suitable vessels are 500 cc wide-mouthed perature until fully established. The flask may then conical flasks or 24 oz wide•mouthed bottles. The be brought into the light (subject to the same previous culture vessel is prepared with a cottonwool plug considerations) and fruiting should occur where the through which is inserted a test tube ofdiameter similar moisture content of the 'feeder' strip is at the optimum to or slightly greater than the expected diameter of for the fungus. the twigs which will be used. (ii) Species of twig used should where possible be 3. Damp Chamber Technique (Plate 2(c) Lower). It is related to the timber from which the fungus was sometimes possible to continue the development of a isolated. Alternatively, both hardwood and softwood fungus in wood by the technique known as 'damp twigs should be employed. Beech is a particularly chambering'. This relies on a fruit body being pro­ good timber to use because its bark naturally supports duced on naturally infected material which is main­ the growth of a wide range of fungi. Straight fast-grown tained in a moist condition in a suitably moist coppice material will usually provide twigs which are environment. easier to handle but it will usually be preferable to include some older material with thicker bark as well. Materials Twig thickness can be varied considerably but they (i) Samples of timber of as large a size as is con­ should not be so large that they cannot be cut with venient, taken preferably at the boundary of attack. secateurs. Material of a thickness of or slightly thinner Severely decayed wood which has dried and broken than a thumb is probably best. up is best avoided. Method (ii) Large, wide-necked jars with oversize lids to pro­ vide loosely fitting caps which will reduce drying but (i) When the cultures have fully overgrown their not wholly prevent air movement. Large crystallising medium, suitable branches should be cut from trees dishes make excellent containers if suitable glass lids and immediately brought into the laboratory and cut can be provided. (Polythene bags could be adapted into twigs about 12 inches {30 em) long. Each twig for use if no other container is available, but some should be pruned to remove side branches. Any form of aeration by provision of small holes or by residual snags and major bark irregularities should be opening the mouth of the bag for a short period in carefully smoothed away with a sharp scalpel to each week would be needed to permit continued provide a smooth outer surface without projections growth in a polythene bag.) and without cavities, which could retain fungus spores. (iii) Small basal supports such as petri-dish lids to Twigs are cleaned with soap and water and gently prevent water-logging of samples. scrubbed with a nail brush to remove as much surface debris as possible. Each twig is then held over a sink Method and thoroughly washed with alcohol. Finally, the twig is held in sterile long-handled forceps over the (i) Cut samples to as large a size as is compatible sink, flooded with alcohol from a wash bottle and with the containers available. the alcohol is flamed off the twig. This process is (ii) Soak the samples in cold tap water. The period repeated a second time and the twig is then transferred of soaking can only be determined in the light of into a sterilised container before removal to the experience and depends to a large extent on the con­ culture room. dition and size of individual samples. Soaking periods (ii) In the culture room one end of the twig is cut ranging from two hours to overnight could perhaps away at an angle of 45° using secateurs which have be tried. Four to six hours is probably adequate for been sterilised by being dipped in alcohol and from an average sample of decayed 4 inch x 2 inch (10 x which the final traces of alcohol have been removed 5 em) softwood about 6 inch (15 em) long. by flaming. The prepared culture vessel is held on its (iii) Place water in the bottom of the container and side, the tube running through the plug is removed support the soaked sample out ofcontact with the water and the twig is thrust down through the hole in the to prevent the timber becoming water-logged. Cover cottonwool and down into the agar so that the top the container loosely with the lid and store away of the bevelled cnt lies just below the upper surface from radiators and away from direct sunshine. of the agar. It is convenient to have available supplies I 83 ,! !I il I; Methods for Isolation and Identification of Fungi on Wood. C. Grant and J. G. Savory. of sterilised cottonwool so that new plugs can be Britain. Two of the papers by Dr. Nobles (110 and made up if necessary or so that padding can be 114) are of particular importance not only for the inserted around the twig to make a tight fit with the numbers of species described but also for the clarity cottonwool plug. Finally, the upper end of the twig of the descriptions illustrated by camera-lucida is shortened to a convenient degree using the flame drawings. She has also studied certain cultural sterilised secateurs and the twig is covered with a characters in relation to taxonomy for 252 species of double layer of cellophane tied down around the Polyporaceae (158). neck of the bottle with string. Index of Fungi (iii) The prepared bottles are incubated suitably and kept under observation, possibly for periods of several Reference Number months. Agaricus arvensis 125 (iv) Fruiting may not take place or may be abnormal Agaricus bisporus 125 in the bottle. To obtain normal fruit bodies it may be Amanita muscaria 125 necessary to take the twigs from their bottles, wire Amauroderma rude 133 them loosely together and keep them in long grass Aporpium caryae (Poria canescens) 90, 114 in the shade of a building for a few weeks. Armillaria me/lea 9, 24, 34, 46, 49, 54, 56, 60, 68, (v) As the process carries risks of contamination it is 69, 110, 113, 114, 121 essential to re-isolate from the fruiting bodies and Armillaria mucida 49 confirm that the organism concerned is the correct Boletinus amabilis 83 one. The process of semi,.terilisation which is used Boletinus cavipes 83 is necessary because normal fruit bodies will not Boletinus ochraceoroseus 83 form on autoclave sterilised twigs. Calocybe gambosa 125 Claudopus nidulans 113 Clitocybe il/udens 54 References Clitocybe inversa 125 Badcock, E. C. (1941). New methods for the cultiva­ Clitocybe odora 125 tion of wood-rotting fungi. Trans. Brit. Myco/. Soc., Clitocybe tabescens 54 25: 200-205. Collybia macula/a 125 Badcock, E. C. (1943). Methods for obtaining fructi­ Collybia radicata 35, 110, 114 fications of wood-rotting fungi in culture. Trans. Collybia velutipes 31, 54, 60, 71, 79, 110, 113, 114, Brit. Mycol. Soc., 26: 127-132. (F/ammulina velutipes) 125 Coniophora arida 133 Tamblyn, N. and DaCosta, E. W. B. (1958). A simple Coniophora cerebella (puteana) 11, 42, 44, 46, 49, 54, technique for producing fruit bodies of wood­ 57, 79, 82, 88, 96, 97, IlO, 113, I14, 148 destroying Basidiomycetes. Nature, Lond., 181(46081 · Coniophora suffocata 54 578-9. Coprinus atramentarius 125 Coprinus micaceus 2, 113 APPENDIX IT BffiLIOGRAPHY OF CULTURE Coprinus sterqui/inus 125 Corticium alutaceum (radiosum) 81, 155 DESCRIPTIONS Corticium coeru/eum 54 Most, but not all, of the papers in the numbered Corticium conj/uens 54 list entitled 'REFERENCES' (page 90) relate to Corticium effuscatum 108 wood-destroying species. The majority of the fungi Corticium furfuraceum ll3, 114 dealt with are Basidiomycetes but a number of the Corticium fuscostratum 113 larger Ascomycetes are also listed; 'soft rot' and 'sap Corticium galactinum 54, 113 stain' organisms have not been included. Some papers Corticium hydnans (Basidioradulum radula) 54, 144 relate solely to individual features, for example odour (Radulum orbiculare) (2), extracellular oxidase production (54 and 113), Corticium incrustans 160 growth rate (44), sporophore production (105) and Corticium investiens 54 compatability factors (137). Corticium /aeve 81, 113 Corticium lividum 54, 56 The first 136 papers are listed in alphabetical Corticium polygonium (Peniophora polygonia) 113, 155 sequence of authors; the remainder-under the sub­ Corticium vel/ereum 112, 113, 114 heading 'Additions' (page 94}-are placed in the order Cyathus he/enae 17 5 in which they were brought to attention. Cyathus striatus 175 The alphabetical 'INDEX OF FUNGI' (page 85) Cytidia sa/icina 54 gives the numbers of the papers referring to each Daedalea ambigua 54, 158 organism. The fungus names used by the original Daeda/ea confragosa 54, 56, IlO, I13, 114, 158 authors of papers have been quoted; in a few instances Daedalea juniperina 54, 158 synonyms have also been given. Daedalea quercina 2, 42, 44, 45, 49, 54, 56, 79, 110, Cartwright and Findlay (49), in a work still readily I14, 132, 158 available, have provided descriptions of the wood­ Daeda/ea unico/or (Cerrena unicolor) 54, 79, IlO, destroying fungi of greatest economic significance in I13, 114, 134, 158 84 Methods for Isolation and Identification of Fungi on Wood. C. Grant and J. G. Savory.

Reference Number Reference Number

Daldinia concentrica 49 Fornes rimosus (robiniae) 3, 37, 54, 79, 85, 110, 114, Echinodontium tinctorium 54, 110, 113, 114, !58 158, 174 Favolus a/veolaris (Polyporus a/veolaris) 54, 110, 114, Fornes robustus 9, 37, 54, 56, 120, 158, 174 !58 Fornes roseus 9, 37, 46, 54, 58, 65, 104, 110, Favolus canadensis 54 113, 114, 128, 130, 158, 174 Fistulina hepatica 45, 49, 54, 56 Fornes sanfordii 5, 174 Flammula alnico/a 59, 114 Fornes scruposus 174 Flammula conissans 59, 113, 114 Fornes scrupulosus 5 Fornes a/bomarginatus 174 Fornes scutel/atus 2, 37, 54, 110, 114, 158, 174 Fornes annosus 9, 37, 42, 44, 46, 49, 54, 79, 110, Fomus senex 3, 174 113, 114, 158, 174 Fornes spadiceus !58 Fornes badius 3, 174 Fornes subroseus (cajanderi) 37, 46, 49, 54, 79, 110, Fornes calkinsii 37, 54, !58 (Trametes subrosea) 113, 114, 130, 158 Fornes caryophylli 174 Fornes texanus 85 Fornes cinchonensis 174 Fornes torulosus 37, !58 Fornes clelandi 120 Fornes tricolor 174 Fornes conchatus 37, 54, 110, 113, 114, !58 Fornes ulmarius 49, !51, 158, 174 Fornes connatus 36, 54, 110, 113, 114, !58, 174 Fornes vitico/a (tenuis) (Trametes tenuis) 2, 37, 54, 110, Fornes densus (johnsonianus) 37, 54, 161 114, 158 Fornes demidojfi 46 Ganoderma applanatum (Fornes applanatus) 2, 3, 37, Fornes dochmius 174 42, 44, 45, 48, 49, 54, 65, 76, 79, 91, 110, 113, 114, 120, Fornes durissimus 174 135, 158 Fornes ellisianus (Po/yporus el/isianus) 18, 21, 110, Ganoderma colossum 133, 158 114, 158, 165 Ganoderma /obatum (Fornes /obatus) 37, 54, 56, 110, Fornes everhartii 17, 37, 54, 56, 79, 110, 113, 114, 158 114, 158 Fornes extensus !58 Ganoderma lucidum (sessile) 3, 54, 56, 79, 110, 114, Fornes fastuosus 174 (Polyporus /ucidus) !58 Fornes fomentarius 2, 37, 48, 49, 54, 65, 72, 87, 98, Ganoderma oregonense 2, 54, 110, 113, 114, !58 110, 113, 114, 158, 164, 174 Ganoderma resinaceum 2, !58 Ganoderma tsugae (Polyporus tsugae) 54, 110, 114, !58 Fornes fraxineus 2, 16, 37, 42, 48, 49, 54, 101, 110, 114, 158 Gloeocystidium karstenii 113 Fornes fraxinophilus 18, 37, 54, 110, 114, 158, 165 Grandinia farinacea 81 He/icobasidium corticiodides 155 Fornes geotropus 37, 54, 56, !58 Hirschioporus /aricinus 137 Fornes hemitephrus 120 (Lenzites /aricinus) Fornes igniarius (Phel/inus igniarius) 37, 48, 49, 54, Hydnum abietis (Hericium abietis) 113 65, 75, 79, 110, 113, 114, 131, 143, 158 Hydnum caput-ursi 54 Fornes igniarius var. laevigatus 37, 54, 110, 114, !58 Hydnum coralloides 54 Fornes igniarius var.populinus 37, 54, 110, 113, 114, !58 Hydnum erinaceus 54, 56, 113 Fornes insu/aris 174 (Hericzum ermaceus) Fornes juniperinus 37, !58 Hydnum /aciniatum 113 Fornes lamaoensis 174 (Hericium laciniatum) Fornes /aricis 2 Hydnum pulcherrinum 54 Fornes lignosus 2, 174 Hydnum septentrionale 54, 56, 113 Fornes lividus 174 (Steccherinum septentrionale) Fornes marmora/us 37, 54, !58 Hymenochaete agg/utinans 54 Fornes melanoporus 174 Hymenochaete corrugata 54, 110, 114 Fornes me/iae (Polyporus meliae) 37, 54, !58, 162 Hymenochaete curtisii 54 Fornes nigrolimitatus 79, 110, 114, !58 Hymenochaete rubiginosa 54, 56 Fornes noxius 2 Hymenochaete tabacina 54, 110, 114 Fornes officina/is 37, 46, 49, 54, 61, 79, 110, 113, Hypholoma capnoides 125, 152 114, 158 (Naematoloma capnoides) Fornes ohiensis 37, 54, 110, 114, !58 Hypholoma fasciculare 125, 152 Fornes ostrico/oris 5 (Naematoloma fasciculare) Fornes pachyphloeus 174 Hypoxylon mammatum 176 l: Fornes pectinatus 174 Hypoxylon ustulatum 10 Fornes pinicola 2, 9, 37, 44, 46, 49, 54, 56, 65, 79, 81, Inonotus nothofagi !58 102, 103, 104, 110, 113, 114, 158, 173, 174 lrpex cinnamomeus 54, 56 Fornes pomaceus (fulvus) 18, 37, 48, 49, 54, 63, 110, Irpex fusco-violaceus 137, 138 ,, 114, 158 (Hirschioporus fusco-violaceus) I< Fornes rhabarbarinus 174 Irpex mol/is 54, 56 I Fornes ribis 158, 174 Irpex obliquus !58 85 Methods for Isolation and Identification of Fungi on Wood. C. Grant and J. G. Savory. '

Reference Number Reference Number

Lentinellus montanus 177, 178 Peniophora luna 1!3, 155 Lentinel/us ponderosus 177 Peniophora mutata 1!3, 1!4 Lentinus kauffmanii 25, 1!0, 1!4 Peniophora nuda 54 Lentinus /epideus 2, 42, 44, 46, 49, 54, 58, 91, 110, Peniophora ph/eboides 1!3 1!3, 1!4, 128, 132 Peniophora piceae (separans) 1!4, 142 Lentinus sajor-caju 133 Pen ophora pini 1!4, 154 Lentinus tigrinus 42, 49, 54, 56, !10, 1!4, 127, 129 (Peniophora pini subsp. pini) Lenzites (Gloeophyllum) abietina 2, 46, 49, 100, 158 (Stereum pini) Lenzites betulina 2, 5, 32, 44, 49, 54, 1!0, 1!3, 1!4,158 Peniophora pithya 81, 113, 1!4, 142 Lenzites eximia 5 Peniophora pubera 54 Lenzites flaccida 2, 158 Peniophora resinosa 1!3 Lenzites palisoti 133 Peniophora septentrionalis 1!3, 1!4, 142 Lenzites repanda 2, !58 Phel/inus cryptarum (megaloporus) 49 Lenzites saepiaria 2, 9, 40, 42, 46, 49, 54, 58, 64, 65, Phellinus tremulae 143 79, 81, 100, !04, 110, 1!3, 1!4, 123, 124, 128, 129, 130, Phlebia atkinsoniana 146 158 Phlebia mellea 113, 158 Lenzites striata 2, 9, 54, 158 Ph/ebia merismoides 1!3, 158 Lenzites subferruginea 9, 100 Phlebia strigosazonata 54, 1!3 Lenzites trabea 2, 43, 44, 46, 49, 54, 58, 79, 91, 100, Pholiota adiposa 2, 54, 56, 60, !02, 1!0, 1!3, 1!4, 125, 1!0, 1!4, 128, !58 169 Lenzites tricolar 158 Pholiota aurivella 1!4 Lepista nuda 125 Pholiota heteroc/ita 169 Macrolepiota procera 125 Pho/iota lucifera 2 M arasmius al/iaceus 2 Pho/iota mutabilis 2, 125, 169 M arasmius oreades 125 (Kuehneromyces mutabilis) Merulius ambiguus 15P Pho/iota spectabilis 2, 1!3, 169 Merulius americanus 148 Pholiota squarrosa 2, 125, 169 Merulius aureus 13, 158 Pleurotus corticatus 54 Merulis confluens 2, 13, 54, 1:58 Pleurotus euosmus 2 Merulius corium 13, 158 Pleurotus lignatilis 2 Merulius himantioides 2, 12, 70, 1!3, 158 Pleurotus ostreatus 2, 44, 48, 49, 54, 56, 60, 79, 1!0, Merulius /acrymans 2, 42, 44, 46, 49, 60, 70, 79, 110, 1!3, 1!4, 125 1!3, 1!4, 148, 158 Pleurotus sapidus 2 Merulius niveus 158 Pleurotus serotinus 54, 1!3 Merulius pinastri 158 Pleurotus subareolatus 113 Meru/ius rufus 2, 158 Pleurotus ulmaris 2, 54, 60, 110, 1!4 44, 158 Merulius sylvester Polyporus abietinus 46, 49, 54, 65, 66, 67, 79, 81, 89, Merulius tremellosus 2, 5, 54, 56, 158 (Polystictus abietinus) 1!0, 1!3, 1!4, 123, 124, Naematoloma sublateritium 125 (Hirschioporus abietinus) 137, 138, 152, 158, 174 Ill, 1!3, 1!4 Odontia bicolor Polyporus adiposus 158 Omphalia campanel/a 1!0,113, 1!4 Polyporus adustus 2, 5, 27, 42, 43, 44, 49, 54, 1!0, (Xerompha/ina campanella) 113, 1!4, 158, 174 Oudemansiella mucida 125 Polyporus albellus 54, 110, 1!3, 1!4, 158 Oudemansiella radicata 125 Polyporus albidus 113, 158 Panellus rupico/a (Collybia rupicola) 6 Polyporus alboluteus 54, 158 Panus fragi/is 177 Panus laevis 54 Polyporus amorphus 14, 81, 110, 113, 114, 158, 174 Panus stipticus (Polyporus stipticus) 54, 81 Polyporus amygdalinus 54 Paxillus artrotomentosus 2, 149 Polyporus anceps 2, 21, 54, 104, 110, 113, 114, 158 Paxillus panuoides 2, 44, 46,49 Polyporus anebus 174 Pellicularia filamentosa 179 Polyporus anthracophilus 120, 158 Pellicularia praticola 179 Polyporus arcularis 54, 91, 110, 1!4, 120, 133, 158, Peniophora allescheri 54, 159 174 Peniophora aspera 113 Polyporus aerofulvus 158 Peniophora cinerea 54, 1!3 Polyporus australiensis 92, 97, 158 Peniophora coccineo-fulva 54 Polyporus balsameus 54, 57, 65, 77, 110, 113, 114 Peniophora duplex 1!4, 154 Polyporus benzoinus 2, 46 (Peniphora pini subsp. duplex) Polyporus berkeleyi 54, 56, 110, 114, 158 Peniophora jilamentosa 5 Polyporus betulinus 2, 48, 49, 54, 86, 110, 113, 1!4, Peniophora gigantea 26, 46, 49, 54, 79, 81, 1!0, 1!3, 1!4 158 Pen'ophora heterocystidia 114 Polyporus biennis 174 Peniophora incarnata 54 Polyporus biformis 14, 174 86 I· ii r·'

Methods for Isolation and Identification of Fungi on Wood. C. Grant and J. G. Savory.

Reference Number Reference Number

Polyporus borealis (Spongipellis borealis) 46, 49, 54, Polyporus /aetus !58 65, 81, llO, ll3, ll4, 158, 172 Po/yporus lapponicus !58 Polyporus brumalis 54, llO, ll4, 158, 174 Polyporus /eoninus 174 Polyporus caesius 81, !58 Polyporus leucospongia 156, 174 Polyporus caperatus 174 Polyporus lignosus 30 Polyporus catervatus !58 Polyporus ludovicianus 54, 56 Polyporus cervinogilvus 174 Polyporus melanopus !58 Polyporus cinnabarinus 54, 94, llO, ll3, ll4, 140, Polyporus mikadoi 2 (Pycnoporus cinnabarinus) 141, 158, 174 Polyporus mol/is 2, llO, 114, 158 Polyporus circinatus see Polyporus Polyporus montanus llO, 113, ll4, 158 tomentosus var. circinatus Polyporus mutabilis 54 Polyporus coccineus 114, 141 Polyporus abducens 2 (Pycnoporus coccineus) Polyporus obtusus 2, 54, 56, 110, 114, 158, 174 Polyporus co/ensoi 92,97 Polyporus occidentalis !58 Polyporus compactus 54, 56, ll 0, ll4, !58 (Po/ystictus occidentalis) Polyporus conchifer 54, llO, ll3, 114, 158 Polyporus oclrro/eucus !58 Polyporus conchoides 14, 174 Polyporus oregonensis 54 Polyporus consors 5, 174 Polyporus osseus 54, 158 Polyporus croceus 2, 54, 56, ll3, 158 Polyporus ostreiformis 28, 174 Polyporus cotoneus 14, 174 Polyporus palustris 6, 54, llO, ll4, 158, 162, 174 (Polystictus cotoneus) Polyporus peckianus !58 Polyporus curtisii 54 Polyporus pergamenus (pargamenus) 5, 54, 56, 89, Polyporus cuticu/aris 48, 49, 54, llO, ll3, ll4, !58 lPo/ystictus pergamenus) llO, 113, 114, 122, 158, Polyporus cutifractus 158 174 Polyporus delectans 54, !58 Polyporus picipes 54 Polyporus destructor var. resupinatus !58 Polyporus pinsitis !58 Polyporus dichrous !4, 54, llO, ll3, ll4, 158, 174 Polyporus platensis !58 : Polyporus distortus (biennis) 54, llO, ll4, !58 Polyporus portentosus !58 Polyporus dryadeus 42, 45, 49, 54, 56, 91, 110, Polyporus pubescens 54, llO, ll3, 114, !58 ll4, 158 Polyporus pu/cherrimus !58 Polyporus dryophilus 8, 54, 56, 85, llO, ll4, !58 Polyporus radiatus 48, 49, 54, 79, llO, ll4, !58 Polyporus dryophilus var. vulpinus llO, ll3, !58 Polyporus radicatus !58 Polyporus durus (Fornes durus) 2, 158, 174 Polyporus resinosus 46, 54, 104, 110, 113, ll4, !58 Polyporus e/egans !58 Polyporus rheades 8 Polyporus elongatus 174 Polyporus rhipidium 120, !58 Polyporus fagico/a !58 Polyporus robiniophilus 54, !58 Polyporus farlowii 85 Po/yporus rubidus !0, 174 Polyporusjibrillosus 54, llO, ll4, 158 Polyporus rugulosus !58 Polyporus jissilis 56, 54, !58 Polyporus rutilans (nidu/ans) 2, llO, ll4, 158 Polyporus flabelliformis 174 Po/yporus sanguineus 94, 97, ll3, 114, 133, 140, 141, Polyporusfragilis !4, 49, 54, llO, ll4, 158, 174 (Polystictictus sanguineus) 158, 168, 174 Polyporus frondosus 2, 47, 49, 54, 56, llO, ll3, ll4, (Pycnoporus sanguineus) (Trametes sanguinea) I !58 Polyporus schweinitzii 2, 9, 42, 44, 46, 49, 50, 54, 65, 'I Polyporus fuliginosus 81 79, llO, ll3, ll4, 158, 174 Polyporus fumidiceps 54, 158 Polyporus scruposus 95,97 Polyporus fumosus 2, 43, 44, 49, 54, llO, ll3, ll4, Polyporus secernibilis 174 !58 Polyporus semipi/eatus 14, 110, 114, 158, 174 Polyporus ga/actinus 54, llO, ll4, !58 Polyporus semisupinus !58 Polyporus giganteus 49, 54, !58 Polyporus serice

Reference Number Reference Number

Polyporus supinus 54 Poria injlata 56 Polyporus tabacinus 2, 158, 174 Poria laevigata 113, !58 (Polystictus tabacinus) Poria lenta 113 Polyporus tamaricis !58 Poria luteoalba 84, 126, !58 Polyporus tasmanicus 158 Poria mappa !58 Polyporus tenuis 54 Poria medulla-panis 49, 54 Polyporus tephroleucus 54, 158 Poria metamorphosa !58 Polyporus texanus 85 Poria mollusca 81 Polyporus tomentosus 9, 113, 114, 150, 158, 174 Poria monticola (microspora) (placenta) 9, 46, 49, 91, Polyporus tomentosus var. circinatus 49, 51, 54, 78, 109, 110, 113, 114, 129, 152, 158 81, 110, 113, 158, 174 Poria mutans 54, 158 Polyporus tuberaster 54, 110, 114, 158 Poria mutans var. tenuis 54 Polyporus tuckahoe 104 Poria nigra 54, 56 Polyporus tulipferus (tulipiferae) 10, 53, 54, 110, 113, Poria nigrescens 54, 113, 158 114, 158, 174 Poria notata 113, 158 Polyporus tumu/osus 92, 97 Poria obliqua 38, 48, 49, 54, 62, 110, 113, 114, 158 Polyporus umbellatus 54, 110, 114, 158 Poria odora 113, 158 Polyporus undosus (Tyromyces undosus) 158, 171 Poria oleracea 58, 158 Polyporus varius 158 Poria overholtsii 158 Polyporus ve/utinus 54, 158 Poria pannocincta 158 Polyporus versatilis 114 Poria pearsonii 158 Polyporus versicolor 2, 5, 23, 42, 44, 48, 49, 54, 56, Poria pereffusa 158 (Polystictus versicolor) 65, 79, 80, 110, 113, 114 Poria prunicola 54, 158 120, 152, 158, 174 Poria pulchella 54 Polyporus vinosus 174 Poria punctata 54, 110, 114, 158 Polyporus volvatus 54, 110, 113, 114, 136, 158 Poria radiculosa 181 ( Cryptoporus volvatus) Poria rancida 158 Polyporus vulpinus 174 Poria reticu/ata 181 Polyporus xanthopus 174 Poria rhodelia 181 Polyporus xeranticus 174 Poria rixosa 9, 84, 158 Polyporus zona/is 158, 174 Poria rubens 158 Polyporus zonatus 54, 56, 110, 113, 114, 158 Poria selecta 54 Polystictus-see Polyporus Poria semitincta !58 Poris spp. 167 Poria sequoiae 109, 114, 158 Poria aestivale 158 Poria sinuosa 158 33, 110, 113, 114, 158 Poria a/bipellucida Poria sitchensis 158 Poria a/bobrunnea 158, 181 Poria spissa 158 Poria albolutescens 181 Poria subacida (colorea) 54, 65, 79, 110, 113, 114, 158 Poria alpina 181 Poria subcrassa 158 Poria ambigua 151, 158 Poria subincarnata 158 Poria andersonii 39, 54, 56, 158 Poria aneirina 158, 181 Poria taxicola (rufa) 110, 114, 158, 181 Poria asiatica (sericeomollis) 33, 110, 113, 114, 158 Poria tenuis 158 Poria bombycina 158, 181 Poria tenuis var. pulche/la 158 Poria borbonica 54 Poria tsugina 37, 54, 110, 113, 114, 158 Poria callosa 118 (Fornes robustus var. tsugina) Poria candissima 158 Poria undata 54, 158 Poria carbonico 109, 110, 113, 114, 158, 181 Poria unita 158 Poria cinerescens 110, 114, 126, 158 Poria vaillantii 2, 42, 44, 46, 49, 54, 91, 110, 113, 114, Poria cocos 54, 56, 113, 158 132, 133, 158, 181 Poria contigua 158 Poria vaporaria 44, 54, 181 Poria corticola 113, 118, 158 Poria versipora 54, 118, 158 Poria crustulina 158 Poria vincta 126 Poria decolorans (sanguinolenta) 113, 158 Poria viridans 158 Poria eupora 84, 126, 158 Poria weirii 54, 91, 107, 110, 113, 114, 158 Poria ferrea 19, 20, 110, 113, 114, 158 Poria xantha 2, 46, 49, 57, 79, 110, 113, 114, 118, 158, Poria ferrugineo-fusca 110, 113, 114, 158 181 Poria ferruginosa 110, 114, 158 Poria zonata 153 Poria fissiliformis 158 Radulum casearium 113, 158 Poria griseoa/ba 158 Rhizoctonia dichotoma 163 Poria hypolateritia 158 Rhizyctonia endophytica 163 Poria incrassata 54, 148, 158 Rhizoctonia so/ani 163 88 Methods for Isolation and Identification of Fungi on Wood. C. Grant and J. G. Savory.

Reference Numher Reference Numher

Sclzizophyllum commune 44, 54, 56, 71, 92, 97, IIO, Trarnetes sepiurn IIO, 113, II4, 157, 158, 174 Il3, Il4, Il9, 125, 133, 180 (Coriole/lus sepium) Stereum abietinum IIO, Il3, Il4 Trarnetes serialis 41, 44, 46, 49, 54, 57, 81, 99, 109, Stereum albobadium 54 (Coriolleus serialis) IIO, Il3, 114, 123, 124, 128, Stereum chailetii Il3, 145 132, 158, 174 Stereum fasciatum 5, 54 Trametes serpens !58 Stereum frustulosum 2, 44, 45, 49, 54, 56 Trametes sinuosus II4 Stereum Juscum 54 ( Coriolel/us sinuosus) Stereum gausapatum 49, 54, 56 Trarnetes spongipellis 174 Stereum hirsutum 2, 5, 44, 45, 49, Il3, 133 Trametes suaveolens 2, 54, 74, 104, IIO, 114, 158 Stereum illudens 2, 120 Trarnetes trogii !58 Stereum lobatum 2, 120 Trarnetes variiforrnis 106, IIO, Il3, 114, 158 Stereum murrayi 2, 54, 55, IIO, Il3, II4 l Coriol/elus variiformis) Stereum ochraceoj/avum Il3 Trechispora brikrnanni 81, Il3 Stereum pini (Peniphora pseudopini) 54, Il3, II4, 142, Trechispora raduloides Il3, II4 !54 Ustulina vulgaris (vulgare) 2, 49, 54, 56 Stereum purpureum 2, 44, 49, 54, II3, 133 Vararia granulosa Il3, 114 Stereum rameale 54,56 Veluticeps berkeleyi 166 Stereum roseo~carneum 54 Xylaria hypoxylon 2 Stereum rufurn (Peniophora rufa) Il3, 155 Xylaria polyrnorpha 2, 139 Stereum rugosurn 2, 5, 44 Stereurn rugosiusculum 54 References Stereum sanguinolenturn 2, 46, 49, 65, IIO, Il3, II4, 124, 133, I 52 I. Abbott, F. H. (1915). The red rot of conifers. Bull. Vt agric. Exp. Stn., No. 191. Stereum sericeum 54 Stereum subpi/eatum 54, 56 2. Badcock, E. C. (1939). Preliminary account of Stereum spadiceum 2, 44, 45, 54 the odqur of wood•destroying fungi in culture. Stereum sulcatum 54, II3, Il4, 147 Trans. Br. mycol. Soc., 23, (2): 188-198. Stereum taxodii 147 3. Bagchee, K., and Bakshi, B. K. (1950). Some Stereum wnbrinum 54 fungi as wound parasites on Indian trees. Indian Stereum vellereum 120 Forest Rec. (N.S.) Mycology, 1 (1). Stropharia aeruginosa 125 4. Bagchee, K. (1954). The fungal diseases of sal. Thelephora terrestris I 70 III. The root rot disease of sal due to Polyporus Trametes spp. 167 shoreae Wakef. (Partridge wood rot). Indian Trametes americana 54, IIO, Il3, II4, 130, !58 Forest Rec. (N.S.) Mycology, 1 (9): 185•197. Trametes carbonaria I 58, I 74 Trametes cingula/a 133, I 74 5. Bagchee, K., Puri, Y. N., and Bakshi, B. K. Trametes cinnabarina 91, 94, 97 (1954). Principal diseases and decays of oaks Trametes corrugata I 74 and other hardwoods in India. II. Indian Phyto­ Trametes cubensis 54, I 74 path., 7: 18-42. Trametes dickinsii I 74 6. Bagchee, K. (1955). New and noteworthy dis­ Trametes gibbosa 44, I 74 eases of forest trees and decay in timber in India. Trametes heteromorpha 54, 81, 106, II I, Il3, II4, !58 (i) Penellus rupicola (Mass.) Singer ( Collybia (Coriolellus heteromorphus) rupicola Mass.), (ii) (a) Polyporus palustria Berk. Trametes hispida 54, 158 and Curt. Indian Forest Rec. (N.S.) Mycology, Trametes hydnoides 54 1 (4): 47-52. Trametes incerta 174 7. Bagchee, K. (1963). Fungal diseases of sal. Part Trametes /actinea 174 IV. Fornes caryphylli. A destructive heart-rot of Trametes lilacino-gilva 93, 97, 120, 158 sal. Indian Forest Rec. (N.S.) Mycology, 2 (3). Trametes malicola 2, 54, Il4, 158 8. Bailey, H. E. (1941). Contributions to the biology (Coriolellus malicola) of Polyporus rheades (Pers.) Fr. Bull. Torrey bot. Trametes meyenii 133, I 74 Club., 68: 198-201. Trametes mol/is 54, !58, 174 9. Bakshi, B. K. (1955). Diseases and decays of Trametes ochroleuca 120 conifers in the Himalayas. Indian Forest Rec. Trametes odorata 2, 114 1 (6). Trametes pini I, 2, 3, 37, 42, 46, 49, 54, 65, 79, 81, (N.S.) Mycology, (Fornes pini) IIO, Il3, II4, II6, II7, !58, 174 10. Bakshi, B. K., Puri, Y. N., Sharma, R. P., Singh, Trarnetes proteus 133 S. and Singh, B. (1956). New and noteworthy Trametes protracta !04 diseases of trees in India. Diseases due to Trametes ravida I 74 Polyporus tulipiferae, Polyporus lignosus, Hypoxy­ Trametes rigida 54 lon ustulatum and Polyporus rubidus. Forest Bull., Trarnetes rubescens 2 Dehra Dun, No. 209. 89 Methods for Isolation and Identification of Fungi on Wood. C. Grant and J. G. Savory.

II. Bakshi, B. K., Singh, B., and Rehill, P. S.l!957). 30. Bose, S. R., and Bakshi, B. K. (1957). Polyporus Coniophora cerebella Pers., a dry rot fungus on lignosus Klotzsch and its identity. Trans. Br. building timber in India. Indian J. mycol. Res., mycol. Soc., 40: 456460. 3: 5!-53. 31. Brodie, H. J. (1936). The occurrence and 12. Bakshi, B. K., Singh, B., and Choudhury, T. function of oidia in the Hymenomycetes. Am. J. (1958). Occurrence of Merulius himantiodes in Bot., 23: 309-327. India. Indian Phytopath, 11: 25-28. 32. Brodie, H. J. (1936). The barrage phenomenon 13. Bakshi, B. K., and Singh, B. (1958). The Indian in Lenzites betulina. Genetica, 18: 61-73. species of Merulius.lndianPhytopath., 11:70-75. 33. Buckland, D. C. (1946). Investigations of decay 14. Bakshi, B. K., and Singh, B. (1961). New and in western red cedar in British Columbia. Can. noteworthy records of Hymenomycetes in India. J. Res., C, 24: 158-181. Forest Bull., Dehra Dun, No. 235. 34. Campbell, A. H. (1934). Zone lines in plant 15. Bavendamm, W. (1928). Neue Untersuchungen tissues. II. The black lines formed by Armillaria iiber die Lebensbedingungen holzzerstOrender me/lea (Yah!) Que!. Ann. appl. Biol., 21: 1-22. Pilzen. New investigations of the conditions of life of wood-destroying fungi. Zentbl. Bakt. 35. Campbell, A. H. (1938). Contribution to the ParasitKde Abt. II., 75:426-533, and 76: 172-227. biology of Collybia radicata (Relh.) Berk. Trans. Br. mycol. Soc., 22: 151-159. 16. Baxter, D. V. (1924). Fomesfraxineus in culture. Pap. Mich. Acad. Sci., 4: 55-66. 36. Campbell, W. A. (1937). The cultural character­ 17. Baxter, D. V. (1925). The biology and pathology istics of Fornes connatus. Mycologia, 29: 567-571. of some of the hardwood heart-rotting fungi. 37. Campbell, W. A. (1938). The cultural character­ I. Am. J. Bot., 12: 522-552. istics of the species of Fornes. Bull. Torrey bot. 18. Baxter, D. V. (1925). The biology and pathology Club, 65: 31-69. of some of the hardwood heart-rotting fungi. II. 38. Campbell, W. A., and Davidson, R. W. (1938). Am. J. Bot., 12: 553-576. A Poria as the fruiting stage of the fungus 19. Baxter, D. V. (1933). Some resupinate polypores causing the sterile conks on birch. Mycologia, from the region of the Great Lakes. IV. Pap. 30: 553-560. Mich. Acad. Sci., 17: 421439. 39. Campbell, W. A., and Davidson, R. W. (1939). 20. Baxter, D. V. (1936). Some resupinate polypores Poria andersonii and Polyporus glomeratus, two from the region of the Great Lakes. VII. Pap. distinct heart-rotting fungi. Mycologia, 31: 161- Mich. A cad. Sci., 21: 243-267. 168. 21. Baxter, D. V. and Manis, W. E. (1939). Polyporus 40. Cartwright, K. St. G. (1929). Notes on Basidio­ ellisianus (Murr.) Sacc. and Trott. and Polyporus mycetes grown in culture. Trans. Br. mycol. anceps Pk. in culture; a study of isolates from Soc., 14: 300-305. , widely separated forest regions. Pap. Mich. 41. Cartwright, K. St. G. (1930). A decay of Sitka Acad. Sci., 24: 189-195. Spruce timber caused by Trametes serialis Fr. 22. Baxter, D. V., and Varner, R. W. (1942). Im­ Forest Prod. Res. Bull. No. 4. portance of fungi and defects in handling 42. Cartwright, K. St. G., and Findlay, W. P. K. Alaskan airplane spruce. Circ. Univ. Mich. Sch. (1930). The diagnosis of decay in timber. Emp. For. Conserv., No. 6. For. J., 9: 190-203. 23. Bayliss, J. S. (1908). The biology of Polystictus 43. Cartwright, K. St. G. (1932). Further notes on versicolor (Fr.). J. econ. Biol., 3: 1-24. Basidiomycetes in culture. Trans. Br. mycol. 24. Benton, V. L., and Ehrlich, J. (1941). Variations Soc., 16: 304-307. in culture of several isolates of Armillaria mellea 44. Cartwright, K. St. G., and Findlay, W. P. K. from western white pine. Phytopathology, 31: (1934). Studies in the physiology of wood­ 803-811. destroying fungi. II. Temperature and rate of 25. Bier, J. E., and Nobles, M. K. (1946). Brown growth. Ann. Bot., 48: 481495 pocket rot of Sitka spruce. Can J. Res., C. 24: 115-120. 45. Cartwright, K. St. G., and Findlay, W. P. K. 26. Biggs, R. (1938). Cultural studies in the Thele­ (1936). The principal rots of English oak. phoraceae and related fungi. Mycologia, 30: H.M.S.O., London. 64-78. 46. Cartwright, K. St. G., and Findlay, W. P. K. 27. Bose, S. R. (1930). On the biology of wood­ (1938). Principal decays of softwoods used in rotting fungi. J. Linn. Soc. London, 48: 417438. Great Britain. MH.M.S.O., London. 28. Bose; S. R. (1934). Sexuality of Polyporus 47. Cartwright, K. St. G. (1940). Note on a heart ostreiformis and Polystictus hirsutus. Cellule, 42: rot of oak trees caused by Polyporus frondosus 249-266. Fr. Forestry, 14: 3841. 29. Bose, S. R. (1943). Binucleate oidia on binucleate 48. Cartwright, K. St. G., and Findlay, W. P. K. mycelium of Polystictus hirsutus Fr. Nature, Land. (1942). Principal decays of British hardwoods. 152: 508-509. Ann. appl. Biol., 29: 219-253. 90 Methods for Isolation and Identification of Fungi on Wood. C. Grant and J. G. Savory.

49. Cartwright, K. St. G., and Findlay, W. P. K. 66. Garren, K. H. (1938). Studies on Polyporus (1958). Decay of timber and its prevention. abietinus. I. The enzyme-producing ability of Second edition, H.M.S.O., London. the fungus. Phytopathology, 28: 839-845. 50. Childs, T. W. (1937). Variability of Polyporus 67. Garren, K. H. (1938). Studies on Polyporus schweinitzii in culture. Phytopathology, 27: 29•50. abietinus. II. The utilization of cellulose and lignin by the fungus. Phytopathology, 28: 875- 51. Christensen, C. M. (1940). Observations on 878. Polyporus circinatus. Phytopathology,30:957-963. 68. Gibson, I. A. S. (1961). A note on variation I 52. Da Costa, E. W. B., Matters, C. N., and Tam­ between isolates of Armillaria me/lea (Vahl. ex blyn, N. (1952). The identification of Basidio­ Fr.) Kummer, Trans. Br. mycol. Soc., 44: 123- mycete fungi in culture. Progress Report No. I. 128. Methods of describing the morphological charac­ 69. Hamada, M. (1940). Physiologisch-morpholo­ teristics of Basidiomycete fungi in culture. Prog. gische Studien tiber Armillaria me/lea (Vahl) Rep. Forest Prod. Lab. Me/b., Sub-project p. Quel., mit besonderer RUcksicht auf die Oxal­ 11-12. saurebilding. Bin Nachtrag zur Mykorrhiza 53. Darley, E. F., and Christensen, C. (1945). The von Galea/a septentrionalis. Jap. J. Bot., 10: culture designated Madison 517 identified as 387-463. Polyporus tulipiferus. Phytopathology, 35: 220- 70. Harmsen, L. (1960). Taxonomic and cultural 222. studies on brown spored species of the genus 54. Davidson, R. W., Campbell, W. A., and Blais­ Merulius. Friesia, 6: 233-277. dell, D. J. (1938). Differentiation of wood­ decaying fungi by their reactions on gallic or 71. Heldmaier, C. (1929). Ober die Beeinftussbar­ tannic acid medium. J. agric. Res., 57: 683-695. keit der Sexualitat von Schizophyl/um commune Fr. and Col/ybia velutipes Curt. Z. Bot., 22: 55. Davidson, R. W., Campbell, W. A., and Lorenz, 161-220. R. C. (1941). Association of Sterewn murrayi 72. Hilborn, M. T. (1942). The biology of Fornes with heart rot and cankers of living hardwoods. fomentarius. Bull. Me agric. Exp. Stn., No. 409. Phytopathology, 31: 82-87. 73. Hirt, R. R. (1928). The biology of Polyporus 56. Davidson, R. W., Campbell, W. A., and Vaughn, gilvus (Schw.) Fries. Tech. Pubis NY St. Col/. D. B. (1942). Fungi causing decay of living oaks For., 22: 1-47. in the Eastern United States and their cultural 74. Hirt, R. R. (1932). On the biology of Trametes identification. Tech. Bull. U.S. Dept. Agric., suaveolens (L) Fr. Tech. Pubis NY St. Coli. For., No. 785. 37: 1-36. 57. Davidson, R. W. and Campbell, W. A. (1943). 75. Hopp, H. (1936). Appearance of Fornes igniarius Decay in merchantable black cherry on the Allegheny National Forest. Phytopathology, 33: in culture. Phytopathology, 26: 915-917. 965-985. 76. Hopp, H. (1938). Sporophore formation by 58. Davidson, R. W., Lombard, F. F., and Hirt, R. Fornes applanatus in culture. Phytopathology, R. (1947). Fungi causing decay in wooden boats. 28: 356-358. Myco/ogia, 39: 313-327. 77. Hubert, E. E. (1929). A butt-rot of balsam fir caused by Polyporus balsameus Pk. Phytopatho­ 59. Denyer, W. B. G. (1960). Cultural studies of logy, 19: 725-731. Flammula alnico/a (Fr.) Kummer and Flammula conissans (Fr.) Gillet. Can. J. Bot.; 38: 909-920. 78. Hubert, E. E. (1929). A root and butt rot of conifers caused by Polyporus circinatus Fr. 60. Edgecombe, A. E. (1941). The growth rate of Phytopathology, 19: 745-747, several wood-inhabiting fungi. Phytopathology, 79. Humphrey, C. J., and Siggers, P. V. (1933). 31: 825-831. Temperature relations of wood-destroying fungi. 61. Faull, J. H. (1916). Fornes ojjicianalis (Viii.), a J. agric. Res., 47: 997-1008. timber-destroying fungus. Trans. R. Can. Inst., 80. Jay, B. A. (1934). A study of Polys/ictus versi­ 12: 185-209. color. Bull. misc. Jnf. R. bot. Gdns Kew, 10: 62. Findlay, W. P. K. (1939). Note on an abnormal 409-424. fungus on birch. Trans. Br. mycol. Soc., 23: 81. Kiiiirik, A., and Rennerfelt, E. (1957). Investiga­ 169-170. tions on the fungal flora of spruce and pine 63. Fisher, E. (1934). Observations on Fornes stumps. Meddn St. Skogsforsk Inst., 47 (7). pomaceus (Pers.) Big. and Guill. infecting plum trees. Trans. Br. mycol. Soc., 19: 102-113. 82. Kemper, W. (1937). Zur Morphologic und Zytologie der Gattung Coniophora, insbesondere 64. Fries, N. (1936).) Ober die Sexualitate einiger des sogenannten Kellerschwamms. Zentbl. Bakt. Polyporacean. Svensk bot. Tidskr., 30: 355-361. Parasit Kde, 97: 100-124. 65. Fritz, C. W. (1924). Cultural criteria for the 83. Laut, J. G. (1966). Cultural characteristics of distinction of wood-destroying fungi. Trans. R. three species of Boletinus. Can. J. Bot., 44: Soc. Can., Report No. 13. 395-402. 91 Methods for Isolation and Identificatiod of Fungi on Wood. C. Grant and J. G. Savory.

84. Lombard, F. F., and Gilbertson, R. L. (1966). 100. Mizimoto, S. (1963). Studies on wood-rotting Poria /uteoa/ba and some related species in Gloeophyllum from Japan. Spec. Rep., Shizuoko North America. Myco/ogia, 58: 827-845. Prefecture For. Exp. Stn, Hamakita, Shizuoka 85. Long, W. H., and Harsch, R. M. (1918). Pure Prefecture, Japan. cultures of wood-rotting fungi on artificial 101. Montgomery, H. B. S. (1936). A study of media. J. agric. Res., 12: 33-82. Fornes fraxineus and its effects on ashwood. 86. Macdonald, J. A. (1937). A study of Polyporus Ann. appl. Bioi., 23: 465486. betulinus (Bull.) Fries. Ann. app/. Bioi., 24: 102. Mounce I. (1926). A preliminary note on Fornes 289-310. pinico/a Fr. and Pholiota adiposa Fr. two 87. Macdonald, J. A. (1938). Fornes fomentarius heterothallic species of wood-destroying fungi. (Linn.) Gill. on birch in Scotland. Trans. Proc. Phytopathology, 16: 757. bot. Soc. Edinb., 32: 396-408. 103. Mounce, I. (1929). Studies in forest pathology. 88. Macdonald, J. A. (1939). Coniophora puteana II. The biology of Fornes pinico/a (Sw.) Cooke. (Schum.) Karst. on living Sequoia gigantea. Bull. exp. Fms Brch. Dep. Agric. Can., No. Ill Ann. appl. Bioi., 26: 83-86. (N.S.). 89. Macrae, R. (1941). Genetical and sexuality 104. Mounce, I. (1929). Notes on sexuality in Fornes studies in some higher Basidiomycetes. Thesis, pinicola (Sw.) Cooke, Fornes roseus (Fr.) Cooke, Univ. Toronto, Canada. Polyporus tuckahoe (Giissow) Sacc. et Trott., 90. Macrae, R. (1955). Cultural and interfertility P. resinosus (Schrad.) Fr., P. anceps Peck, studies in Aporpium caryae. Myco/ogia, 47: Lenzites saepiaria Fr., Trametes protracta Fr., 812-820. and T. suaveo/ens (L) Fr. Proc. Can. phytopath. Soc., pp. 27-28. 91. Matters, C. N., Da Costa, E. W. B., and Tamblyn, N. (1952). The identification of 105. Mounce, I. (1932). Microscopic characters of Basidiomycete fungi in culture. Progress Report sporophores produced in culture as an aid to No. 2. Morphological characters of 14 cultures identifying wood-destroying fungi. Trans. R. Soc. of Basidiomycetes. Prog. Rep. Forest Prod. Lab. Can., 26 (section 5): 177-181. Me/b., Sub-project p. 11-12. 106. Mounce, I. (1935). Sexuality and cultural studies 92. Matters, C. N., Da Costa, E. W. B., and of wood-destroying fungi. Rep. Dep Agric. Can., Tamblyn, N. (1953). Progress Report No. 3. 1931-34, Ottawa, 1935. Morphological characters of Polyporus australen­ 107. Mounce, I., Bier, J. E., and Nobles, M. K. (1940). sis, P. colensoi, P. tumulosus and Schizophyllum A root-rot of Douglas fir caused by Poria weirii. commune. Prog. Rep. Forest Prod. Lab. Me/b., Can. J. Res., C, 18: 522-533. Sub-project, p. 11-12. 108. Nobles, M. K. (1942). Secondary spores in 93. Matters, C. N. (1953). Progress Report No. 4. Corticiurn effuscaturn. Can. J. Res., 20: 347-357. Morphological characteristics of 9 isolates of Trametes li/acino-gilva Berk. Prog. Rep. Forest 109. Nobles, M. K. (1943). A contribution toward a Prod. Lab. Me/b., Sub-project p. 11-12. clarification of the Trametes serialis complex. Can. J. Res., C, 21: 211-234. 94. Matters, C. N. (1955). Progress Report No. 5. 110. Nobles, M. K. (1948). Studies in to rest pathology Morphological characters of Trametes cinna­ VI. Identification of cultures of wood•rotting barina, Polyporus cinnabarinus and Polystictus fungi. Can. J. Res., C, 26: 281-431. sainguineus. Prog. Rep. Forest Prod. Lab. Me/b., Sub-project p. 11-12. Ill. Nobles, M. K. (1953). Studies in wood· inhabiting Hymenomycetes. I. Odontia bico/or. Can. J. Bot., 95. Matters, C. N. (1955). Progress Report No. 6. 31: 745-749. Morphological characters of Polyporus gilvus and Polyporus scruposus. Prog. Rep. Forest Prod. 112. Nobles, M. K., and Nordin, V. J. (1955). Studies Lab. Me/b., Sub-project p. 11-12. in wood-inhabiting Hymenomycetes. II. Corti­ ciurn vellereum Ellis and Cragin. Can. J. Bot., 96. Matters, C. N. (1955). Progress Report No. 7. 33: 105-112. Morphological characters of six isolates of Coniophora cerebella Pers. Prog. Rep. Forest 113. Nobles, M. K. 0958). A rapid test for extra­ Prod. Lab. Me/b., Sub-project p. 11-12. cellular oxidase in cultures of wood-inhabiting Hymenomycetes. Can. J. Bot., 36: 91-99. 97. Matters, C. N. (1955). Progress Report No. 8. Oxidase reactions of ten species of wood­ 114. Nobles, M. K. (1965). Identification of cultures destroying fungi. Prog. Rep. Forest Prod. Lab. of wood-inhabiting Hymenomycetes. Can.J.Bot., Me/b., Sub-project p. 11-12. 43: 1097,1139. 98. Meyer, H. (1936). Spore formation and dis­ 115. Nutman, F. J. (1929). Studies of wood-destroying charge in Fornes fomentarius. Phytopathology, fungi. I. Polyporus hispidus (Fries). Ann. appl. 26: 1155-1156. Bioi., 16: 40-64. 99. Mez, C. (1908). Der Hausschwamm un die 116. Owens, C. E. (1936). Studies on the wood-rotting iibrigen holzzerst6renden Pilze der menschlichen fungus, Fornes pini. II. Cultural characteristics. Wohnungen. Dresden. Am. J. Bot., 23: 235-254. 92 i I Methods for Isolation and Identification of Fungi on Wo~d. C. Grant and J. G. Savory. I I, 117. Percival, W. C. (1933). A contribution to the 134. Van der Westhuizen, G. C. A. (1963). The biology of Fornes pini (fhore) Lloyd (Trametes cultural characters, structure of the fruit body, pini (fhore) Fries). Tech. Pubis NY St. Col/. For., and type of interfertility of Cerrena unicolor No.40. (Bull. ex Fr.) Murr. Can. J. Bot., 41: 1487-1499. 118. Puri, Y. N. (1956). Studies on Indian Poria. 135. White, J. H. (1920). On the biology of Fornes I I. J. Indian bot. Soc., 35: 277--283. app/anatus (Pers.) Wallr. Trans. R. Can. Inst., 12: 133-174. 119. Putterill, V. A. (1922). The biology of Schizo­ phyllum commune Fries with special reference to 136. Zeller, S. M. (1915). Notes on Cryptoporus " its parasitism. Bull. Dep. Agric. For. Un. S. Afr., volvatus. Myco/ogia, 7: 121-124. I No. 25. 120. Refshauge, L. D., and Procter, E. M. (1936). Additions The diagnosis of some wood,destroying Austra­ 137. Macrae, R. (1967). Pairing incompatability and lian Basidiomycetes by their cultural characters. and other distinctions among Hirschioporus Proc. R. Soc. Viet., 48 (N.S.): 105-123. (Polyporus) abietinus, H. fusco-vio/aceus, and 121. Reitsma, J. (1933). Studien iiber Armillaria H. laricinus, Can. J. Bot., 45: 1371-1398. me/lea (Vahl.) Que!. Phytopath. Z., 4: 461-522. 138. Raestad, R. (1940). The relation between 122. Rhoads, A. S. (1918). The biology of Po/yporus Polyporus abietinus (Dicks. ex Fr.) Fr. and pargamenus Fries. Tech. Pubis NY St. Col/. For., Irpex fusco-violaceus (Ehren b. ex Fr.) Fr. Nyt No. II. Mag. Naturvid., 81: 208-231. 123. Robak, H. (1936). Studies in the biology ofwood­ 139. Campbell, A. H. (1933). Zone lines in plant destroying Hymenomycetes. I. Contribution to tissues. I. The black lines formed by Xy/aria the knowledge of homothally and heterothally polymorpha (Pers.) Grev. in hardwoods. Ann. in some species of Thelephoraceae and Poly­ Appl. Bioi., 20: 123-144. poraceae. Nyt Mag. Naturvid., 76: 5-13. 140. McKay, H. H. (1959). Cultural basis for main­ taining Polyporus cinnabarinus and Polyporus 124. Robak, H. (1942). Cultural studies in some sanguineus as two distinct species. Myco/ogia, Norwegian wood-destroying fungi. Meddr Vest­ 51:465473. land. forst/. ForStn, 7 (3). 141. Nobles, M. K., and Frew, B. P. (1962). Studies 125. Semerdzieva, M., and Cejp, K. (1966). Investiga­ in wood-inhabiting Hymenomycetes V. The tion of mycelial growth in some gill fungi under genus Pycnoporus Karst. Can. J. Bot., 40: 987- laboratory conditions. Folia microbiol. Praha, 1016. 11: 146-154. 142. Nobles, M. K. (1956). Studies in wood-inhabiting 126. Singh, B. (1966). Studies on Indian Poria. II. Hymenomycetes. III. Stereum pini and species Diagnoses of five species as new records. Indian of Peniophora sect. Colaratae on conifers in Forester, 92: 680-683. Canada. Can. J. Bot., 34: 104-130. 127. Skoric, V. (1938). Zilavka tigrasta - Lentinus 143. Roll-Hansen, F. (1967). Phellinus tremulae tigrinus (Bull.) Fr. Studije o biologiji, razvoju i (Bond.) Bond. and Boriss. and Pile/linus igniarius patolaskom djelovanju gljive. Studies on the (L. e:X. Fr.) Que!. on Populus tremula L. Meddr biology, development and pathogenic properties norske SkogsforsVes., 23: 243-263. of the fungus. Glasn. sum. Pok., 6: 99-126. 144. Nobles, M. K. (1967). Conspecificity of Basidio­ 128. Snell, W. H. (1922). Studies of certain fungi of radu/um (Radulum) radula and Corticium hydnans economic importance in the decay of building Myco/ogia, 59: 192-211. timbers. Dept. Bull. US Dep. Agric., No. 1053. 145. Basham, J. T. (1959). Studies in forest pathology 129. Snell, W. H. (1923). Occurrence and identity of XX. Investigations of the pathological deteriora­ cotton mill fungi. Myco/ogia, 15: 153-165. tion in killed Balsam Fir. Can. J. Bot., 37: 291- 326. 130. Snell, W. H., Hutchinson, W. G., and Newton, 146. Cauchon, R. and Oullette, G. B. (1966). Cultural K. H. N. (1928). Temperature and moisture characters of Phlebia atkinsoniana W. B. Cooke. relations of Fornes roseus and Trametes subrosea. Can. J. Bot., 44: 527-528. Mycologia. 20: 276-291. 147. Davidson, R. W., Lentz, P. L., and McKay, ' . 131. Verrall, A. F. (1937). Variation in Fornes H. H. (1960). The fungus causing pecky cypress. igniarius (L) Gill. Tech. Bull. Minn. agric. Exp. Myco/ogia, 52: 260-279. Stn., No. 117. 148. Davidson, R. W., and Lombard, F. F. (1953). ,, 132. Walek,Czernecka, A. (1933). Sur les champig­ Large-brown-spored house"rot fungi in the ' nons destructenrs des traverses de chemins de United States. Mycologia, 45: 88-100. fer en Pologne. Acta. Soc. Bot. Pol., 10: 179-290. 149. Davidson, R. W., and Patton, R. F. (1961). 133. Van der Westhuizen, G. C. A. (1958). Studies Paxillus atrotomentosus causes brown root rot of wood-rotting fungi. I. Cultural characteristics in dead jack pine in plantations in Wisconsin. of some common species. Bothalia, 7: 83-107. Pl. Dis. Reptr., 45: 836-838. 93 Methods for Isolation and Identification of Fungi on Wood. C. Grant aud J. G. Savory.

150. Dimitri, L. and Zycha, H. (1968). Der Erreger 167. Balla!, S. (1964). Comparative studies of resu­ der 'Bienenrosigkeit' der Kiefer. Allg. Forst-u. pinate species of Poria and Trametes (Polypora­ Jagdztg, 139: 115-117. ceae) in cultures. Ph.D. Thesis, University of 151. Lombard, F. F., Davidson, R. W., and Lowe, Tennessee. In-Diss. Abstr., !965, 25 (12): 6909. J. L. (1960). Cultural characteristics of Fornes 168. Nagatomo, I. (1964). On Trametes sanguinea ulmarius and Poria ambigua. Mycologia, 52: (L. ex Fr.) Lloyd causing the white-rot of 280-294. coniferous and broadleaved woods. Bull. Kyoto 152. Zycha, H. and Knopf, H. (1966). Cultural Gakugei Univ. Ser. B, 25: 45-70. characteristics of some fungi which cause red­ 169. Balko, S. (1946). Biometrical researches of stain of Picea abies. Lloydia, 29: 136-145. secondary spores and a study of the mycelium 153. Gilbertson, R. L. (1960). Studies on the western of Pholiota adiposa Fr., P. heteroclita Fr., wood-rotting fungus Poria zonata Bres. Can. J. P. mutabilis (Schaeff.) Fr., P. spectabilis Fr. and Bot., 38: 87-91. P. squarrosa (MUll) Fr. Trans. Br. myco/. Soc., 29: 242-249. 154. Weresub, L. K., and Gibson, S. (1960). 'Stereum pini' in North America. Can.J. Bot., 38:833-867. 170. Vaartaja, 0. (1968). Isolation of Thelephora terrestris from soil and hypha! strands on 155. McKay H. H., and Lentz, P. L. (1960). Descrip­ seedlings. Bi-mon Res. Notes Dept. For. Rural tions of some fungi associated with forest tree Dev. Ottawa, 24: 26-27. decay in Colorado. Mycopath. Myco/. appl., 13: 265-286. 171. Brotzman, H. G. and Gilbertson, R. L. (1967). Tyromyces undosus in North America. Mycopath. 156. Bakshi, B. K., Singh, B., and Puri, Y. N. (1958). Mycol. appl., 33: 33-42. Occurrence of Polyporus /eucospongia in India. Can. J. Bot., 36: 363-365. 172. Fidalgo, 0. (1958). Studies on Spongipellis borealis (Fr.) Pat. Mycopath. Myco/. appl. 10: 157. Bakshi, B. K., Singh, B., and Gibson, S. (1958). 1-18. Occurrence of Trametes sepium in India. Can. 173. Suvorov, P. A. (1967). Biology of Fornes J. Bot., 36: 603:606. pinicola (Fr.) Cke. Byu/1. mosk. Obshch. Ispyt. 158. Nobles, M. K. (1958). Cultural characters as a Prir., 72 (6): 80-92. guide to the taxonomy and phylogeny of the 174. Sehgal, H. S., Sen, M. and Bakshi, B. K. (1966). Polyporaceae. Can. J. Bot., 36: 883-926. Temperature relations of Indian species of 159. Nobles, M. K. (1935). Conidial formation, Polypores. Indian Forest Rec. (N.S.) Forest Path., mutation and hybridization in Peniophora 2 (7): 131-137. allescheri. Mycologia, 27: 286-301. 175. Olchowecki, A. and Brodie, H. J. (1968). Sexu­ 160. Nobles, M. K. (1937). Production of conidia by ality and mycelial characteristics of Cyathus Corticium incrustans. Myco/ogia, 29: 557•566. helenae and the related fungus Cyathus striatus. Can. J. Bot., 46: 1423-1429. 161. Davidson, R. W., Lombard, F. F., and Campbell, W. A. (1959). A white heart rot of Fraxinus 176. French, D. W., Hodges, C. S. Jr. and Froyd, caused by Fornes johnsonianus (M urr.) Lowe. J. D. (1969). Pathogenicity and taxonomy of Pl. Dis. Reptr., 43: 1148-1149. Hypoxylon mammatum. Can. J. Bot., 47: 223-226. 162. McKay, H. H. (1967). Cultural studies of 177. Miller, 0. K. Jr. (1965). Three new species of Polyporus meliae and two similar species. ligoicolous agarics in the Tricholomataceae. My­ Myco/ogia, 59: 1050-1058. cologia, 57: 933-945. 178. Hinds, T. E., Davidson R. W. and Lombard, 163. Gomez Nava, M. S. (1967). Examen morpholo­ gico comparativo de especimenes de Rhizoctonia F. F. (1968). Decay of Engelmann spruce by DC, aislados de semilleros forestales. Boln Tee Lentinellus montanus in Colorado. Pl. Dis. Reptr., 52: 820-821. Inst. Nac. Invest. For., Mexico, No. 21. 179. Papavizas, G. C. (1965). Comparative studies of 164. Suvorov, P. A. (1967). Biological characteristics single-basidiospore isolates of Pellicularia fila­ of Fornes fomentarius found on spruce and mentosa and Pellicularia praticola. Mycologia, birch. Can. J. Bot., 45: 1853-1857. 57: 91-103. 165. Pandila, M. M. (1965). Cultural studies of 180. Parag, Y. (1965). Common-B heterokaryosis and Fornes fraxinophilus (Peck) Cooke and Fornes fruiting in Schizophyl/um commune. Myco/ogia, ellisianus Anderson. N.Y. St. Col/. For. 57: 543-561. 166. Gilbertson, R. L., Lombard, F. F. and Hinds, 181. Lombard, F. F. and Gilbertson, R. L. (1965). T. E. (1968). Veluticeps berkeleyi and its decay Studies on some western Porias with negative of pine in North America. Myco/ogia, 60: 29-41. or weak oxidase reactions. Myco/ogia, 57:43-76.

94 !'

'I

' 'I 1;. LETTERS TO THE EDITOR i li Sir, We have noted in the article by D. G. Parbery (Int. Biodetn Bull., 4 (2), 79-81 1968) an opinion that he attributes to us in his discussion of the significance of microbial counts in a fuelfwater system: "Consequently, the opinion held by Rogers and Kaplan (Dev. Ind. Microbial., 6, 80-94, 1964) that bacteria grow better than the Cladosporium resinae could be re-interpreted if the meaning of the relative numbers of materials and fungal colonies in their experiment was known." No such opinion was made or implied in·our paper. We did do bacterial and fungal counts but stated quite clearly that the exact significance ofthe counts obtained in the surveywasnotknown for reasons enumerated in the paper. We can find no statement that could be interpreted as indicating that bacterial growth was better than Cladosporium resinae. We merely reported that bacteria were present in the fuels in higher numbers than fungi. We further included in the article the following: "In the quantitative studies, five different media were used to count fungi and two different media were used for bacterial counts. No definite recommenda­ tions as to which media are superior can be made at this point. Indeed, fungal counts, although expressed quantitatively, should be considered to be only qualitative. This is because a single fungal hypha may be fragmented during culturing procedures, giving rise to a number of fungal colonies from the fragments and thereby indicating a count higher than the true count. Or conversely, one mycelial mass may have formed from many spores but would only be counted as one colony." Actually, during the entire study eleven different media were used as noted: "The II media used in these studies were selected on the basis of recommendations from the Society for Industrial Microbiology Committee on Microbiological Deterioration of Fuels (1963) and from Dr. Emory Simmons, Pioneering Research Division, U. S. Army Natick Laboratories for the culturing of fungi. These include Tryptone Glucose Extract Agar and American Petroleum Institute (API) Agar for total bacterial plate counts. API media for Sulfate Reducing Bacteria, Waksman's media for Sulfur-Oxidizing Bacteria, Waksman's Thiosulfate Agar, Waksman's Iron-Depositing Bacteria Medium, Hay Decoction Agar (designed as an isolation medium which permits early sporulation with minimal mycelial production,) Mycophil Agar (designed for organisms requiring low pH), Czapek Agar plus Yeast Extract (specially advantageous for the isolation of Penicillia and Aspergilli), Rose Bengal-Streptomycin Agar (useful in controlling bacteria and permitting fungal growth on mixed plates), and .Potato-Dextrose Agar (useful for certain organisms that won't grow well on the other media specified)." Parbery notes that "no one has yet made detailed studies of the effects of oxygen levels, temperature or pH, or the effect of other organisms." The importance of ecological studies was recognized when the problem first came under investigation and such studies have indeed been made but have not been published in the open literature. Very truly yours, ARTHUR M. KAPLAN MORRIS R. ROGERS Pioneering Research Laboratory U. S. Army Natick Laboratories Natick, Mass.

Sir, This is to thank Drs. Rogers and Kaplan (Int. Biodetn Bull. 5: this issue) for drawing attention to my error in quoting them in a recent issue of this bulletin (Parbery, Int. Biodetn Bull., 4 (2), 79-81, 1968). I wish to apologise for this and give the assurance that I did not intentionally misrepresent their findings. I would like, therefore, to recast and express my ideas differently so as to remove any doubts about the meaning of my article. Since Myoshi (Jb. wiss. Bot. 28: 269, 1895) first described the utilisation of hydrocarbons by microorganisms, there have been many biological problems recognised in the Petroleum and dependent Industries as resulting from the activities of bacteria. The extent of the importance of bacteria has been such that many Oil Companies have appointed their own bacteriologists to deal with them. There is no doubt about the importance of bacteria in regard to biological contamination of, and attendant problems in, petroleum. I wish to suggest, however, that because of the historical importance of bacteria in biological problems in the Petroleum Industry, there is an ingrained bias against the consideration that other groups of microorganisms may also be important in certain situations, let alone that they may be even more important. Because of its insidious nature this bias is likely to go unnoticed and to be expressed negatively in emphasising the role of bacteria while ignoring or minimising the role of other organisms. Consequently, the presence of the filamentous fungus, Cladosporium resinae (Lind~u) de Vries, in aviation kerosene would have been against the traditional experience of petroleum microbiologists. Since the fungus was observed in company with bacteria, it was most likely assumed that the traditional organisms, bacteria, were the more important (Donahue, Lockheed Field Service Digest, 7 (3); 3-13, 1961). This is understandable but it is, nevertheless, biased. 95 In the initial stages of the investigations of the problem of biological sludge in aircraft fuels, this bias would have gained support from results based on methods which were unsuitable for determining the presence oi fungi but which were quite suitable for estimating the quantitative presence of bacteria. In regard to fungi, neither the primary sampling method used, nor the isolation procedures followed were suitable for showing more than the presence of the fungus or fungi. Current work suggests that they may not always do even this. A negative result does not necessarily mean that C. resinae is not present or even abundant, since a positive result depends on the fungus either producing spores or mycelial fragments into the water or fuel phase from which the original sample is taken. Some isolates do not produce spores at aU, others sporulate sparsely, while yet others sporulate irregularly as the conditions change in the water-kerosene medium. These changes are so far undefined. Isolation methods used are frequently the best available at the time of investigation but become suspect later as the result of further research by the same or other researchers. Some methods suitable only for the study of one particular group of organisms have been used for other groups. Techniques used in bacteriology for instance such as estimating the quantitative presence of an organism by making colony counts based on streak plates, dilution plates or from spores trapped in filters, have been applied to the study of fungi. Methods like this can be unsuitable for revealing the presence of fungi in mixed cultures (Parbery foe. cit.). Thus, for this very reason, the results of Hedrick, Carroll, Owen and Pritchard* (Appf. Microbiol., 11, 472-475, 1963) are doubtful. They showed that C. resinae could survive for at least 126 days in a water-kerosene medium in mono- and mixed fungal culture. but it could not survive in mixed bacterial and fungal culture. Contrary results of Nicol and Zakartchenki (Rev. Mycol., 31, 48-74, 1966) showed that C. resinae could survive for 30 months in a different water-kerosene medium in company with bacteria. After that time, however, the fungus was difficult to reisolate by conventional methods and its sporulation was reduced. Unfortunately reports based on unsuitable methods are not easily recognised and may lead to an erroneous conclusion by both the researcher and subsequent reader. Rogers and Kaplan (Dev. Ind. Microbiof., 6, 80-94, 1964) did not express an opinion that bacteria grew better than fungi in kerosene, nor did they express an opinion on the relative importance of these organisms in fuel problems. I did believe, however, that they did imply a belief that the bacteria were the important organisms to consider, since in discussing the significance of their colony counts, they did not refer to the fungus at all, but considered only the numerical levels of bacteria constituting a problem. Rogers and Kaplan (foe. cit.) realised the quantitative meaninglessness of their fungal counts and consequently they could not be expected to try to draw conclusions from them. That they did discuss the significance of the bacteria to the point of what numbers constituted a problem, while completely ignoring the presence of the fungus, indicated to me that they considered the fungus as of little or no significance in their problem. I apologise for concluding that this was their opinion. I reaffirm my initial statement regarding ecological studies (Parbery, /oc. cit.). Surveys of organisms present in fuel and some studies on the influence of·physical factors on growth have been reported. I know of few, if any studies dealing with the influence of oxygen levels, temperature, pH or the interactions with other microbes, on the behaviour of C. resinae, particularly in regard to sporulation in fuels. Unless scientific reports are published in the open literature, where they are available to others interested in their content, they are to all intents and purposes not published. Thank you, Yours faithfully, D. G. PARBERY, School of Botany, University of Melbourne, Parkville. 3052. Australia. *I must apologise also to Crum, Reynolds, Pritchard and Hedrick (Dev. Ind. Microbial., 8, 253-9, 1967) to whom I attributed this work in the previous paper (Parbery foe. cit.). They used a different method of showing the quantitative presence of the fungus in the systems.

BOOK REVIEWS WAYSIDE AND WOODLAND FUNGI W. P. K. Findlay (Frederick Warne & Co., Ltd., London, New York, 1967. xi + 202 pp., 50 plates + 7 figures. Price £3.5.0) Beatrix Potter is well know as a writer of stories for children. She was also a talented artist, whose illustrations of toadstools provided the majority of the 40 coloured plates, and also the stimulus for Dr. Findlay to write this book. R. B. Davies and E. C. Large provided the other coloured plates, and these collectively give an excellent impression of the toadstools and some of the other larger fungi. There are also seven line drawings and ten black and white plates which do not match the quality of the colour plates. 96 The book falls naturally into two parts. In Chapters 1-5, Dr. Findlay mtroduces the fungi and discusses their tmpact on man and the difficulties of finding a "correct" name; gives hints on the collection and field identification of toadstools, and deals with environmental factors, spore distribution and ecological habitats. He also gives an I interesting "potted history" of the activities of amateur mycologists, and this leads up to the collection of illustra­ i tions by Beatrix Potter, around which this book has been written. Chapter 6 forms a link between the wide-ranging, informative early chapters and the more detailed descriptions of species in Chapters 7-17. There is an outline classification of the fungi as a whole, followed by three keys for the identification of toadstool genera. The only use of microscopic characters in identification appears in the second key. The remaining chapters are devoted to straightforward descriptions of fungi, with the Ascomycetes, Tremellales and Gasteromycetes forming Chapters 7-9, and the Boletaceae {including Paxillus), Polyporaceae, other Hymen­ omycetes, and a brief mention of Myxomycetes and Lichens forming Chapters 14-17.

The toadstools form the bulk of the book (72 out of 128 pages given to descriptions). Dr. Findlay's effective account of them reflects his great knowledge and regard for them. He divides them into four chapters, 10-13, using spore colour as his main criterion. Thus the white-spored types (Leucosporae) are found in Chapter 10, with keys to the common species of the major genera. These are followed by those with pink, brown and finally purple-black spores, where there is a key to the main genera. No grouping is perfect. While this arrangement separates genera of similar form, it has the great merit for the beginner, of appearing to get somewhere as soon as the spore colour has been determined. The great majority of the names used are based on the New Check List (Dennis, Orton & Hora, Suppl. Trans. Br. myco/. Soc. 1960), although a few still retain their old names (e.g. Clitocybe gigantea is preferred to Leuco­ paxil/us, presumably because the separation is based on microscopic spore characters; Amanitopsis is retained as it is so well defined.) The book was written to help the amateur naturalist in his walks through the fields and woods. It amply fulfills this purpose, and there is much in it which will add to the enjoyment of the more dedicated agaricologists, amateur and professional. However, it is not, and does not claim to be, a treatise for the separation of critical species, so the virtual absence of microscopic characters such as spore size and features is to be expected. There are some weaknesses: the quality of the line drawings and plates has been 'mentioned ; an indication of scale would be helpful in Figs. I and 4, while the volva in Fig. 6 seems to refer to the base of the stipe. The glossary could perhaps be extended to cover terms in the second and third keys which do not appear to be defined (e.g. amyloid, trama), and a few more references in the rather sparse bibliography might encourage the interested reader to delve further, for example into the description of Dr. Hesseltine's (not Haseltine) tasty fermented foods. These, however, are relatively minor matters. This is a book which will provide pleasure to those who can just enjoy looking at toad­ stools and other large fungi. Unfortunately, it is so expensive that many will regard it as a luxury which they cannot afford. G. J. F. Pugh

THE PROBLEMS OF BIRDS AS PESTS

Edited by R. K. Murton and E. N. Wright

(Institue of Biology Symposia No. 17. Academic Press, London, New York. xiv + 254 pp. Price £3.10.0)

This book comprises the papers and following discussions presented at a Symposium of The Institute of Biology, held in September 1967, on "Birds as Pests" in the fields of aviation, agriculture and public health. It highlights the growing recognition of certain species as agents of biodeterioration of economic importance, as well as the tight-rope walked by those intent on resolving the conflict between birds and man in the face of those who regard all species of birds as being in need of conservation. Six papers under the heading "Birds and Aircraft" draw attention to the steps which might be taken to mini• mise the hazard of bird strikes. These are: research into windscreen and jet engine design; techniques of acoustic bird scaring to move birds from airfields during periods of high air traffic, and to portable battery operated equip­ ment already in use for this purpose on a number of civil airfields. Two papers deal with the use of radar; for quantitative analysis of bird migrants and species identification, as well as its use as an aid to flight planning and air traffic control. Alteration of the environment of airfields to discourage birds, and thus reduce their numbers, is suggested as the "best hope for a long term solution to this problem". The introduction to six papers on "Birds and Agriculture" emphasises the changing pattern of agriculture that is "undoubtedly increasing the area of conflict between birds and man". A paper on the problem of Rooks (Corvusfrugilegus) in North East Scotland provides a preliminary report on the potential harm by this species to cultivations in a part of Aberdeenshire. In the summary of the paper on "The Oystercatcher-A pest of Shell-

97 fisheries" the conclusion is reached that,.''if the fishery is to maintain a steady level of production, control of the Oystercatchers is necessary", a conclusion to be set against the aesthetic needs of people who enjoy watching wild life, the value of which cannot be provided in the form of a monetary estimate. Man as a predator on birds could not be seen by one contributor, concerned with the depredations of wood pigeons, as acting as a regulatory agent of bird populations. The present level of cropping pest birds, by currently used techniques, did not appear sufficient to reduce their numbers below the level of natural fluctuations: "killing may remove an expendable surplus without affecting population size". One paper deals with the urban bird problem of feral pigeons and starlings and their fouling and defacement of buildings. It is concerned also with feral pigeons and house sparrows within premises and their association with food. The success of treatments involving the use of alphachloralose and other stupefying baits is reviewed in detail. Data is presented which show the number of birds of various species caught with stupefying baits in different environments. Damage on a large scale by Bullfinches to the flower buds of fruit trees is recognised in the United Kingdom as one of the greatest problems of the fruit growing industry. This paper emphasises the availability of seeds, notably of Ash, in determining whether fruit bud damage is likely to be severe. In the final paper, Que/ea, a highly greaga­ rious sparrow-sized weaver, is noted for its descructive attacks on various cereal crops in the semi-arid grasslands of Africa-a problem only recently recognised-but again it is believed that the hundreds of millions of birds killed each year probably constitute only part of the "doomed reproductive excess". This Symposium emphasised the tremendous advance made during recent years in our knowledge and thinking of avian ecology. Nevertheless, the warning was given: techniques of control must perforce make haste slowly, notably when ornamental and attractive species are involved, for fear of man's activities culminating in extinction instead of control. P. B. Cornwell

THE COCKROACH. VOLUME 1. A LABORATORY INSECT AND AN INDUSTRIAL PEST P. B. Cornwell (Hutchinson, London, 1968. 391 pp. Price £3.3.0) This is the first of two volumes on the cockroach in the Rentokil Library by the Director of Research. at Rentokil Laboratories. Volume II will be concerned specifically with "Insecticides and Cockroach Control" but volume I is of more general interest and provides an account of the biology of the more common species, including details of their structure, physiology, behaviour and ecology. Cockroaches may occur in such numbers as to cause considerable damage and spoilage by contamination of stored food products. They have biting mouthparts and will eat materials of both animal and vegetable origin. Because they are so easy to feed and to rear in large numbers, cockroaches have been drawn and dissected more than any other insect in school and college laboratories and it is probably because of this that the cockroach is one of the most quickly recognised stored product pests. There are however a number of different kinds of cock­ roaches and this book provides descriptions of species associated with man, a key for their identification and maps showing the occurrence of Blatella germanica (L.) and Blatta orienta/is L. as pests in different parts of Britain. The distribution of the different species throughout the world has resulted from the transport of food materials containing either insects or egg cases but perhaps the deliberate actions of biologists in sending insects by post for use in teaching establishments, has also played a part. The use of cockroaches in teaching has certainly made these insects readily available for research and an account is given of the behaviour and physiology of cockroaches, with particular emphasis on aspects which are of most interest in relation to biodeterioration problems including the cuticle and the gut, through which insecticides enter the body, sex attractants which may provide new ways of catching and killing cockroaches, natural enemies, movement and climatic factors affecting activity and survival. The occurrence of cockroaches in kitchens and hospitals is usually taken to indicate poor standards of cleanliness and hygiene and a review is provided of the evidence against the cockroach as a potential carrier of disease. There is a subject index and an author index and also a bibliography which includes references to both original papers on all aspects of cockroach biology and to review articles, Papers published as recently as 1967 are included: they are not arranged alphabetically but are numbered in the order in which they are first used in the text. Refer­ ences are also cited by the same number in the text. The book is well illustrated and interesting to read and will be used by both students and teachers as well as those requiring an introduction to recent research on cockroaches or a background to control measures. This is a very useful reference book and a welcome addition to the series. R. Barrass 98 KERATIN AND CHITIN (Coni), LEATHER, LIGNIN, METALS

69/1- GRAY, T. R. G. & BAXBY, P. 69/l- ANDERSEN, J. E.; HERWIG, G. L. & 6721 6729 MOFFITT, R. B. Transactions British Mycological Society, 51 (2): 293-309, (1968). Australian Mining, 35-39, (15 April1966). Chitin decomposition of soil. II. The ecology of Heap leaching at Rum Jungle. [Copper. Thiobacillus.] chitinoclastic micro-organisms in forest soil. [Fungi. Bacteria. Actinomycetes.] 69/1- LOBL, M. 6730 Australasian Corrosion Engineering, 12 (6): 3-12, (1968). LEATHER The choice of materials for gas mains and modifications of existing systems. [Soil. Corrosion. Resistance.] 69/1- ORLITA, A. 6722 Proceedings International Biodeterioration 69/1- IVERSON, W. P. Symposium, 1st, Southampton, 9th-14th Sept., 6731 1968; 297-301, {1968). Nature, 217 (5135): 1265-1267, (1968). Biodeterioration in the leather industry. [Fungi.] Corrosion of iron and formation of iron phosphide by Desulfovibrio desulfuricans.

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Tappl, 51 (7): 298-300, {1968). Metal-organic acid corrosion and some mechanisms Improved ball m11ling in the Isolation of milled associated with these corrosion processes. (Metal.] wood lignin. [Grinding. Technique.]

69/l- GUPTA, S. K.; SANYAL, B. & NANDA, J. N. METALS 6736 Labdev J. Science Technology, 6-A (1): 1-13, (I968). Underground corrosion: Pt. I - Factors affecting 69/1- GOLOMSIK, A. I. & IVANOV, V. I. corrosion and methods of assessment. 6727 [Metals. Chemical deterioration. Bacteria. Testing.] Mikrobiologiya, 34 (3): 465468, (1965). Adaptation of Thiobaci/lus ferroxidans to increased concentrations of hydrogen nnd 69/1- GUPTA, S. K.; SANYAL, B. & NANDA, J. N. iron ions. [In Russian; English summary.] 6737 LabdevJ. Science Technology, 6-A (2): 5I-59, (1968) Underground corrosion: Pt. II - Methods of 69/1- ANON. protection. 6728 Newsletter Ontario Research Foundation, 9 (7): 1p. (1966). Corrosion engineering. [Metal.] • When you need a paint I I' fungicide, you should I look to Nuodex. I

II • Why? I Because we have the. most versatile line around.

SUPER AD·IT The recommended universal paint fungi· NUODEX products are available from the following cide for both oil and aqueous systems. affiliates and licensees: PM0·1 0 For mildew resistant oil, oleoresi nous and ARGENTINA • Plastlca Bernabo ·S.A .• Terrada 658/664, Buenos Aires: AUSTRALIA • Nuodex IAust.) Pty. Ltd., 49-61 Stephen Road, alkyd paints. Botany, N. S. W.: BRAZIL • Nuodex S. A, Industria, E, Comercio de PMA-18 For use in standard aqueous systems as a fungi· Secantes, Rua Dom Gerardo 80·1' and., Rio de Janeiro; ENGLAND • cide and/or preservative. Nuodex Limited, Birtley, County Durham; FRANCE • Nuodex France S. A. R. L., 14, Rue de Moscou, Paris 8e, France; WEST GERMANY • PMA-60 For use in emulsion paint systems to prevent Gebr. Borchers A. G., Elizabethstr. 14, Dusseldorf; G. Siegle & Co, GmbH, Sieglestrausse 25, Stuttgart-Feuerbach !Stabilizers only); bacterial spoilage during liquid paint storage and to pro· HOLLAND • N. V. Chemische Fabriek Servo, Del den: ITALY • Nuodex teet the applied paint film against mildew attack. Packaged ltaliana, S. P. A., Piazza Della Republica 11/A, Milan; JAPAN • . ' in 4 oz. and 8 oz. water-soluble packages. Harima, Kasei Kogyo Ltd., Mizuashi, Noguchi·cho, Kakogawa, Hyogo; MEXICO • Nuodex Mexicana, S. A., Durango 209, Desp. 101, Mexico 1. '~ FUNGITROL 11 Fungicide-bactericide for non-aqueous 7 D. F.; NEW ZEALAND • A. C. Hatrick IN. Z.) Ltd., 66 Main Road, ' ' paint systems. Effective for meat-packing plants, breweries, Tawa, Wellington; SOUTH AFRICA • Poly-Resin Products, Ltd., dairies, etc. Also imparts fungus resistance to baking Durban; SPAIN· Nuodex Espanola, S. A., Av. Jose Antonio 55, Madrid. enamels. In any language NUODEX is your symbol for quality and service. TENNECO CHEMICALS, INC. NUODEX DIVISION INTERNATIONAL DEPT. e P.O. Box 2, Piscataway, N.J.

Published by the Biodeterioration Information Centre. £4 per annum, Printed by Suttons (Printers) ltd.