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MICROBIOLOGICAL CORROSION OF METALS~ MARINE WOOD BORERS- RODENT ATTACKS ON STORED PRODUCTS- FOULING OF SHIPS BY BARNACLES- DETERIORATION OF STONE BY BACTERIA­ROTTING OF WOOD BY FUNGI- BACTERIAL BREAKDOWN OF ASPHALT- MILDEWING OF LEATHER -INSECT DAMAGE TO BOOKS -BIRD HAZARDS TO AIRCRAFT- FUNGI IN JET FUEL TANKS­TERMITES IN TIMBER --MICROBIOLOGICAL ATTACK ON RUBBERS PLASTICS AND PAINTS,- FUNGAL ETCHING OF GLASS o 0 0 0

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INTERNATIONAL

BIDDETERIORATION

BuLLETIN

A QUARTERLY JOURNAL OF BIODETERIORATION

BIODETERIORATION INFORMATION CENTRE

THE UNIVERSITY OF ASTON IN BIRMINGHAM

'ENGLAND

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CATOMANCE LIMITED

manufacturers of

mystox* for the preservation of

timber, textiles paper, cordage

plastics and specialised applications

* mystox is the registered trade mark of

Catomance Limited 94 BRIDGE ROAD EAST, WELWYN GARDEN CITY,

HERTS., ENGLAND. Telephone: Welwyn Garden 24373/8

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Volume 7 Winter 1971 Number4

INTERNATIONAL

BIODETERIORATION

BuLLETIN

CONTENTS

BIODETERIORATION SOCIETY NEWSLETIER 145

INDUSTRY AND COMMERCE 146

METHYLENE BLUE REDUCTION FOR RAPID INPLANT DETECTION OF COOLANT 147 BREAKDOWN H. W. Rossmoore

MODEL TESTS ON THE MECHANISMS OF MICROBIAL DETERIORATION OF FILLED 155 VULCANIZATES: I. INFLUENCE OF PSEUDOMONAS SP. ON THE SYSTEM PARAFFIN OIL-CARBON BLACK B. J. Zyska, P. S. Fuda1ej aud B. J. Rytych

THE SELECTIVE ISOLATION OF THE "KERSOENE FUNGUS" CLADOSPORIUM RESINAE 161 FROM THE AIR J. E. Sheridan and Jan Nelson

A SIMPLE TECHNIQUE FOR ESTIMATING MICROBIAL ACTIVITY BY TOTAL CARBON 163 DIOXIDE EVOLUTION AND ITS APPLICATION TO THE ATTACK OF PLASTICISER-TREATED COTTON YARN C. S. Stewart and J. H. Walsh

PRELIMINARY INVESTIGATIONS INTO THE USE OF A TIDN STRIP TENSILE STRENGTH 169 TEST FOR THE RAPID EVALUATION OF WOOD PRESERVATIVES AGAINST BASIDIOMYCETE FUNGI A. F. Bravery and C. Grant

BOOK REVIEWS 174

INDEX 178

TABLE DES MATIERFS

BULLETIN DE LA SOCIETE DE BIODETERIORATION 145

L'INDUSTR!E ET LE COMMERCE 146

UTILISATION DE LA REDUCTION PAR MOYEN DU BLEU DE METHYLENE POUR LA DECOUVERTE 147 RAPIDE, DANS LES LABORATOIRES LOCAUX, DE LA RUPTURE DES L!QUIDES REFRIGERANTS H. W. Rossmoore ESSAIS DE MAQUETTE APPLIQUES AUX MECHANISMES DE LA DEGRADATION MICROBIALE DES 155 VULCANISES REMPL!S. I. L'INFLUENCE DES PSEUDOMONAS SP. SUR LA SYSTEME HUILE DE PARAFFINE-NUIR DE CARBONE B. J. Zyska, P. S. Fudalej et B. J. Rytych

L'ISOLATION SELECTIVE DANS L'AIR DU "KEROSENE-FONGUS" CLADOSPORIUM RESINAE 161 J. E. Sheridan et Jan Nelson UNE TECHNIQUE SIMPLE POUR ESTIMER L'ACTIVITE MICROBIALE PAR MOYEN DEVELOPPEMENT 163 TOTALE DU CARBONE DIOXIDE, ET SON APPLICATION A L'ATTAQUE AU F!L DE COTON PLASTICISE C. S. Stewart et J. H. Walsh

LES INVESTIGATIONS PREL!MINAIRES DANS L'USAGE D'UN MORCEAU MINCE DE BOIS POUR FAIRE 169 UN ESSAIS DE FORCE DE TENSION, POUR L'EVALUATION RAPIDE DES PRESERVATIVES DUBOIS CONTRE LES BASIDIOMYCETES A. F. Bravery et C. Grant

REVUES DES LIVRES 174

INDICE 178

INHALT

RUNDSCHREIDEN DER BIO-VERSCHLECHTERUNGSGESELLSCHAFf 145

INDUSTR!E UND HANDEL 146

DIE REDUKTION MIT METHYLENBLAU ALS EINE METHODE FOR DIE SCHNELLE ORTSBETRIEBL!CH 147 ENTDECKUNG DES KUHLMITTELSZERFALL H. W. Rossmoore

MODELLVERSUCHE AUF DEN MECHANISMUS DER MIKROB!ALISCHEN VERSCHLECHTERUNG IN DEN 155 GEFOLLTEN VULKANISIERTEN. I. DER EINFLUSS DER PSEUDOMONAS SP. AUF DIE ANORDNUNG "PARAFFIN0L-RUSS". B. J. Zyska, P. S. Fudalej und B. J. Rytych.

DIE SELEKTIVE ISOL!ERUNG VON DER LUFT DES "KEROSENENSCHWAMM" CLADOSPORIUM RESINAE 161 J. E. Sheridan und Jan Nelson

EINE EINFACHE TECHNIK FOR DAS VERMESSEN DER MIKROBIALISCHEN TATIGKEIT, MITTELS DER 163 GANZEN ENTWICKLUNG DES KARBONDIOXYD UN SEINES GEBRUACHES GEGEN DEN ANGRIFF AUF DEN PLASTIFIZIERTEN BAUMWOLLGARN C. S. Steward und J. H. Walsh

EINLEITENDE ERFORSCHUNGEN AUF DEN GEBRAUCH EINER ZUGSPANNIJNGSPROBE MIN EINEM 169 DUNNEN HOLZSTREIF, UM DIE WIRKUNEN DER HOLZKONSERVIERUNGSMITTEL GEGEN BASIDIOMYCETEN ZU BEWERTEN A. F. Bravery und C. Grant

BUCHBESPECHUNGEN 174

REGISTER 178

CONTENIDO

CARTA DE NOTICIAS DE LA SOCIEDAD DE BIODETERJORACI6N 145

INDUSTRIA Y COMERCIO 146

LA REDUCCI6N METHYLENE AZUL PARA LA DETECCI6N RAPIDA DENTRO DE LAS PLANTAS DEL 147 TRASTORNO DEL ENFRIANTE H. W. Rossmoore

PRUEBAS MODELOS SOBRE LOS MECANISMOS DE DETERIORACI6N M!CR6BICA DE LOS VULCANIZATOS ISS LLENADOS. I. INFLUJO DEL PSEUDOMONAS SP. SOBRE EL SISTEMA DEL ACEITE DE PARAFINA-CARB6N NEGRO B. J. Zyska, P. S. Fudalej y B. J. Rytych

EL AISLAMIENTO SELECTIVO DEL HONGO DE KEROSINA CLADOSPORIUM RES/NAE DEL AIRE 161 J. E. Sheridan y Jan Nelson

UNA TECNICA SENCILLA PARA CALCULAR LA ACTIVIDAD MICR6BICA POR LA EVOLUCI6N TOTAL 163 DEL DlOXIDO DE CARB6N Y SU EMPLEO PARA EL ATAQUE DEL HILO DE ALGOD6N TRATADO CON UN PLASTICER C. S. Stewart y J. H. Walsh

INDAGACIONES PREL!M<NARIAS EN EL EMPEO DE UNA CORREA DELGADO PARA LA PRUEBA DE LA 169 RESISTENCIA DE TENS16N PARA LA EVALUACI6N RAPIDA DE LOS PRESERVATIVOS DE LA MADERA CONTRA LOS HONGOS BASIDIOMYCETE A. F. Bravery y C. Grant

REV!STAS DE L!BROS 174

INDICE 178

ii

ACKNOWLEDGMENTS TO SUSTAINING ORGANISATIONS

Financial support for the Biodeterioration !Information Centre from the following organisations is gratefully acknowledged:

ABBOTT LABORATORIES, Commercial Dev., Chemical Division, North Chicago, Illinois, 60064, U.S.A.

ALBRIGHT & WILSON (MFG) LTD., Oldbury Division, P.O. Box 3, Oldbury, Warley, Wares.

B.D.H. CHEMICALS LIMITED, Laboratory Chemi­cals Division, Poole, Dorset; manufacturers of laboratory chemicals, biochemicals, industrial fine chemicals and microbiocides.

BRITISH INSULATED CALLENDERS CABLES LIMITED, 38 Wood Lane, London, W.l2.

THE BRITISH PETROLEUM COMPANY LIMITED.

CATOMANCE LIMITED, Welwyn Garden City, Hertfordshire; manufacturers of speciality chemi­cals for the textile, paper, timber, leather industries, etc., including fungicides, bactericides and insecti­cides.

CENTRAL LABORATORY TNO, Delft, The Netherlands; research, analysis and testing facilities in materials science and technology, including bio­deterioration and marine research. Sponsored by government agencies and by international industries.

CIBA-GEIGY LIMITED, CH-4002 Basle, Switzer­land; manufacturers of dyestuffs, industrial chemi­cals, plastic additives, photochemicals, pharmaceu­tical and agricultural chemicals.

CIBA-GEIGY (U.K.) LIMITED, Dyestuffs Division Clayton, Manchester, Mil 4AR,

COURT AULDS LIMITED, Coventry.

THE DUNLOP COMPANY LIMI'IE6.

Ill

FARBENFABRIKEN BAYER A.G., Leverkusen, Germany; manufacturers of dyestuffs, industrial chemicals, synthetic fibers, pharmaceutical and agricultural chemicals and preservatives for wood, foodstuffs and technical products.

GRAYLABORATORIESINTERNATIONAL LIM· ITED, Marshall Road, Hampden Park, Eastbourne, Sussex.

HALDANE LABORATORIES LIMITED, Haldane Place, London S.W. 18, consultants in industrial microbiology and microbiological dete· rioration.

HICKSON & WELCH (HOLDINGS) LTD., Ings, Lane, Castleford, Yorkshire.

IMPERIAL CHEMICAL INDUSTRIES LIMITED, Agricultural Division, Billingham, Co. Durham.

LUCAS GAS TURBINE EQUIPMENT LTD., Shaftmoor Lane, Birmingham, 28.

MAY & BAKER LIMITED, Dagenham, Essex; chemical manufacturers.

MOLECULAR CONSERVATION LIMITED, Harrogate, Yorkshire; manufacturers of speciality products for conserving metals, roofing, engineering equipment and other man-niade resources.

RENTOKIL LABORATORIES LIMITED, East Grinstead, Sussex.

REVERTEX LIMITED, Harlow, Essex.

SCIENTIFIC CHEMICALS INC., 1637 South Kil­bourne Ave., Chicago, lllinois, 60623, U.S.A.; leading manufacturers of industrial fungicides and bactericides who maintain substantial research and development facilities to assist customers in the development of final products geared to meet government and industry standards.

INTERNATIONAL BIODETERIORATION

BuLLETIN Editors Dr. H. 0. W. Eggins and Mr. M. J. D. Willsher,

Biodeterioration Information Centre, Department of Biological Sciences, The University of Aston in Birmingham, 80 Coleshill Street, Birmingham, England, B4 7PF.

Editorial Board Mr. G. Ayers!,

Technology, England.

Department of Biology, College of Wolverhampton, Staffordshire,

Professor G. Becker, Bundesanstalt fur Material­prufiing, Berlin-Dahlem 45, Unter den Eichen 86/87, Germany.

Dr. D. S. Belford, Hickson & Welch (Holdings) Limited, Ings Lane, Castleford, Yorkshire, England.

Mr. D. G. Coursey, Tropical Products Institute, 56/62 Gray's Inn Road, London, W.C.l., England.

Mr. J. J. Elphick, C.N.R. Dept., RM. 0173, M.O.D" Main Building, Whitehall, London, S.W.l., England"

Dr. J. Garrido, Departmento de Fermentaciones Industriales, Castello 25, Madrid, Spain.

Dr. N. E. Hickin, Rentokil Research Laboratories, Felcourt, East Grinstead, Sussex, England.

Dr. H. J. Hueck, Central Laboratory TNO, Schoe­makerstraat 97, P.O. Box 217, Delft, The Nether­lands.

Dr. A. M. Kaplan, Fungicides and Germicides Laboratory, Pioneering Research Division, U.S. Army Natick Laboraiories, Natick, Mass. 01760, U.S.A.

Monsieur Y. Le Grand, Centre d'etudes du Bouche!, Vert-le-Petit, (S & 0), France.

Mr. A. 0. Lloyd, Catomance Limited, 94, Bridge Road East, Welwyn Garden City, Herts., England.

Dr. G. J. F. Pugh, Department of Botany, University of Nottingham, Nottingham, England.

Professor R. W. Traxler, Department of Plant Patho­logy, Entomology, University of Rhode Island, Kingston, Rhode Island, 02881, U.S.A.

Dr. R. Zinkernagel, Ciba-Geigy, Ltd., CH-4002 Basle, Switzerland.

Dr. B. J. Zyska, Gl6wny Instytut Gornictiva, Kato­wice, Plac Gwarkow I, Poland.

Advertising Manager Mr. D. Allsopp

Biodeterioration Information Centre, Department of Biological Sciences, The University of Aston in Birmingham, 80 Coleshill Street, Birmingham, England, B4 7PF.

NOTES FOR CONTRffiUTORS

The International Biodeterioration Bulletin is pub­lished four times per year (Spring, Summer, Autumn and Winter). Typescript contributions in triplicate should be sent to the Editors, Dr; H. 0. W. Eggins, or Mr. M. J.D. Willsher at the above address.

The Bulletin acts as a vehicle for the publication of works on all aspects of biodeterioration, i.e. the deterioration of materials of economic importance by micro-organisms, insects, rodents, etc.

Contributions may be in English, French, German or Spanish and should be submitted in triplicate on international A4 size paper (21.0 em x 29.7 em or 8.27 in. X 11.69 in.); typewritten on one side of the paper only. A summary of 25-100 words should accompany each contribution.

Illustrations should be clearly drawn in Indian ink or should be photographed. The reduction desired should be clearly indicated and illustrations when reduced are not to exceed 17 em X 26 em. Where figures are to be inserted in the text the approximate position for each one should be clearly marked in the typescript.

IV

The bibliographic references are to be indicated in the text as, e.g.:

Reese and Levison (1952).

and in the bibliography:

Reese, E. T. and Levison, H. G. (1952) Comparative study of the breakdown of cellulose bymicroorganisms. Physiol. Plant., 5, 354-366.

Authors are requested to abbreviate journal titles according to the conventions of the World List of Scientific Periodicals.

Proofs will not be sent to authors before final publication.

25 reprints will be sent free of charge to each author. Additional reprints are obtainable: scale of charges available on application to the Editors.

BIODETERIORATION SOCIETY NEWSLETIER

Scientific Meeting - Preliminary Announcement

"Biodeterioration-The Role of Higher Organisms"

A one-day meeting at the Uni­versity of Aston in Birmingham, Friday 25th February 1972.

It is hoped to include papers on insects, birds, rodents and marine organisms.

Further details will be sent to members nearer the date. Non­members should write to the Biodeterioration Information Cen­tre for details.

News of Members

The Programme Secretary of the Society, Mr. A. 0. Lloyd, officially retired from Catomance Limited on November 30th 1971. He is continuing his long associa­tion with the Company as a consultant until' his full retirement in 1976.

Mr. Lloyd will not only be acting as a consultant for Cato­mance Limited, but will also be working as an independent con­sultant. Mr. Lloyd has a long and wide experience in the field of industrial textile microbiology and has recently been engaged in the study of lichens as deteriogens.

One-day Meeting at Hatfield Polytechnic

The Biodeterioration Society and the Hatfield Polytechnic held a joint symposium on Techniques in Biodeterioration at the Hatfield Polytechnic on Friday 3rd Decem­ber, 1971. The Chairman of the meeting was Mr. A. 0. Lloyd and the speakers were Mr. D. Allsopp of the Biodeterioration Informa­tion Centre and Mr. A. R. M. Barr of Catomance Limited who lec­tured and presented a film on textile testing and perfusion tech­niques; Dr. R. N. Smith of the Hatfield Polytechnic on the biology of paper mill deteriogens; Miss. J. Taylor of the Forest Products Research Laboratory on termite testing; Mrs. C. E. Skinner of the

Paint Research Station on labora­tory tests for biocidal paints; Mr. J. G. Savory of Forest Products Research Laboratory, on accelera­ted testing of wood; and Mr. J. Mills of the Department of Medi­cal Biochemistry, University of Manchester, and Mr. A. E. Darby of Ciba-Geigy Limited on tech­niques in plastics degradation.

The evening session consisted of demonstrations of equipment by the speakers, which proved extremely absorbing.

The friendly and lively atmos­phere at this meeting contributed to its success, and thanks are due to Dr. R. N. Smith and his staff at the Hatfield Polytechnic for their efforts in making the meeting such an enjoyable and worthwhile event.

Biodeterioration Research Titles

From volume 8, number I onwards the name IBBRIS (Inter­national Biodeterioration Bulletin Reference Index Supplement) is to be changed to Biodeterioration Research Titles.

The name International Rio­deterioration Bulletin Reference Index Supplement was originally chosen because the first issue appeared in 1966 as part of the International Biodeterioration Bull­etin and was retained when it became a separate publication the following year. Later the acronym IBBRIS was used in preference to the cumbersome original title but this had the disadvantage of giving no indication of the nature or content of the publication.

It has therefore been decided to select a new title which, while remaining fairly simple, will give a clue to the type of publication it is and the field which it covers.

Along with the change o.f title there will be some other altera­tions in the layout of the publica­tion but the coverage of the biodeterioration literature will not be altered.

Timberlab Courses 1972

Details of the 1972 courses to be held by the Forest Products

14§

Research Laboratory of the U.K. Department of the Environment have been announced. Of particu­lar interest is the three-day course on "Control of insects and rot in buildings" which is to be held from 31 January to 2 February, from 20 to 22 March, from 18 to 20 September and from 30 October to I November. The course is particularly aimed at surveyors, supervisors and foremen concerned with the eradication and control of wood-destroying insects and fungi.

The fee for the course will be £19.50 and fnrther details may be obtained from Mrs. J. Marshall, Forest Products Research Labora­tory, Princes Risborough, Ayles­bury, Buckinghamshire, England.

International Symposiom on the Deterioration of Building Stone

The Department of the Study of Materials of the Centre de Re­cherches et d'Etudes Oceano­graphiques, Paris is organising the 1st International Symposium on the Deterioration of Building Stones to be held in La Rochelle, France from 11 to 15 September, 1972.

Details of the Symposium can be obtained from the Secretariat, Centre de Recherches et d' Etudes Oceanographiques, I Quai Branly, Paris 7e, France.

Materials in the Sea

The Third International Con­gress on Marine Corrosion and Fouling, "Materials in the Sea" will be held at the National Bureau of Standards, Gaithersburg, Mary­land, U.S.A. from 2 to 6 October, 1972. The Congress is held under the auspices of the Permanent International Committee for Re­search on the Preservation of Materials in the Marine Environ­ment and is sponsored by the Maritime Administration, the Na­tional Bureau of Standards, the Naval Oceanographic Office and the Office of Naval Research.

INDUSTRY AND COMMERCE

Fumite pyrotechnic smokes

The Fumite Division of Pains­Wessex Ltd. has been manufactu­ring pesticidal smokes since 1946. Their uses include disinfestation of silos, ships' holds and ware­houses and control of wood­boring beetles as well as control of glass-house pests and insect vectors of disease.

The smokes are available either as generators or as pellets and are calculated to treat a given capacity

depending on the amount of active pesticide evolved in the smoke. The smoke cloud expands to fill the space being treated and settles as a fine deposit on upward facing surfaces. Normally the pesticides deposited in this way have little persistency although more persistent formulations are available for use against wood­boring beetles for example.

No mixing of toxic chemicals is involved in the use of the smokes

146

as each unit is complete in itself and requires only to be ignited.

The present Fumite range in­cludes DDT/lindane, lindane, diel­drin/lindane, bromophos/lindane, tecnazene and tecnazeneflindane (Tecnalin).

The manufacturers are Pains­Wessex Ltd., Fumite Division, High Post, Salisbury, Wiltshire, England.

'Abstracted from PANS.'

., '

Rossmoore, H. W. Int. Biodetn Bull., 7 (4), 147-154, (1971). Methylene blue reduction for rapid inplant detection of coolant breakdown.

METHYLENE BLUE REDUCTION FOR RAPID INPLANT DETECTION OF COOLANT BREAKDOWN1

H. W. Rossmoore2

Summary. A method is described for the detection of micro­bial breakdown of cutting fluids and coolants which can be easily perfonned in local plant chemical laboratories without the need for specially trained personnel.

Utilisation de Ia rMuction par moyen du b1eu de miithylEme pour Ja diicouverte rapidc, dans les Iaboratoires locaux, de Ia rupture des liquides riifrigiirants. On decrit une methode de dlkouvrir Ia rupture rnicrobiale des ftuides de coupe et refrigc!rants, qui peut facilcment s'accomplir dans les laboratoires chimiques des outillages locaux, sans avoir besoin d'employer les personnes specialement instruitcs.

Introduction

This investigation was undertaken not only for the purpose of more quickly detecting bacterial breakdown of water-soluble cutting fluids but also to make detec­tion less dependent on a central bacteriology laboratory. Most large users of cutting fluids either have no facilities for adequately performing routine bacterio­logical tests or must rely on a central company laboratory or on an outside contractor. In either, case, there is more delay than is tolerable in the initiating of tests and relaying of results to interested individuals in the plant. There is ample evidence that both quantitative and qualitative changes in the bacterial flora take place within 24 hours after collection and we have evidence in this report that recommendations based on bacterial counts are often too late to be useful.

Thus, we looked for a test procedure that could easily be performed in existing plant chemistry laboratories without the necessity of training or employing personnel in specific bacteriological tech­niques. Although it does have recognized limitations, there is sufficient background on methylene blue reduction correlating microbial metabolism in a particular milieu with the total active bacterial popu­lation. It has been recommended for the screening of raw milk samples {ALPHA, 1967) and, though no longer official, for the measurement of the relative stability of sewage effluents (ALPHA, 1960). Pivnick and Fabian (1953) reported the use of methylene blue reduction in soluble oil emulsions for the purpose of screening inhibitors. However, they were measuring growth from small inocula rather than the results of growth and their control systems were reduced before 36 hours. In addition, Senez and Azoulay (1961) reported that methylene blue was the most effective electron acceptor in the oxidation of n-heptane by

Die Reduktlon mit Methylenblau nls eine Methode filr die schnelle ortsbetrieblich Entdekung des Kiihlmittelszerfall. Eine Methode fUr die Entdeckung des mikrobialischen Zerfalls der Schneide- und KOhlungsftussigkeiten wird beschrieben, die sich ohne ausdrficklich erzogenes Personal in den chemischen Laboratorien der Ortsanlage sehr Ieicht ausfUhren kann.

La reduccl6n methylene azul para Ia detecciOn rBpida dentro de Ins plnntas del trastorno del enfriante. Se describe un mCtodo de descubrir un trastorno micr6bico de los ftuidos cortantes y enfriantes. metoda que se puede emplear facilmente en los laboratories quimicos para las plantas sin necesidad de emple­ados ensenados especialmente para esto.

Pseudomonas aeruginosa suggesting the potential utility of dye reduction for the detection of hydro­carbon breakdown.

It was obvious that if methylene blue reduction was to be used for the purposes stated previously, that we would have to increase sensitivity of the test beyond that reported by Pivnick and Fabian. In this context sensitivity is stated to mean not only rapid results which can be related to total viable population but also and perhaps more important a high degree of cocrelatability with the condition or the coolant; i.e., the faster the reduction, the poorer the overall quality of the coolant.

Methods and Materials

The study was divided into three parts: the first was aimed at establishing parameters for the test system which would enable an operator to arrive at results that were repeatable and that were also obtainable in a short period of time. The test itself was based npon the ability of biological systems to reduce methylene blue to a colorless compound, a process that only goes to completion in the absence of oxygen.

The effects of the following variables were explored: pH, oxidation-reduction potential, temperature, meth­ylene blue concentration, the need for exogenous energy source, the concentration of exogenous energy source, light, the addition of iron, and the addition of tramp (hydraulic) oil, order of reagent addition and relationship of bacterial count to reduction time. These studies were made with representative members of the soluble cutting fluid family.

In addition to the above-described vanat10ns the advisability that standards would be necessary for the evaluation of results was considered. Unused coolants with amounts of methylene blue equal to zero to 90%

!Contribution No. 260, Department of Biology, Wayne State University. 2Department of Biology, Wayne State University, Detroit, Michigan 48202, U.S.A. (Copy received April1971).

147

Methylene blue reduction for rapid inplant detection of coolant breakdown. H. W. Rossmoore.

reduction were prepared and subjected to the following temperature and storage conditions:

I. Steam sterilization, 121°C, 15 pounds pressure for 15 minutes and stored at room temperature for one month.

2. Storage for one month at 60°C in the dark.

3. Storage at room temperature (25°C) and exposed to the diurnal conditions of the laboratory.

4. Storage at refrigeration temperatures (4°C) for one month.

Results with these presumptive standards indicated that exposure to light for long periods of time and exposure to temperatures of sterilization destroyed the color of the methylene blue•coolant combination. Since the reliability of any standard is related to its continuing dependability and since it would have been necessary to heat treat systems to some extent to destroy the bacteria present in them, we decided to abandon standard comparison and instead direct our attention to 100% reduction systems.

The second portion of this program was aimed at correlating the test system that was proven most reliable in laboratory pilot samples with regularly collected cutting fluid samples from central systems in a selected manufacturing operation. We were able to collect samples from many central systems either weekly or semi•weekly. We compared reduction times and bacterial plate counts, with the subjective evalua­tion of the cutting fluid by plant personnel.

The third part of this program involved two con­comitant studies: the utilization of the methylene blue reduction procedure in the plant by inhouse personnel, modified to suit their schedules, at selected central systems for a period of five weeks; and the determina­tion of possible reasons for some of the inconsistencies in the results obtained in the initial pilot study. Since we found that relatively low bacterial counts with fast reduction time oftentimes correlated with an unsatis­factory rating by personnel, we thought it would be profitable to examine the effect of the suspending media on reduction time of a microbial population centrifuged from a representative cutting fluid. This, in effect, might reveal the contribution of an old or new coolant on the metabolic activity of the microbial flora. In addition, several experiments were conducted on the reduction proclivities of the two major species involved in cutting fluid deterioration.

Pseudomonas oleovorans ATCC 8062 was grown as described (Rossmoore & Brazin, 1968), washed three times in M/15 Na2HP04 buffer and resuspended in a final concentration of 8 x JOIO cells/mi. Aliquots of this suspension were lysed by freezing and thawing at -70°C to ambient reducing the viable population by 99.9%- Binary dilutions of the original suspension and its lysed derivative were used as the inoculum in the previously described reduction protocol. Desulfo-

148

vibrio desu/furicons A.T.C.C. 7757, was grown as described (Rossmoore et a!, 1963) washed as above and resuspended at a concentration of 10 mgm of cells in 15 mi. An aliquot of this suspension was completely lysed in a Raytheon 10KC sonic oscillator for 30 minutes. The sonicated suspension was fraction­ated at 33,000 g in a Servall RC-2 centrifuge. Aliquots of the original suspension, its lysed derivative and fractions were used as inoculum in the previously described reduction protocol except that acetate buffer, pH 5.2 was substituted for the M/15 phosphate buffer.

Results

Part One

I. pH: The studies on pH revealed that the fastest reduction times were obtainable at alkaline pH. Twelve pH variations from 4 through 8 were run with the coolant varieties and no reduction was obtained at pH 5.4 or lower. In addition, reduction time at pH 8 was the fastest obtained, a fortuitous result since the ambient pH of most cutting·operations is above 8.5.

2. Oxidation Reduction Potential: Oxidation values varied from -20 millivolts to +100 millivolts. The values were not repeatable and no statistical signific­ance could be attributed to this function. We therefore abandoned control and/or measurement of oxidation reduction potential as part of the test system.

3. Temperature: The preliminary tests were carried out at four temperatures: 25°C; 37°C; 45°C; and 4°C. No results were detectable within reasonable time limits when the incubation temperature was either 45°C or 4°C. In addition, 37°C did not prove better than 25°C; therefore, all subsequent testing was most conveniently done at ambient laboratory temperature.

4. Methylene Blue Concentration: A concentration of methylene blue was sought that would give a detectable color in any coolant base and which would undergo 100 per cent reduction when the other variables were controlled. Methylene blue isothio­cyanate tablets in a recommended dilution of one tablet to 200 ml of water was found to be the optimal concentration.

5. The need for and concentration of exogenous energy source: It was found that reduction time was much too slow without the addition of a readily utilized carbohydrate. Sodium lactate was the energy source of choice. Initial concentrations of 5, 10, and 20% showed the 10% to be most effective, while the 5% was slightly less so, and 20% was, in fact, inhibitory to reduction.

6. Light: Because of the results obtained with the standards stored at room temperature and the feeling that the color fading was due to light, duplicate sets were carried out at room temperature in the dark and

Methylene blue reduction for rapid inplant detection of coolant breakdown. H. W. Rossmoore.

in the laboratory light. Although no significant differences were noticed, it is recommended that the test system not be exposed to direct light.

7. The addition of iron: Up to 3 parts in 100 of iron fine was added to duplicate systems without significantly affecting reduction results.

8. The addition of tramp oil: Up to 25% by volume of oil was added to duplicate systems. While the presence of oil did extract methylene blue from unused coolant, it did not significantly affect reduction times in contaminated coolant.

9. Order of Reagent Addition: In order to eliminate as many variables as possible we recommend the following order of mixing of test components.

One methylene blue isothiocyanate (8.8 mgmftablet) tablet is dissolved in 200m! of distilled water; 60% sodium lactate (USP) diluted to 10% in distilled water. The methylene blue and sodium lactate solutions were mixed in the ratio 1 :1 and stored in the cold in an amber glass bottle.

Four screw cap tubes (Falcon plastic 2027) were labeled 2, 4, 6, and 8ml., respectively. To each vial was added 4 ml of methylene blue-lactate mixture and then the appropriate amount of coolant was intro­duced. The two ingredients were mixed gently and then buffer (M/15 Na2HP04) was added to the very top of the vial. The cover was secured tightly and the contents were gently mixed by inverting several times. Results were read after 3, and 6 hours. The following key (Table 1) is presented to indicate the quantitation that was used in evaluating reduction.

Table !-Recommended Key for Evaluating Methylene

Blue Reduction in Oil/Water Cutting Fluids.

Size of Sample in ml Time of Reduction

8 6 4 2 3 hours 6 hours

R R R R S+ S+

R R R N 4+ 3+

R R N N 3+ 2+

R N N N 2+ I+

N N N N

R = 100% reduction

N = less than 50% reduction

149

10. There is no doubt that increase in bacterial viable count parallels a decrease in methylene blue reduction time (Table 2). However, this parallelism was not always so regular a phenomenon indicating that the relationship between counts and reduction is a multifaceted one.

Table 2-The Relationship of Total Aerobic Count to

Methylene Blue Reduction time in Soluble Oil.

Viable Bacterial Max% Countfml %Reduction Time (min.)

7 X JOB 100 15

3.5 X 108 100 30

1.8 X JOB 100 60

9 X 107 100 75

4.5 X 107 45 120

0 0 120 (7 X JOB

heat-killed)

Part Two

Of major interest in any test system geared to co-relatability are the actual results carried out on site. Over 100 samples from more than 20 central systems were run in this series. The results indicated that a very high degree of repeatability and co-relatability existed. The reduction times were fastest in those systems that were rated the poorest by plant personnel. Since we have not yet arrived at quantitation, it is difficult to set the percentage of co-relatability. In Table 3 you can see a selection of results from five central systems for a three week period. No attempt had been made to confer previously with plant person­nel on the relative incanings of their subjective evaluations. However, a better clue to the condition of the fiuid is the regularity of biocide addition. This was done sometimes with recommendation of the central bacteriology laboratory and sometimes on the subjective evaluation of the operator. The adding of an appropriate biocide at the appropriate time (prior to deterioration) would be the most rational deterrent to premature cutting fluid disposal. We have used methylene blue reduction to evaluate the biocides used in the plant (Table 4). Even at levels four .to eight times those used in the plant neither inhibitor does spectacularly in lowering biological activity. This is a brief for a reasonable program of control although it does not in itself demonstrate the effective­ness of the methylene blue reduction test.

Methylene blue reduction for rapid in plant detection of coolant breakdown. H. W. Rossmoore.

Table 3-Correlation of Methylene Blue Reduction, Bacterial Count and on Site Evaluation

of Cutting Fluid Quality.

Week I

Size in Methy. Bact. Germicide Operator Gallons Blue Count Addition Appraisal

35,000 2+ 15 X J06 Day Insol. Prior High

21,000 - 48 X 106 2 weeks High Prior Insol.

60,000 4+ 65 X J06 4 days Fair-Prior poor

High In sol.

36,000 - 28 X 106 I week Good Prior

26,000 3+ 41 X 106 3weeks Poor Prior

Part Three

Week2

Methy. Bact. Germicide Ope rat or raisal Blue

3+

-

4+

+

4+

Count Addition App

J06 5 days

2.5 X J06 3 weeks Prior

3 X J06 6 days Prior

16 X J06 I week Prior

32 X J06 4weeks Prior

I Fair nsol.

High

Fair High nsol. I

Fair­poor

Fair

Fair­poor

Week 3

Methy. Bact. Germicide Op era tor raisal Blue

4+

2+

4+

2+

3+

Count Addition App

I X 104

5 X 106

I X 104

2 X J06

2.6 X 107

6 days Prior

4weeks Prior

4 days Prior

I day Prior

5 weeks Prior

Fair

Fair

Fair

Good

Fair­poor

Although we had recommended that a 4-tube test be used and that at least two readings (e.g. 3 and 6 hour) be taken during the 8 hour day, there was a reluctance on the part of management to increase

either work load or work force. As a result, the procedure adapted did not reflect the soundest possible approach for evaluating a new test. For the most part, readings were made at 5 hours and 20 hours, (the next day), with only the 5 hour reading on day 5 (Friday).

Table 4-Biocide Selection Using M~thylene Blue Reduction.

Sample

I. From plant (untreated)

2. With 100 ppm 0-phenylphenol

3. With 2000 ppm 0-phenylphenol

4. With 1000 ppm dichlorophenol

5. With 2000 ppm dichlorophenol

Size of Central System 20,000 gal

0-phenylphenol added: 350 ppm 4 day prior

% Methylene Blue Reduction

3 hrs 100

100

0

100

50

6 hrs 100

100

50

100

50

Bacterial Countfml

1.6 X lOB

71. X 107

3.6 X !OS

3.4 X 107

4.6 X 107

Bacterial counts taken immediately on sample from plant; No. 2, 3, 4, 5 taken after 6 hour contact with biocide.

!50

Methylene blue reduction for rapid inplant detection of coolant breakdown. H. W. Rossmoore.

Nevertheless, it was possible to monitor change in each system (Table 5 and 6). In system No. I (Table 5) in week I between day 3 and 4 there was a change in reduction time from 20 hours No Reduction to 20 hours Reduction. At the same time, the condition of coolant according to the operator was severe enough

to require the addition of 500 ppm o-phenyphenol on day 4. The report from the Bacteriology laboratory based on the samples of day I and day 3 did not arrive until day 2, week 2, and stated "system bears watching." Needless to say, this advice was a little late.

Table 5---Inplant Evaluation of Methylene Blue Reduction for Cutting Fluid Maintenance-Central System No. 1.

. On Site

Week Day Methylene Blue Reduction!

I I 20 hours N. R. 2 20 hours N. R. 3 20 hours N. R. 4 5 hours N. R.- 20 hours R 52 5 hours N. R.

2 I 20 hours N. R. 2 5 hours N. R. - 20 hours R 3 5 hours N. R. 4 Holiday 5 5 hours N. R.

3 I 20 hours N. R. 2 20 hours N. R. 3 20 hours N. R. 4 20 hours N. R. 5 5 hours N. R.

4 I 20 hours N. R. 2 20 hours N. R.

5 I 20 hours N. R. 2 5 hours N. R., 20 hours R 3 5 hours N. R., 20 hours R 4 5 hours N. R., 20 hours R 5 5 hours N. R.

lR = 100% Reduction of Methylene Blue NR = No reduction of Methylene Blue

Central Bacteriology Laboratory

Total Sulfate Count Reduction

3 X !()6 none

43 X !06 none

24 X 104 none

13 X JQ6 none

2.8 X JQ6 none

3.6 X !06 none

4.5 X !06 none 2.8 X !06 none

. 6 X 106 none

II X !()6 none

1.7 X !06 none

2Week I, Day !-No recommendation from Bacteriology Iab-"bears watching" Day 2-No recommendation from Bacteriology Iab-"bears watching"

Both reports arrived Day I, Week 2. Operator added 50# 0-phenylphenol Day 5, Week I.

The final series of experiments were related to explaining some reasons for the lack of correlation between viable count and reduction. We replaced the spoiled emulsion (Table 7) with phosphate buffer, fresh emulsion and pasteurized spoiled emulsion. Both

!51

the original suspension and the pasteurized suspension had faster reduction times than buffer or fresh emul­sion. This suggests that a deteriorated millieu could contribute to the metabolic pool, thereby shortening reduction time.

Methylene blue reduction for rapid in plant detection of coolant breakdown. H. W. Rossmoore.

Table 6--lnplant Evaluation of Methylene Blue Reduction for Cutting Fluid Maintenance-Central System No. 2.

Total Sulfate Week Day Methylene Blue Count Reduction

1 1 5 hours N. R., 20 hours R 20 X 1()6 trace 2 4 hours R 3 5 hours N. R., 20 hours R 20 X 106 none 4 3 hours R 5 5 hours N. R.

2 1 5 hours N. R., 20 hours R 9 X 106 none 2 5 hours N. R., 20 hours R 3 5 hours N. R. 30 X 1()6 none 4 holiday 5 5 hours N. R.

3 I 5 hours N. R., 20 hours R 28 X 106 none 2 5 hours N. R., 20 hours R 3 5 hours N. R., 20 hours R 53 X 106 none 4 5 hours N. R., 20 hours R 5 5 hours N. R.

4 1 5 hours N. R., 20 hours R 36 X 106 trace 2 5 hours N. R., 20 hours R 32 X 106 none

5 1 5 hours N. R., 20 hours R 35 X 106 none 2 5 hours N. R., 20 hours R 3 5 hours N. R., 20 hours R 35 X 106 none 4 5 hours N. R., 20 hours R 5 5 hours N. R.

System dumped, cleaned and recharged Day 6, Week 1

Table 7-The Effect of Suspending Medium on Methylene Blue Reduction Time.

Viable Bacterial pH7 Emulsion Culture Pasteurized Countfml Fresh 5% oil/water Phosphate Buffer Supernatant Culture

Max. Time Max. Time Max. Time Max. Time Red. Min. Red. Min. Red. Min. Red. Min.

1.5 X 109 100 60 100 45 100 30 100 45

7.5 X lOB 65 105 60 105 100 75 100 60

3.8 X lOB 35 105 35 105 75 90 100 120

1.9 X JOB 35 105 25 120 50 90 50 120

9.5 X 107 15 105 5 150 25 90 30 120

0 5 150 5 150 5 150 5 150

152

r

I '

r

I I r

l

Methylene blue reduction for rapid in plant detection of coolant breakdown. H. W. Rossmoore.

Table 8-The Effect of Cell Lysis on Methylene Blue Reduction of Pseudomonas oleovorans ATCC 806~.

Whole Cell Time for Viable count/ml 100% Reduction

8 X JOIO spontaneous

4 X 1010 spontaneous

2 X JOIO spontaneous

5 X 109 5 min.

2.5 X !09 10 min.

1.3 X !09 30 min.

6.5 X JOB 60min.

3.3 X 10B 105 min.

1.7 X JOB 210 min.

Finally, (Table 8 and 9) we looked at the effects of cell lysis on reduction time in Ps. oleovorans and D. desulfuricans. It is readily seen that destruction of cell integrity did not destroy enzymatic activity associated with methylene blue reduction, suggesting that total viable cell population might not realistically correspond to reduction activity.

Table 9-The Effect of Cell Lysis on Methylene Blue

Reduction of Desu/fovibrio desulfuricans 7757.

Time for 100% Reactants Reduction

1. Methylene blue & lactate None

2. Methylene blue & lactate & pasteurized cell suspension None

3. Methylene blue & lactate & unheated cells 2 min.

4. Methylene blue & lactate & lysed cells 2 min.

5. Methylene blue & lactate & supernatant from #4 4 min.

6. Methylene blue & lactate & pellet from #5 None

!53

Remaining Viable Time for Count/ml after lysis 100% Reduction

5 X 107 spontaneous

2.5 X 107 spontaneous

1.3 X 107 5 min.

6.5 X 1()6 10 min.

3.3 X 106 45 min.

1.7 X 106 90 min.

8.5 X !OS 210 min.

Summary and Conclusion

Although there is a certain degree of positive correlation between extent of reduction and viable cell population within each cutting fluid system, it was readily apparent that it was not possible to make any extrapolations from one system to another. For example, 3+ reduction might be equivalent to 20 million cells/ml in one place and 200 million cells/ml in another. In addition, quite often these were a drastic drop in total viable bacteria without a concomitant drop in reduction time. As a rule this inconsistency followed after treatment of the system with one of several biocides. However, despite this drop in count more often than not the operator agreed with the methylene blue reduction level as evidenced by the subjective evaluations we received. In the laboratory it is much easier to control many of the extraneous factors that interfere with perfect biological correla­tion. Nevertheless, the important fact to be noted is that the operator could have much closer control over the rational addition of biocide and the subsequent prevention of dumping if he utilized the results of the methylene blue reduction test rather than rely on either his own organoleptic evaluation or that of a distant (in time and space) bacteriology laboratory. Although it seemed incongruous for total bacterial levels to drop without affecting reduction time, results in our laboratory showed Ps. o/eovorans was killed by freezing and thawing without an equivalent lengthening of methylene blue reduction time. At the time this study was conducted, the biocides used on site were all phenol derivatives. Hugo (1956) has demonstrated that one mode of action of these

Methylene blue reduction for rapid inplant detection of coolant breakdown. H. W. Rossmoore.

compounds when used at low concentrations is to cause membrane disruption. Thus, it is entirely possible that loss in viability due to membrane destruction did not completely impair metabolic activity. In fact, you will note that the addition of those biocides to the same cutting fluids samples not only did not lengthen reduction time, but actually permitted cell growth in their presence over a 24 hour incubation period. There is another dye reduction test (Hill eta!., 1967) which is more directly correlated with viable cell population. This involves the incorporation of a tetrazolium salt in a nutrient agar medium. To the medium in a petri dish is added between 0.05 and 0.1 ml of suspect cutting fluid. After an overnight incubation period, heavily infected fluids will produce a red colored spot of growth. Eventually most levels of contamination will reduce to a red color. However, I believe even this test must be checked with each fluid since the breakpoint microbial load will vary from one system to the next. The value of dye reduction not only is due to its simplicity and rapidity but in the main in its use as a prognostic tool. That is to ascertain the norms for a particular system and to take proper preventive measures if there is a change for the worse in reduction time. Unfortunately, while this test was being evaluated in a large plant, the personnel were either indifferent or cavalier about interpreting the results during the trial period (Table 5 & 6). Thus, methylene blue reduction lasted only 5 weeks as a viable test procedure. This fact points out the necessity of getting complete cooperation from all levels of individuals if a new procedure is being examined. In the final analysis, prevention of cutting fluid deteriorations is a function of this kind of total cooperation.

154

References

American Public Health Association. (1960). Standard methods for the examination of water and waste water. II th ed. New York.

American Public Health Association. (1967). Standard methods for the examination of dairy products. 12th ed. New York.

Hill, E. C.; Davies, I.; Pritchard, J. and Bryom, D. (1967). The estimation of microorganisms in petroleum products. J. Inst. Petrol. 53: 275-279.

Hugo, W. B. (1956). The action of phenol and 2-phenoxyethanol on the oxidation of various sub­stances by Escherichia coli and by a disrupted cell preparation of the organism. J. Gen. Microbial. 15: 315-323.

Pivnick, H. and Fabian, F. W. (1953). Methods for testing the germicidal value of chemical compounds for disinfecting soluble oil emulsions. Appl. Micro­bioi. 1: 204-207.

Rossmoore, H. W. and Brazin, J. G., (1968). Control of cutting oil deterioration with gamma radiation. Biodeterioration of Materials: Microbiological and Allied Aspects pp. 386401. Proc. 1st int. Biodetn Symp. Elsevier Pub. Co. Ltd., England.

Rossmoore, H. W., Shearer, M. E. and Schearer, C. (1964). Growth studies on Desulfovibrio desulfuricans Devel. ind. Microbial. 5: 334-342.

Senez, J. C. and Azoulay, E. (1961). Des hydrogenation d'hydrocarbures paraffiniques par les suspensions non-proliferantes et les extraits de Pseudomonas aeruginosa. Biochem. Biophys. Acta 47: 307-316.

I 1

j

i

i· i

I

r

I I I I

I l !

Zyska, B. J.; Fudalej, P. S. & Rytych, B. J. Int. Biodetn Bull. 7 (4) 155-159, (1971). Model tests on the mechanisms of microbial deterioration of filled vulcanizates. I. Influence of Pseudomonas sp. on the system paraffin oil•carbon black.

MODEL TESTS ON THE MECHANISMS OF MICROBIAL DETERIORATION OF FILLED VVLCANIZATES.

1. INFLUENCE OF PSEUDOMONASSP. ON THE SYSTEM PARAFFIN OIL-CARBON BLACK.

B. J. Zyska,t P. S. Fudalejl and B. J. Rytychl

Summary. carbon black suspension in paraffin oil was used in investigation on the mechanisms of microbial degradation of filled vulcanizates. Using electrical conductivity technique the kinetics of the structure formation and the thixotropic pheno­mena were examined. The value of the maximwn specific conductivity for samples inoculated with pseudomonads shows a continuous drop, reduced after a period of 75 days to about 40%. Measurements of the thixotropic properties of the system paraffin oil-carbon black indicate that all the samples retain their ability to regenerate the structure after removing the mechanical stimulus, but there are significant differences in the values between the control and samples influenced by the bacteria.

Essais de rnaquette appliques nux mecani.smes de Ia degradation rnicrobiaJe des vulcanises remplls. L'influence des Pseudomonas sp. sur le systeme bnile de paraffine-nair de carbone. Une suspension de nair de carbone dans I'huile de paraffine s'employ­ait dans !'investigation sur les mecanismes de Ia dCgr.adation rnicrobiale des vulcanises remplis. Utilisant Ia technique de Ia conductibilite Clectrique, on exarninait les Clements cm·etiques de Ia formation de Ia structure et les phCnomCnes thixotropiques. On decouvrit que Ia valeur de Ia conductibilit6 spOCifique maximum dans les exemples inocules des pseudomonades montrait une descente continue, rCduite aprCs une pCriode de 75 jours a 40% environ. Les rnesurages des elements thixotro­piques du systCme huile de paraffine-noir de carbone indiquent que tous Jes exemples retiennent son pouvoir de regenerer Ia structure, apres s•etre enleve Je stimulant mecanique. mais il y a des differences irnportantes entre les valeurs du contr6le et des exemples influences par les bacteries.

Introduction

Knowledge of the mechanism of microbial deteriora­tion of rubber has lagged far behind that of other classes of compounds such as carbohydrates. Certain investigations on this subject have been reviewed in the past (Blahnik and Zanova, 1963; Blake and Kitchin, 1949; Blake, Kitchin and Pratt, 1955; Heap and Morrell, 1968; Leeflang, 1963; MacLachlan, Heap and Pacitti, 1966; Rock, 1955; Rytych, 1969; Schwartz, 1963). The mechanisms involved in micro­bial rubber deterioration have hitherto been tested on specimens of compounded vulcanizates causing diffi­culties in the interpretation of results.

To improve the commercial properties of vulcanized rubber several inorganic and organic additives are incorporated during processing. Among the principal

Modellversuche auf den Mechanismus dcr mikrobialischen Verschlecbterung in den gcfiillten Vulkanisierten. Der Einftuss der P.seudomonaden sp. auf die Anordnung "ParaffinOI-Russ.'' Eine Russ-Suspension in ParaffintH wurde in einem Experiment auf den Mechanismus der mikrobialischen Verschlechterung in den fegiillten Vulkanisierten gebraucht. Mit dem Gebrauch, der Stromlcitungsfahigkeit-Technik, wurden die kinetischcn Elementen des Anordnungsaufbau und die thixotropiken Erscheinungen untersucht. Der Wert der hOchsten spezifischen Leitfahigkeit in den mit Pseudomonaden geimpften Mustern zeigt einen ununterbrochenen Fall, der sich nach ciner Zeitspanne von 75 Tagen zu ungefiihr 40% vermindert. Beim vermessen der thixotropiken Elementen des Systemes ParaffinOI-Russ wird gezeigt, dass aile Muster ihre FB.higkeit behalten, nach der Entfernung des mechanistischen Antrieb, den Aufbau wiederzubilden; aber es gibt zwischen der Kontrolle und den von den Bakterien beeinftussten Mustern bedeutende Wertsun­terschiede.

Pruebas modclos sobre los mecanismos de deterioraciOn rnlcrOblca de los vulcanizatos llenados. lnflujo del Pseudomonas sp. sobre el sistema del aceite de parafina.carbOn negro. Una suspensi6n del carb6n negro en el aceite de parafina se emple6 indagando los mecanismos de Ia degrad.aci6n rnicr6bica de los vulcanizatos llenadcs. Empleando Ia tecnica de Ia conductividad elb:trica se examin6 Ia cinCtica de Ia formaci6n estructunil y los fen6menos thixotr6picos. El valor de Ia conductividad especifica mAxima de los muestras inoculadas con los pseudomonads indica una caida continua, reducida despues de un periodo de 75 dias a unos 40%. Las rnedidas de las propiedades de thixotropicas del sistema aceite de parafina-negro de carb6n indican que tod.as las muestras conservan su capacidad de regenerar Ia estructura despues de apartado el estimulo rnecAnico, pero hay diferencias significantes en los valores entre las muestras inftuidas por los bacterios y las rnuestras de mando.

additives are reinforcing agents, mainly carbon black. The influence of reinforcing agents on the susceptibility of vulcanizates to the attack by microorganisms has not been investigated. Petrujova and Zanova (1960) and Ritzinger (1959) tested the ability of certain microorganisms to grow in mineral salts agar medium on some of the materials used in compounding rubber, but not the ability of microorganisms to attack vulcanized rubber with added fillers.

The mechanisms of the property enhancement and control obtained by the use of fillers in rubber vulcani­zates is only approximately known due to the complica­ted interaction between the elastomer and the filler. To simplify this problem a great amount of work has been done by the investigation of models which gave a better knowledge of some aspects of the carbon black-elastomer interaction. One of the models used

!Central Mining Institute, Plac Gwark6w I, Katowice, Poland.

(Copy received April 1971).

!55

Model tests on the mechanisms of microbial deterioration of filled vulcanizates. 1. Influence of Pseudomonas sp. on the system paraffin oil-carbon black. B. J. Zyska, P. S. Fudalej and B. J. Rytych.

was a carbon black suspension in paraffin oil (Lipatov, 1967; Voyutskii et a/.,1951; U-Shu -Tsyu eta/., 1956; Yampolskii et a/., 1962 a, b) that made possible a study of the formation of spatial carbon black struc­tures, similar to the structures formed during the interaction of the elastomer and carbon black. The kinetics of the structure formation and the thixotropic phenomena were examined using electrical conductivity techniques. The phenomenon of the structures (Lipatov, 1967) is of great importance in the interpre­tation of the function of the filler in the reinforcement of the elastomer properties.

The model using paraffin oil-carbon black appears to be promising for investigating the mechanisms of the microbial deterioration at the boundary filler­elastomer phase. In the relevant literature the degrada­tion of paraffin oil by bacteria and fungi has been reported several times (Davis, 1967; Malek and Schwartz, 1966; Mavrina, 1966; Traxler and Flannery, 1968). The paraffinolytic bacteria used in our investiga­tions are pseudomonad strains.

This work reports investigations on the influence of these Pseudomonas sp. strains on the order of the structure in a paraffin oil-carbon black model and the changes in thixotropic properties.

Experimental

Erlenmayer flasks of 100 ml capacity, each contain­ing 40 ml of Bushnell-Haas medium (Bushnell and Haas, 1941), were sterilized at 121'C for 0.5 hrs. A paraffin oil-carbon black suspension (V oyutskii et a/., 1951; U-Shu -Tsyu et a/., 1956), containing 10 parts by weight channel carbon black SAO (manufac­turer Carbochem, Gliwice, Poland, specific surface 90.88 m2fg evaluated in argon by the BET method) in I 00 parts by weight of paraffin oil was prepared and sterilized separately under the same conditions. Ten g. of cooled suspension was added to each Erlenmayer flask under aseptic conditions. The sterile flasks containing the medium and the suspension were each inoculated with 0.5 ml of a 14 day culture and shaken at 30'C for up to 75 days. Strains of Pseudomonas sp. 35, 37, 105 and Ill, obtained from the Petroleum Institute in Cracov, Poland, were used in these tests. Bottles containing only medium and suspension were set up as controls under the same conditions.

On removal from the flasks after asettimethesuspen­sion was placed in the Petri dishes and dried at 50'C for 48 hrs in air and in a vacuum drying oven at 50'C and 60 mm Hg until constant weight was attained. The process was defined as finished when consecutive weighings showed no differences. The specific conduct­ivity !.. (ohm-'cm-1) of the paraffin oil-carbon black suspension was measured by the compensation method using Weatstone-Thomson bridge 0. I%; MWT 77 a (manufacturer ZOMP Polit. Sl, Gliwice, Poland).

156

The suspension sample was placed in a glass cell I 0 X 18 x 25 mm and I 0 X 25 mm copper electrodes were used. The measurements were made at 30'C. The conductivity constant of the glass cell was measured using 0. 01 n KCL on a Kohlrausch bridge. The suspension was poured into the glass cell and left until the measured specific conductivity showed no further change. The highest level of specific conduct­ivity found was taken as the maximum specific conductivity !.. m.

In order to examine the changes of thixotropic properties of the system the balance was disturbed by stirring. The increments in specific conductivity were measured during the following 60 minutes and repeated similarly after a second disturbance of the system during the same period. The choice of 60 minutes as a measurement time unit, during which the increase of the specific conductivity was measured, is a matter of convention. This period appears to be a sufficient measure for changes in the regeneration properties of the investigated structures. The per­formance of the full kinetics seems to be aimless, because the achieved values of the specific conductivity in the state of balance, are given in the first series of measurements.

The magnitude of the mechanical stimulus was such that the minimum specific conductivity was 0. 9 X J0-7 ohm-1 cm-1.

Discussion of Results

Taking maximum specific conductivity !..m (Figure I) as a measure of ability of structure formation in the suspension, a comparison of values for samples inoculated with Pseudomonas sp. and the controls clearly indicated the role of the bacteria in deterioration of the material. The value of the maximum specific conductivity !..m shows a continuous drop and after a period of 75 days is reduced to about 40%. This is evidence of the decrease of structural properties in the used filler carbon black SAO. According to La Riviere (1955), during the deterioration of paraffin oil pseudomonads are able to form, among other com­pounds, high molecular surface-active organic acids. It is possible that these acids may react with carbon black and be adsorbed on its surface. Contact between the particles of carbon black is then more difficult, resulting in the decrease of the maximum specific conductivity !..m of the system. Similar results were obtained by U-Shu-Tsyn et a/. (1956) when a surface­active compound such as oleic acid was added to a suspension of carbon black in oil.

Figure I shows the relation between the maximum specific conductivity and the period of the influence of Pseudomonas sp. It may be seen that the maximum specific conductivity !.. m decreases in the first stage very quickly and is followed by slight changes for as

Model tests on the mechanisms of microbial deterioration of filled vulcanizates. I. Influence of Pseudomonas sp. on the system paraffin oil•carbon black. B. J. Zyska, P. S. Fudalej and B. J. Rytych.

long as measurements were made. The changes of the maximum specific conductivity may be taken as a measure of the deterioration rate of paraffin oil by the strains used.

20 80 TIME, days

Figure 1. Effect of Pseudomonas sp. strains on the maximum specific conductivity of the paraffin oil­carbon black suspension

x- control; t;. - strain 35;

0- strain 37; 0- strain III;

Measurements of the thixotropic properties of the paraffin oil-carbon black system shown in fig. 2-4 indicate, that despite the action of Pseudomonas sp. all the samples retain their ability to regenerate the structure after removal of the mechanical stimilus. There are however significant diffierences in the values found for specific conductivity at a given mo­ment between the control and samples influenced by the bacteria. The values of the instantaneous specific conductivity in the control are much higher than in the deteriorated samples. The results obtained in deteriorated samples give evidence of the decrease in regenerative ability of the spatial structure formed. It may be concluded that the decrease in the structure­forming ability of these system has the effect of decreasing the reinforcement effect obtained through the use of filler in the rubber vulcanizates.

157

It was indicated in the introduction, that the paraffin oil-carbon black suspension approximately represents the model of interaction between carbon black and rubber. Hence the results obtained provide a basis for concluding the microbiological degradation causes not only degradation of chains responsible for trans­mission of stresses but also influences the ability for interaction between filler and rubber. The decrease in the number of effective crosslinkings responsible for transmission of stresses and the drop in the inter­action of filler and rubber together form the total attenuating effect in the properties of the rubber. A better knowledge of the mechanisms involved in microbiological degradation of the system requires further tests on the changes appearing on the polymer­filler boundary phase.

QgL---~----4~0~--~~~--~2~0--~~--~~

TIME, min

Figure 2. Change in instantaneous specific conduct­ivity of the paraffin oil-carbon black suspension after 12 days action of Pseudomonas sp.

X- control; 0- strain III; I - first set of measurements 2 - second set of measurements

Model tests on the mechanisms of microbial deterioration of filled vulcanizates. 1. Influence of Pseudomonas sp. on the system paraffin oil-carbon black. B. J. Zyska, P. S. Fudalej and B. J. Rytych.

1,7

1 I 2 I I I

/T * X I x/1

/ I / I X I I i X I

1 :I i

IX I I I X :t I

I

~L----2~0--~40,---~~n---~20,---4t0~--6~0~ TIME, min

Figure 3. Change in instantaneous specific conduct­ivity of the paraffin oil-carbon black suspension after 43 days of Pseudomonas sp.

X -control; 6 -strain 35;

D- strain 37; 0 - strain lll; I - first set of measurements

2 - second set of measurements

References

Blahnik, R. and Zanova, V. (1963). Mikrobialni koroze. SNTL, Praha, 222 pp.

Blake, J. T. and Kitchin, D. W. (1949). Effect of soil microorganisms on rubber insulation. Ind. Engng Chern. 41 (8), 1633-1641.

Blake, J. T., Kitchin, D. W. and Pratt, 0. S. (1955). The microbiological deterioration of rubber insula­tion. Appl. Microbial. 3 (!), 35-39.

Bushnell, L. D. and Haas, H. F. (1941). The utilization of certain hydrocarbons by microorganisms. J. Bact. 41, 653-673.

!58

~r---.---.---~---.---,---.-,

?1 X I 2/f I

j

I /X

!

1,1

I

I

60 TIME, min

Figure 4. Change in instantaneous specific conduct­ivity of the paraffin oil-carbon black suspension after 75 days action of Pseudomonas sp.

x - control; 6 -strain 35;

D-strain37; o-strain 111; e-strain 105; I - first set of measurements

2 - second set of measurements

Davis, J. B. (1967). Petroleum microbiology. Elsevier, Amsterdam-Loudou-N. York, 604 pp.

Heap, W. M. and Morrell, S. H. (1968). Microbiologi­cal deterioration of rubbers and plastics. J. appl. Chern. 18 (7), 189-194.

La Riviere, J. W. M. (1955). The production of surface active compounds by microorganisms and its possible significance in oil recovery. I. Some general observations on the change of surface tension in microbial cultures. Antonie van Leeuwen· hoek, J. Microbial. Sera/. 21, 1-8.

Leellang, K. W. H. (1963). Microbiologic degradation of rubber. J. Am. Wat. Wks. Ass. 55 (12), 1523-35.

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.I I

i•

I Model tests on the mechanisms of microbial deterioration of filled vulcanizates. 1. Influence of Pseudomonas sp. on the system paraffin oil-carbon black. B. J. Zyska, P. S. Fudalej and B. J. Rytych.

Lipatov, Yu.S. (1967). Fiziko-Khimiya napolnennykh polimerov. Izd. "Naukova Dumka", Kiev, 232 pp.

MacLachlan, J., Heap, W. M. and Pacitti, J. (1966). Attack of bacteria and fungi on rubbers and plastics in the tropics. In: Microbiological deterioration in the tropics. S.C./. Monograph No. 23, London, 185-200.

Malek, I. und Schwartz, W. Heraugeber, (1966). Vortriige des internationalen Symposiums "Edrol­mikrobiologie". Abhandl. dt. Akad. Wiss. Berlin, Klasse fur Chern., Geo/. u. Bioi. Berlin Akademie­Verlag, 1965, Nr. 2, 240 pp.

Mavrina, L. A. (1966). Okislenie uglevodorodov mikroorganizmami. Trudy Moskovskogo Obshestra Ispitateli Prirody, XXIV, 192-202.

Petrujova, A. and Zanova, V. (1960). Soviet Rubb. Techno/. 19 (2), 16.

Ritzinger, G. B. (1959). Rubb. Plast. Age 40, 1067. Rook, J. J. (1955). Microbiological deterioration of

vulcanized rubber. Appl. Microbial. 3 (5), 302-309. Rytych, B. J. (1969). Fungicides and bactericides for

use in rubber. Int. Biodetn Bull. 5 (1), 3-8. Schwartz, A. (1963). Mikrobielle Korrosion von

Kunststoffen und ihren Bestandteilen. Abhandl. dt. Akad. Wiss. Berlin, Klasse fiir Chern, Geol., u. Bioi. Berlin Akademie-Verlag, Nr. 5, 138 pp.

Traxler, R. W. and Flannery, W. L. (1968). Mechan­isms of hydrocarbon degradation. In: Biodeteriora­tion of Materials. Walters, A. H. and Elphick, J. J. (Editors), Elsevier, Amsterdam-Loudou-N. York, pp. 44-54.

U Shu-Tsyu, Yampolskii, B. Ya., and Voyutskii S. S. (1956). Issledovanie struktur v suspenziakh sazhi. 2. Vliyanie temperatury i dobavok poverkhno­stno-aktivnykh veshchestv na strukturirovanie sazhevy)ch suspenzii. Ko/1. Zh. XVII (6), 748-754.

Voyutskii, S. S., Zaionchkovski, A. D., Kargin, V. A. and Rubina, S. I. (1951). Issledovanie struktur v suspenziakh sazhi. 1. Vlyanie skorosti techeniya, vremeni, temperatury i kotsentratsii sazhi na strukturu sazhevykh suspenzii. Ko/1. Zh. XIll (5), 333-338.

!59

Yampolskii, B.Ya., U Shu-Tsyu and Akademik Rebinder, P. A. (1962). 0 mekhanizmie strukturo­obrazovanya v uglevodorodnykh suspenziakh napo­lnitelei kauchukov. 1962 Dok/ady AN SSSR, 142 (3), 633-636.

Yampolskii, B.Ya., U Shu-Tsy\1, (1962). 0 mekhaniz­mie strukturoobrazovanya v suspenziakh sazhi v uglevorodnoi srede. Ko/1. Zh. XJUV,.(3), 348-354.

I

I

I I

·I

Sheridan, J. E. & Nelson, J. Int. Biodetn Bull. 7 (4) 161-162, (1971). The selective isolation of the "kerosene fungus" Cladosporium resinae from the air.

THE SELECTiVE ISOLATION OF THE "KEROSENE FUNGUS" CLADOSPORIUM RESINAE FROM THE AIR

J. E. Sheridan! and Jan Nelson!

Summary. A method is described which selectively isolates the "kerosene fungus" Cladosporium resinae (Lindau) de Vries from the air where it occurs. Three standard type spore traps have proved suitable for monitoring air one of which being light, portable and battery-operated should be ideal for use at oil installations, airports and within aircraft during flight.

L'isolatlon sCiective dans l'air du "kl'!rosene-fongus" Clado­sporium resinae. On decrit une methode qui isote setectivemcnt le "kerosene-fongus" Cladosporium resinae (Lindae) de Vries, de l'air oil il croit. Trois modCles standardises de trappe de spores se sont montres efficaces a faire examiner I" air minutieuse~ ment, dont l'une, etant Iegere, portable et operee par moyen d'une batterie electrique, devrait Ctre idCale pour !'utilisation dans les installations huilieres, les aeroports et dans les avions pendant le vol.

Introduction

The "kerosene fungus" Cladosporium resinae (Lin­dau) de Vries, best known as a contaminant of aviation jet fuel (Hendey, 1964), has recently been shown to be widely distributed in soil (Parbery, 1969; Sheridan, Steel & Knox, 1971). This fungus sporulates profusely on soil contained in Petri dishes, in the presence of creosote, and in pure culture. The spores are readily dislodged by air currents (Harvey, 1970). Further, it has been found in our laboratory that C. resinae can become a nuisance as a contaminant. Apparently spores escape during examination of plates and so become airborne yet there appear to be only four reports of the successful isolation of this fungus from the air (Harvey, 1967; Parbery, 1969). It seems difficult to believe therefore, that the scarcity of reports of the isolation of C. resinae from air bears any relation to the true situation.

Because this fungus grows readily on a wide variety of agar media containing creosote and few other fungi (or bacteria) will tolerate the latter (Sheridan, Steel & Knox, 1971), we experimented with the use of a selective medium containing creosote for isolation of C. resinae. The method described below is a simple one and has proved suitable for isolating C. resinae from the air in New Zealand.

Method

The selective medium is prepared as follows. Forty grams of Davis agar (other brands .should also be suitable) is added to 1500 mi. tap water in a glass

Die sclekth·c Isolienmg von der Loft des uKerosenenschw::tmm" Cladosporium resinae. Man bcschrcibt cine Methode, womit man den "KerosenenschWamm" Cladosporium resinae (Lindae) de Vries von der Luft, wo er wachst, setektivartig isoliert. Drei normaltypischen Sporefalle haben sich als fdhig erwiesen, die Luft zu untersuchen. Weil der Eine Ieicht, tragbar und Batterie­angetrieban ist, sollte er zum Gebrauch bei den Olaulagen, Flughafen und in Flugzcugcn wiihrend des Fluges, ideal sein.

El nislamiento selective del bongo de kcrosina Cladosporium resinae del aire. Se describe un metoda que aisla, del aire donde se halla, de rnanera selectiva el hongo de kerosina Cladosporium resinae (Lindae) de Vries. Tres tipos normales de trampas de esporas se han rnostrado adecuados para examinar el aire, y uno de Cstos, si:ndo ligero, portAtil y funcionando par pita, podrA bien ser perfecto para crnplear el las instalaciones de petr61eo, los aeropuertos y dentro de los aviones en vuelo.

vessel which is heated in a boiling water bath until the agar melts. To this is added 350 mi. of V-8 juice (Campbell's Soups Ltd.). The medium is mixed gently, distributed into medicine flats and autoclaved at 103kNm..2 (15 p.s.i.) for 10 minutes. When the medium has cooled to about 50'C. sterile creosote (sterilized by autoclaving) is added aseptically to give a final concentration of 0.1 %. The concentration is not critical since all strains of the fungus studied by us will tolerate creosote up to I%. However, a lower concentration is . undesirable because the volatile components of the creosote disappear rather rapidly when plates containing the selective medium are exposed to the air.

Plates of this selective medium are exposed in an Air Slit Sampler with a throughput of 18 litres of air per minute, for periods of up to 30 minutes. An ex­posure of less than 30 minutes is uneconomical; a longer exposure,results in growth of contaminants and unreliable results. After exposure plates are incubated at 25'C. for 5 days. Colonies of the fungus can be recognised visually and by the characteristic smell produced on this medium and confirmed by micro­scopic examination.

For continuous monitoring of the air we use a Hirst spore trap, 2 metres above the ground with a throughput of 10 litresfmin., carrying a microscope slide coated with vaseline. When the slide is removed after exposure it is placed into a sterile Petri dish and sterile selective medium at 50'C. is gently poured over it. The preparation is incubated as above, and colonies of C. resinae identified after 5 days (Fig. 1).

!Botany Dept. Victoria University of Wellington, New Zealand. (Copy received April 1971).

161

The selective isolation o f the " kerosene fungus" Cladosporium resinae from the air. J. E. Sheridan and J. Nelson.

Figure 1. Colonies of C. resinae (arrowed) trapped from air, growing on V-8 juice agar containing 0.1 % creosote.

TOP :

BOTTOM :

Personal Dust Sampler (7 days)

Hirst Spo re Trap (5 days).

Because the a mo unt o f air entering the trap and the rate of movement of the slide past the orifice is known, the concentratio n of the fungus in the air and the time at which the spore of C. resinae landed on the slide can be calculated.

For isolation of the fungus from air a t o il installa­t io ns, airports and o n aircraft during flight, trappings are made on a ste rile glass fibre paper in a Personal Dust Sampler. After each run (l-8 hrs.) the paper is removed aseptically and placed inside a sterile dis­posable plastic Universal container for transport to the Laboratory. On arrival it is placed inside a sterile Petri dish, correct side up, and selective medium at 50°C. is poured over it. Incubatio n is as above (Fig. 1).

Discussion

The key factor in the isolation of C. resinae from a1r is the use of a selective medium. V-8 juice agar containing 0.1 % creosote has proved ideal. The acid medium suppresses growth of bacteria and the creosote suppresses growth of most fungi. Certain precautions

162

must be taken to avoid contaminatio n with C. resinae from within the laboratory. Where possible preparation of materials, pouring of media and incubation should be do ne in a place remote from the laboratory where pure cultures of the fungus are being studied. Air in our laborato ry has yielded one spore (based on the assumption that each colo ny a rises from a single spore) per cubic metre. The air above Wellington (at Brooklyn) during a trapping schedule in November and December, 1970, yielded 1 spore per 20 cuM.

Each of the spore traps serves a diiTercnt purpose. The Hirst spore trap is ideal fo r continuous mo nitoring. The Personal Dust Sa mpler shou ld prove useful for monitoring air inside aircraft, at airports, and a t oil installations. The Air Slit Sampler is used as a standard against which the o thers ar\! compared periodically to check on their efficiency.

It is suggested the reasons fo r infrequent isola tio n in the past arc firstly that spores of C. resinae are difficult to distinguish from those of other species and secondly, in the absence of creosote vapour, the presence of C. resinae is masked by the growth of other fungi.

Acknowledgments

We would like to thank Mr. R. Hoverd, Mrs. I. Labonc and Mr. H . Christophers for technical assistance, and the Operations Manager, o f N.A.C. Wellington, for permission to sample at airports and inside aircraft.

References

Hendey, N. l. (1964). So me observations on Cladospor­ium resinae as a fuel contaminant and its possible ro le in the corrosion o f aluminium alloy fuel tanks. Trans. Br. myco/. Soc., 47: 467-475.

Parbery, D. G. (1969). The Natural occurrence of Cladosporium resinae. Trans. Br. mycol. Soc., 53 (1): 15-23.

Sheridan, J. E., Steel, Jan & Knox, M . D . E. (197 1). The natural occurrence of the "kerosene fungus" Amorphotheca resinae in New Zealand soils. N.Z. Jl. Sci .. 14 (1): 147-160.

Harvey, R. ( 1967). Air-spora studies at Cardiff. I. Cladosporium. Trans. Br. myco/. Soc., 50 (3): 479-495.

Harvey, R. (1970). Spore productivity in Cladosporium. Mycopath. Mycol. appl. 41: 251-256.

!I ' I Stewart, C. S. & Walsh, J. H. Int. Biodetn Bull. 7 (4) 163-167, (1971).

A simple technique for estimating microbial activity by total carbon dioxide evolution and its application to the attack of plasticiser-treated cotton yarn.

A SIMPLE TECHNIQUE FOR ESTIMATING MICROBIAL ACTIVITY BY TOTAL CARBON DIOXIDE EVOLUTION AND ITS

APPLICATION TO THE ATTACK OF PLASTICISER-TREATED COTTON YARN

C. S. Stewart! and J. H. Walsh2

Summary. A modification of a simple method for measuring C02, evolution by fungi is described. The level of replication obtained compares favourably with traditional respirometric methods. The application of the method to studies of the cellulolytic activity of fungi, and fungal growth on plasticiser treated cotton yarn is assessed. Results suggest that the main application of the method might be in screening a range of substrates for their ability to suport growth of one or a few test organisms.

Une technlque simple pour estimer l'activit6 miaobiale par moyen do dlh'eloppcment totnle du carbone dioxide, et son application a l'attaque au fil de coton plasticis6. Description d·une modification d'une methode simple pour mesurer revolu­tion du C0 2 produit par les fongus. Le niveau de replication obtenu est favorable par comparaison a celui obtenu par les methodes traditionnelles respiromCtriques. L'application de Ia methode aux etudes de l'activitC cellulolytique des fongus, et des excroissances sur le fil de eaton plasticisC, est evatuee. Les rCsultats suggC:rent que l'application principale de Ia methode pourrait consister 3. sCparer une serie d'CICments qui peuvent supporter l'excroissance d'un, ou de plusieurs organismes d'Cpreuve.

Introduction

Various methods have been designed to assess fungal growth or activity by measuring the accompanying oxygen absorption or carbon dioxide e\;'olution. These methods have been of particular use where it is not possible to separate fungal mycelium from the substrate, as in soil, wood and other solids. Early workers (Heukelekian & Waksman, 1925; Dubos, 1928) measured carbon dioxide evolved from soils by chemical absorption and titration, but the Warburg manometer has since provided a more convenient method for measuring rates of gas utilisation or evolution. Originally limited to short term measure­ments as, for example, in the studies of Bunt & Rovira (1955), the technique was modified to measure rates over a period of about 2 weeks by Chase & Grey (1957). A differential manometer measuring total oxygen uptake over several days was used to follow the growth of fungi on cotton (Siu & Mandels, 1950) and on plastic film (Burgess & Darby, 1964, 1965). Finally direct monitoring of carbon dioxide evolution

Einc einfache Technik fiir das Vermessen der mikrobialischen Tiitigkeit, mlttels der ganzen Entwicklung des Knrbondioxyd und seines Gebrauches gegen den AngrifT auf den plastifizierten Baumwollgam. Eine Modifizierung einer einfachen Methode fUr das Vennessen des durch Schwamm hervorgebrachten C02 wird beschrieben. Die entstehenden Entwicklungen zeigen sich erfolgreicher, als bei den traditionellen Atmungsmethoden. Der Gebrauch dieser Methode auf die Studicn der cellulolytischen Tiitigkeit der Schwammen, und der schwammigen Gewiichses auf plastifizierten Baumwol1garn, wird veranlagt. Die Ergebnisse deuten darauf, dass diese Methode am rneistcn gebraucht kl:Snnte, urn eine Reihe von Elementen zu enthOilen, die das Gewiichs eines oder mehrercr Probe-organismen tragcn konntcn.

Una tecnica scncilla pam calcular Ia actividad micrObica por Ia evoluciOn total del dloxido de carbOn y su empleo pam el ataque del hilo de algod6n tmtndo con un plasticcr. Se describe una modificaci6n de un metoda sencillo de medir Ia evoluci6n del C02 por los bongos. El nivcl del de Ia replicaci6n asi obtenida se puede comparar frivorablernente con los metodos respirometricos tradicionales. Se evalUa Ia aplicaci6n del mCtodo a los estudios de Ia actividad celulolitica de los bongos, y tambien el crecimicnto de los bongos en el hilo de a1god6n tratado con un plasticiser. Los resultados sugieren que Ia aplicaci6n principal del metodo bien pudiera haUarse en cribar una variedad de substrates respecto de su habilidad en fomentar el crecimiento de un organismo de prueba o o mAs.

by a 'conductivity cell' was described by Smith (1967) and used by Good & Darrah (1967) in a study of wood decay fungi.

All of these methods require rather specialised apparatus which makes them unsuitable for large numbers of replicate tests. Largely because of this Kaplan (1964), in a review, concluded that traditional respirometric techniques were not suitable for large scale rapid screening of deterioration of materials. To overcome these problems Kibble (1966) reverted to chemical absorption and titration of evolved carbon dioxide in a very simple apparatus, which could be assembled in large numbers.

The present paper describes a modified version of this technique, together with results obtained in a test of replicability. The paper also includes results obtained using this technique in an attempt to discover the mechanism of the effect of plasticisers on the fungal attack of cotton yarn described by Stewart & Walsh (1971).

!Present address: Dept. of Microbiology, Rowett Research Institute, Bucksburn, Aberdeen, AB2 9SB. 2Present address: Dept. of Biochemistry, University of Manchester Institute of Science and Technology, Sackville Street, Manchester, I.

(Copy received July 1971)

163

A simple technique for estimating microbial activity by total carbon dioxide evolution and its application to the attack of plasticiser-treated cotton yam. C. S. Stewart and J. H. Walsh.

These authors found that if yarn is impregnated with plasticisers, as may happen in PVC-coated cotton fabric, those plasticisers which support fungal growth (e.g. butyl acetyl ricinoleate-BAR) reduce yam attack in proportion to the concentration of plasticiser. Plasticisers which do not support fungal growth on the other hand (e.g. dialphanyl phthalate­DAP) caused only a slight reduction of yam attack, indepel)dent of concentration. Respirometric measure­ments have now enabled us to estimate the fungal growth accompanying. loss of yarn tensile strength in each case, and to show that reduction of yarn attack by these two classes of plasticisers is the result of different effects.

Materials

The two types of cotton yarn were used, a 'com­mercial' type supplied by Scandura: Ltd., Cleckheaton, Yorkshire, and a 'purified' type supplied by Dr. K. Selby formerly of the Shirley Institute, Didsbury, Manchester. Analysis of these types of yarn at the Shirley Institute, gave the following compositions: I. 'Commercial' yarn: Nitrogen 0.25%: Mineral Ash 0.93%: Fat -and Wax 3.28%: Cellulose 90.2%: Miscellaneous 5.3% (pectin, organic acids, free sugars etc.). 2. 'Purified' yarn: Mineral Ash 0.3%: Fat and Wax 0.6%: Cellulose 99.2%.

Plasticisers were supplied by Turner Bros. Hindley Green, Wigan, Lanes. and Lankro Chemicals Ltd., Eccles, Lancashire.

Other chemicals were of Analar grade and were obtained from B.D.H. Ltd., Poole, Dorset.

Methods

Estimation of C02 production by fungi. The fuugi were grown either on small heaps of cellulose powder (0.5 g of Whatman CF 11) or small coils (34 em) of cotton yarn contained in specimen bottles. The C02 produced was absorbed by I N NaOH and the Na2C03 content, was determined by titration using the double indicator method of Vogel (1955).

The specimen bottles (Gallenkamp BT265 125 ml) of actual capacity about 150 ml, containing the substrate under study and a 5 ml beaker were auto­claved (104 KNfm2.J5 min) with temporary aluminium foil tops. 2.5 ml sterile I N NaOH was subsequently pipetted into the beaker, through a small hole punched in the foil. The substrate was inoculated with 2.5 ml of a spore suspension of the test organism made up in the mineral salt solution of Marsh, Bollenbacher, Butler & Raper (1949) modified to have greater bl!ffering capacity. This contained (per 100 ml) NH4N03 0.2 g: K2HP04 0.54 g: KH2P04 0.42 g: MgS047H20 0.15 g, pH = 6.2.

The temporary foil tops were then replaced by heat sterilised metal screw caps. The joint between the metal top and the bottle was sealed with 'Parafilm'

164

so that the bottles were airtight. All of the experiments were carried out with incubation at 25°C.

Treatment of cotton yarn with plasticiscrs. Yarn was soaked in an acetone solution of the plasticiser, and the acetone evaporated off, as described by Stewart & Walsh (1971). The final' ratio wt. plasticiser: wt. yarn was 3:4 in each case.

Tensile strength measurements on cotton yam. These were determined as described by Walsh & Stewart (1969). According to tables of the A.S.T.M. (1957), at the level of replication obtained in this study, differences in residual tensile strength of 10-15% are significant.

Results

Test of Replicability of the Method. A test experiment was carried out with 10 replicate flasks, each contain­ing 0.5 g cellulose powder as substrate, inoculated with a spore suspension of Trichoderma kiiningii Oud. as test organism. A similar series of flasks, without cellulose powder, was also inoculated as 'controls' for any traces of growth due to carry over of nutrients in the inoculum, etc. Table I shows the individual C02 values, averages and standard deviations for each set of flasks after incubation for 7 days at 25'.

Test of the correspondence between C02 evolution and tensile strength loss data in the attack of untreated yam. The amounts of C02 produced by fungi growing on lengths of cotton yarn were determined as previously described. The 'purified' cotton yarn was used to eliminate as far as possible interference from fungal growth on fats and waxes present in the commercial yarn. After determination of C02 production, the same pieces of yarn were then tested for tensile strength losses.

The results from this study are shown in Table 2 below.

From Table 2 it is clear that minimum tensile strength losses do correspond to minimum evolution of C02. However the wide range of C02 evolution shown by three fungi causing equal losses of tensile strength indicates that the quantitative relationship between these two measures of fungal activity is very variable.

A comparison of C02 evolution and tensile strength loss on untreated and plasticiser treated yam. C02 produc­tion and tensile strength losses caused by Trichoderma kiiningii growing on untreated and plasticiser treated yarns were used to investigate the effect of the presence of plasticisers on the attack of cotton yarn.

Individual flasks were 'harvested' after 4, 7, and 10 days incubation, and determinations of C02 produced were again compared with tensile strength losses of the yarn. The results are shown in Table 3.

~-------------------------------------------------------------------------------~--~ ·----~ -

A simple technique for estimating microbial activity by total carbon dioxide evolution and its application to the attack of plasticiser-treated cotton yarn. C. S. Stewart and J. H. Walsh.

Table 1-C02 production by T.koningii growing (a) on cellulose (b) control flasks without cellulose. Test ofreplicatiou.

Incubation 7 days at 25°.

SD = Standard deviation

(A) (B) Flask No. mgsC02 Average± SD n:'gs C02 Average± SD

I 20.8 2.8

2 20.8 2.8

3 20.8 2.8

4 20.8 2.8

5 21.5 21.6 ± 0.8 3.0 3.1 ± 0.4

6 21.5 3.0

7 22.2 3.2

8 22.2 3.7

9 22.9 3.9

10 22.9 •

• Alkali spilled

Table 2-C02 production and tensile strength losses caused by fungi grown on 'purified' cotton yarn, over 7 days at 25°C.

Five replicates were used throughout.

Organism mgC02 %loss in tensile strength

Trichoderma koningii 28.5 87.0

Chaetomium globosum 15.8 ~ 84.0

Memnoniella echinatat 19.5 80.0

Aspergillus ustus 10.1 34.0

t 0.1 % wfv Difco yeast extract was included in the mineral salt solution in the case of M. echinata only.

Iu Table 3 the position is best illustrated by a comparison of the amounts of C02 produced with the tensile strength losses of the yarn. This is summarised as an approximate ratio in column (C).

165

This ratio is near unity during the attack of untreated di-alphanyl phthalate (DAP) treated yarn, although both C02 production and losses in tensile strength are slightly depressed by the presence of DAP.

In the case of butyl acetyl ricinoleate (BAR) treated yarn, the high ratio during the initial period (0-4 days) indicates high C02 production in relation to tensile strength losses. In the later stages of attack, the ratio falls progressively as the rate of tensile strength loss increases.

Discussion

In terms of convenience of use, and the level of replication attained it is believ"ed that this method compares favourably with these previously described, and has the particular advantage of involving no expensive or highly specialised apparatus. The level of replication obtained was similar to that reported by Good & Darrah (1967) for C02 production deter­minations with the Warburg manometer, Smith (1967) for the conductivity respirometer, and Burgess & Darby (1965) for differential manometry.

A simple technique for estimating microbial activity by total carbon dioxide evolution and its application to the attack of plasticiser-treated cotton yam. C. S. Stewart and J. H. Walsh.

Table 3-C02 production and tensile strength losses cansed by T. koningii growing on 'commercial' cotton yarn at 2s•.

Five replicates were used throughout.

(a) Plasticiser Time mgC02

(Days)

None 4 28.9

" 7 54.3

" 10 76.7

di alphanyl 4 21.9

phthalate 7 44.7

10 63.6

bntyl acetyl 4 31.7

ricinoleate 7 52.8

10 69.8

The main disadvantage of the method, inherent in its simplicity is that in a sealed system, the level of oxygen will fall as growth proceeds. This effect could be reduced by using larger containers and may be relatively unimportant unless accurate comparisons of one organism with another are required (see below).

Carbon dioxide production data for a range of fungi grown on pure cotton showed no exact relationship to tensile strength losses. The present method cannot differentiate high carbon dioxide levels due to abundant growth from those due to a rise in R.Q. Thus it is impossible to say whether the differences between the various fungi in the comparative levels of carbon dioxide production and tensile strength losses are due to differences in the amount of mycelial growth accompanying cellulolysis or differences in the tend­ency for the R.Q. to rise above I under the conditions used. Calculations suggest that a high proportion of the available oxygen may be used up by the later stages of these incubations.

When the technique was used for comparisons of the growth of one organism, however, results were obtained which in combination with determinations of tensile strength losses, confirm that BAR reduces yam attack by itself providing a carbon source, thus 'sparing' the cellulose. In all cases C02 production over the I 0 days was nearly linear reflecting steady growth, and in the cases of untreated or DAP treated yam this was accompanied by steady losses of tensile strength. Only in the case of BAR treated yarn was

166

(b) (c)

% tensile approx. ratio strength loss a:b

25.2 I :I

48.9 I :I

55.7 4:3

21.1 1 :I

41.9 1 :1

50.0 5:4

6.9 9:2

11.9 4:1

36.5 2:1

rapid C02 evolution initially accompanied by much smaller losses of tensile strength. This is interpreted as indicating that growth was not reduced but that the fungus was initially utilising BAR as the major carbon source, with reduced attack of cellulose. Comparison of figures for untreated and DAP treated yam indicate that in this case the reduction of tensile strength of plasticiser treated yam is accom­panied by, and is presumably due to, a reduction in fungal growth.

Acknowledgments

We are indebted to the National Coal Board for a grant in aid of this research. The views expressed however are those of the authors, not necessarily those of the Board. Gifts of materials from Lankro Chemicals Ltd., Scandura Ltd., the Shirley Institute and Turner Brothers are gratefully acknowledged.

References

A.S.T.M. (1957). Supplement to the book of A.S.T.M. standards including tentatives. Part 7. Methods of test for the resistance of textile materials to micro organisms. D 684-57, pp. 47•53.

Bunt, J. S. and Rovira, A. D. (1955). The effect of temperature and heat treatment on soil metabolism. J. Soil Sci. 6, 129-136.

A simple technique for estimating microbial activity by total carbon dioxide evolution and its application to the attack of plasticiser-treated cotton yam. C. S. Stewart and J. H. Walsh.

Burgess, R. and Darby, A. E. (1964). Two tests for the assessment of microbiological activity on plastics. Br. Plast. 37: 32-37.

Burgess, R. and Darby, A. E. (1965). Microbiological testing of plastics. Br. Plast. 38, 165-169.

Chase, F. E. and Grey, P. H. H. (1957). Application of the Warburg respirometer in studying respiratory activity in soil. Can. J. Microbial. 3, 335-349.

Dobos, R. J. (1928). Influence of environmental conditions on the activities of cellulose decomposing orgaJ)isms in the soil. Ecology 9, 12-27.

Good, H. M. and Darrah, J. A. (1967). Rates of decay in wood measured by carbon dioxide produc­tion. Ann. appl. Bioi. 59, 463-472.

Heukelekian, H. and Waksman, S. A. (1925). Carbon and nitrogen transformations in the decomposition of cellulose by filamentous fungi. J. bioi. Chern. 66, 323-342.

Kaplan, A. M. (1964). Comments on the respirometric technique for assessment of microbiological suscepti­bility of materials. Devs. ind. Microbial. 6, 191-201.

167

Kibble, R. A. (1966). Physiological activity in a pinewood soil. Ph.D. Thesis. University of Liver-pool. ·

Marsh, P. B., Bollenbacher, K., Butler, M. L. and Raper, K. B. (1949). The fungi concerned in fabric deterioration. 2. Their ability to decompose cellu­lose. Text. Res. J. 19, 462-484.

Siu, R. G. H. and Mandels, G. R. (1950). Rapid method "for determining mildew susceptibility of materials and disinfecting activity of compounds. Tex. Res. J. 20, 516-518.

Smith, R. S. (1967). Carbon dioxide evolution as a measure of attack of wood by fungi and its applica­tion to testing wood preservatives and sapstain preventives. Ann. appl. Bioi. 59, 473-479.

Stewart, C. S. and Walsh, J. H. (1971). Effects of plasticisers and plastic coatings on the fungal attack of cotton yarn. (In Press).

Vogel, A. I. (1955). A textbook of quantitative in­organic analysis: theory and practice (2nd Ed.). London, Longmans.

Walsh, J. H. and Stewart, C. S. (1969). A simple method for the assay of the cellulolytic activity of fungi. Int. Biodetn. Bull. 5, 15-20.

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Bravery, A. F. & Grant, C. Int. Biodetn Bull. 7 (4), 169-173 (1971). Preliminary investigations into the use of a thin strip tensile strength test for the rapid evaluation of wood preserva­ti ves against Basidiomycete fungi.

PRELIMINARY INVESTIGATIONS INTO THE USE OF A THIN STRIP TENSILE STRENGTH TEST FOR THE RAPID EVALUATION OF WOOD PRESERVATIVES AGAINST BASIDIOMYCETE FUNGI

A. F. Bravery! and C. Grantl

Introduction

The current British Standard method for assessing the toxicity of wood preservatives to wood rotting Basidiomycetes (BS838: 1961) utilises weight loss as a criterion of attack but specifies test blocks of a size that require an incubation period of 12 weeks to produce satisfactory weight losses. Thus the method is time-consuming and also quite unsuitable fo r the rapid screening of la rge numbers of potential preserving chemicals.

The possibili ties of u ing strength crit eria for making more rapid laboratory assessments of preservative effectiveness against wood destroying fungi have been reviewed by Hartley (1958). Kennedy and lfju ( 1962) described a microtensile test method for assessing fungal decay in thin wood strips, and Brown (1963) used the same criterio n but with thicker samples for assessing wood preservative effect iveness. Brown a lso refers to similar work by Hopkins and Coldwell ( 1944).

Smith (Baker, Savory and Smith 1969; Smith 1970) has developed a refined tensi le test technique with the prediction of field performance from labora­tory tests as his major objective a ltho ugh he a lso considers the potentialit ies of the method as a rapid screening test. Smith's method in volves exposure of

wood strips to an unknown mixed microftora by burial in natural soil. Richardson ( 1968) has applied the thin strip principle to preservative testing against Basidio mycetes in petri-dish mono-culture. Although commendably simple, Richardson's method does not lend itself to quant itative assessment of strength losses.

The present paper descri bes the use of Smith 's thin strips and tensile st rength measurements as the basis of a monoculture test conducted in petri dishes. which might prove suitable as a preliminary screening test for assessing the potential effectiveness of wood preservatives against Ba idiomycete fungi .

Materials and Method Test pieces : Thin strips of wood 100 x 10 x 0 .2 rnm (200f.L thick) were cut with a sledge microtome from the radial face of carefully machined Scots pine sapwood blocks. The blocks from which the strips were cut were very carefully selected for freedom from defects, st raightness of grain, and an even growth rate (approximately 25 rings to the inch).

Treating solutions and impregnation: Tanalith CTI06, a fixed, copper/chrome/arsenic type preservative, was dissolved in de-ionised water in the following per cent w/v concentrations, 0.5, 1.0, 2.0 and 5.0. Thirty

Plate 1.

Method of stacking and drying strips after treatment.

(Crown copyright ).

I Forest Products Research Laboratory, Department of the Environment, Princes Risborough, Aylesbury, Buckinghamshire, England.

(Copy received April 1971).

169

Preliminary investigations into the use of a thin strip tensile strength test for the rapid evaluation of wood preserva­tives against Basidiomycete fungi. A. F. Bravery and C. Grant.

randomly selected strips were vacuum impregnated for 5 minutes in each of the treating solut ions and allowed to soak for a further 30 minutes. Control strips were treated in de-ionised water alone. After soaking strips were removed, lightly dried to remove excess solution and allowed to air dry for three days in partially ventilated covered containers. Care was taken to ensure that the strips were separated from one another during drying (Plate 1) in order to faci litate even drying and prevent cross-transfer of preservative.

When dry identically treated strips were placed 3 mm apart in parallel groups of five and joined together at the ends with folded masking tape on which was written the treatment number of each strip (Plate 2).

P late 2. Exposure group of 5 strips held with masking tape. (Crown Copyright).

These 'exposure groups' of five strips were finally oven d ried at 50°C for three hours and then stcnliscd by exposure to propylene oxide vapour for 24 hours followed by ventilation in a stream of sterile air for 48 hours.

Exposure to test fungus: The test fungus was Conioplwra cerebella II£, growing on 5 per cent malt agar medium in square plastic petri-dishes 100 mm x 100 mm x 18 mm. The fungus mat was overlaid with methanol­sterilised pvc mesh measuring 90 X 90 mm from which a central portion 70 X 25 mm had been removed to

170

facilitate colonisation of the strips by the fungus . The st rips were then laid on the me h transversely to the long axis of the aperture. Four exposure groups (20 strips in total) at each preservative concentration were exposed to fungus attack and two groups (10 strips in total} were exposed under sterile conditions as controls. These latter were placed on similar mesh supports over 5 per cent malt agar medium to which 5 per cent sodium fluoride had been added to preserve sterility.

All the dishes were incubated at 22°C for 5 weeks.

Examination and assessment of strips: After incubation (Plate 3) the extent of overgrowth was noted before the strips were removed from the culture vessels and adhering mycelium gently wiped off. They were then conditioned at 60 per cent RH and 21 °C for approx­imately 5 hours to obtain the equilibrium moisture content of approximately 12 per cent at which all tensile strength measurements were made. Each exposure group was then cut into its component strips which were assessed individually for tensile strength parallel to the grain in a Zwick tensile strength testing machine set with a constant head movement of 2 mm per minute.

Plate 3. Appearance of strips overgrown by C. cerebella II E. (Crown Copyright).

Results

The strips were overgrown by the test fungus at all preservative concentrations. Failure of the strips under test resulting from fungal attack was usually charac­terised by 'brash' fractures; where no fungal attack was present the break presented a splintered appearan­ce (Plate 4). Summaries of the failing loads and calculated strength losses are given in Table I & 2 and presented graphically in Figure I . Differences between the means were assessed for significance by t tests.

Preliminary investigations into the use of a thin strip tensile strength test for the rapid evaluation of wood preserva­ti ves against Basidiomycete fungi. A. F . Bravery and C. Grant.

, 0·0% 5·0%

P late 4. Strips of Scots pine sapwood tested in tension to complete failure after 5 weeks incubation at 22°C.

(Crown copyright)

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Treating solution concentration of Tanalith CT 106 (% wfv)

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Discussion

T he mean failing load of the untreated control strips d id not differ significa ntly (Probability (P) > 0.5) from those of the treated control st rips at any of the treatment levels. The means of the 0.5 per cent and 1.0 per cent CCA treated controls alone showed significant differences ( P = .01) but since these represent the extremes of the variability the result cannot be considered experimentally meaningful. Thus there appears to be no significant loss or gain in tensile strength of the strips due to preservative treatment alone.

0 /0 w/v TREATING SOLUTION CONCENTRATION

F igure J. Loss in tensi le strength of treated strips exposed to C. cerebella for 5 weeks.

After exp osure to fungus sets of treated strips all showed significantly greater failing loads than the set of untreated strips (P < <.001). Furthermore there was a trend for higher fa il ing loads (increasing pro­tection) with increasing preservative concentration. This trend is more clearly seen when the losses in strength of the sets of strips at each treatment level are expressed as a percentage of the mean value for a ll the controls (Table 2, see note t and col (v)). However, a t adjacent concentrations the means were

171

Preliminary investigations into the use of a thin strip tensile strength test for the rapid evaluation of wood preserva­tives against Basidiomycete fungi. A. F. Bravery and C. Grant.

Table I.-Details of failing load (kg) for each replicate set of strips.

1.1 Control strips.

Co ncentration ofC CAper cent 0.0

• 16.5 (±2.9) -X

min/max 8.1 20.5

range 12.4

S.D. 4.0

1.2 Fungus Exposed strips.

c oncentration ofC CAper cent 0.0

•• '0.6 (±0.5) -X

min/max 0.0 2.7

range 2.7

S.D. 0.8

*mean of 10

0.5

14.8 (±2.5)

8.8 19.7

10.9

3.5

0.5

"7.0 (±2.4)

0.0 16.9

16.9

5.1

**mean of 20 except where otherwise indicated eg ' = 15 "= 19

(±) 95 per cent confidence limits for the mean

S.D. standard deviation

not always statistically different, for example the values for 0.5 per cent and 1.0 per cent CCA of 7.0 kg and 9.5 kg respectively (P > .05).

At a preservative concentration of 2.0 per cent CCA the fungus exposed strips have a failing strength that does not differ from that of the sterile controls and the slightly higher failing strength of fungus exposed strips at 5.0 per cent CCA is not significant (P > 0.5). Protection has therefore been conferred at a treatment level of 2.0 per cent wfv CCA-considerably higher than the 0.05 per cent concentration at which Smith (1967) reported protection against the same fungus strain by the same preservative in a BS838 test. However, Levi (1969) has also shown similar disparti­ties between protective levels of CCA on wood veneers and on BS838 size test blocks. Smith (1970) using mixed cultures of microfungi in soil burial techniques has demonstrated interactions between

172

1.0

19.9 (±2.7)

14.3 25.4

11.1

3.8

1.0

9.5 (±2.5)

0.0 24.7

24.7

5.3

2.0

18.5 (± 1.9)

13.5 22.9

9.4

2.6

2.0

16.5 (±2.1)

5.4 25.0

19.6

4.6

5.0

15.5 (±2.6)

10.5 20.7

10.2

3.7

5.0

17.0 (±1.6)

12.5 23.7

11.2

3.4

preservative concentrations, thickness of test sample and period of protection. This indicates that closer matching of results with those of BS838 tests might be achieved by further reducing the exposure period from 5 weeks. However, CCA itself is a highly fixed non-volatile preservative and it will be necessary to ascertain the extent of the disparity between results obtained by the two methods for preservatives which are leachable or somewhat volatile.

Although results have been obtained after a shorter incubation period than is possible in the BS838 tests, the thin strip method as practised has involved careful selection of samples and the use of sophisticated equipment; more time has been spent in making tensile strength determinations than would have been needed for weighings in a comparable BS838 test. Experience gained could permit better sample slection to reduce variability, but for screening tests where speed and simplicity are important it should be possible

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Table 2-Summary of results.

Mean failing load kg Loss in strength per cent Concentration Overgrowth of

ofCCA Sterilet Exposed Compared with Compared with strips per cent wfv controls to fungus replicate control mean of all controls

(i) (ii) (iii) (iv) (v) (vi)

appressed 0.0 16.5 0.6 96 97 mycelium with

fine strands

fluffy mycelium; 0.5 14.8 7.0 53 59 no strands

present

1.0 19.9 9.5 52 44 ditto

ditto 2.0 18.5 - 16.5 11 3 but somewhat

sparse

ditto 5.0 15.5 - 17.0 +10 - 0 very sparse

tMean of all controls 17.0 kg

=Indicates no significant difference (probability (P) > .05)

to obtain adequate results using less stringently selected material and simpler, less sophisticated testing equipment.

Conclusions

In its present form the thin strip method has given a toxic level higher than that previously obtained by BS838 method for a CCA type preservative. This still does not preclude the possible use of the method for rapid comparative evaluations, especially if simplification proves acceptable.

References

Baker, J. M., Savory, J. G. and Smith, D. N. (1969) . Recent work on wood protection at the Forest Products Research Laboratory. Rec. a. Conv. Br. Wood Preserv. Ass., 83-99.

British Standards Institution 1961. Methods of test for toxicity of wood preservatives to fungi. BS 838: 1961.

Brown, F. L. (1963). A tensile strength test for com­parative evaluation of wood preservatives. Forest Prod. J., 13, (9), 405-412. ·

173

Hartley, C. (1958). Evaluation of wood decay in experimental work. Rep. Forest Prod. Lab., Madison, No. 2119.

Hopkins, C. Y. and Coldwell, B. B. (1944). Surface coatings for rot proofing wood. Can. Chern. Process. Ind., NRC No. 1256.

Kennedy, R. W. and Ifju, G. (1962). Applications of microtensile testing to thin wood sections. TAPPI, 45, (9) 725-733.

Levi, M. P. (1969). A rapid test for evaluating the fungicidal activity of potential wood preservatives. J. Ins/. Wood Sci., 23, 45-50, July 1969.

Richardson, B. A. (1968). A new technique for the comparative evaluation of some organo-metallic wood preservatives. Internal. Pest Control, 10 (1), 14-17, 19.

Smith, D. N. (1970). A possible method for the rapid evaluation of wood preservatives; Mitt. dt. Ges. Holzforsch. (in Press).

Smith, R. S. (1967). Carbon dioxide evolution as a measure of attack of wood by fungi and its applica­tion to testing wood preservatives and sapstain preventives. Ann. appl. Bioi. 59, 473-479.

BOOK REVIEWS

AN INTRODUCTION TO INDUSTRIAL MYCOLOGY. 6th EDmON. G. Smith

Edward Arnold (Publishers) Ltd., London, 1969. ix + 390 pp. Price £4.00

The successive editions of "Smith" have been acclaimed for the last three decades. A new edition was always .welcome but the sixth edition, published posthumously, is now the definitive edition.

The arrangement of the book follows the same pattern as the two previous editions but a short chapter has been added on the mycology of soil. Eight years have elapsed since the last edition was prepared, during which time much new work has been published in the field of industrial microbiology as well as some important taxonomic works. The sixth edition represents the author's efforts to integrate this new material into his book, but as the concept of the previous editions was so sound no radical altera­tions were necessary. The changes for the most part are ones of detail. One wishes that names like Aureo­basidium {for Pullularia) had remained buried in taxonomic archives, but on the other hand the revision of the Genus Aspergillus is a great improvement.

Reviews covering previous editions have pointed to an error in attributing the introduction of Bordeaux mixture to Pasteur, an error which still persists (p.301).

"Smith" is a classic. It is an essential companion for all workers in applied mycology and will continue to remain so for the forseeable future.

N. J. Butler.

COOUNG TOWERS: PRINCIPLES AND PRACTICE. 2nd EDmON W. Stanford and G. B. Hill

Carter Thermal Engineering Limited, Birmingham, 1970. xi + 184 pp. Price £1.25

This is a book of 184 pages (2nd edition) written for the user of cooling towers in industry. It falls into three sections.

Section one deals with the terms used, the different types of cooling towers, the materials used for packing, choice of tower, maintenance, water treatment and industrial applications. The second section is concerned with the theoretical aspects, e.g. psychrometry, heat transfer calculations and the determination of cooling duties. The book ends with an appendix, which lists 19 sources of further information {the nearest the book comes to a bibliography), conversion tables ("Celsius to °Fahrenheit), with information on water hardness, background sound pressure levels and water flow capaciiies.

174

The book describes simply and clearly the funda­mentals of cooling towers and is well illustrated. However, the section on materials used as packing, especially the biodeterioration information, is often confusing: Two types of fungal decay of timber are listed, yet the authors describe three. While the dis­advantages of one packing material are given, only the advantages of others are mentioned. The preserva­tion of timber is dealt with briefly, .but no mention is made of the recommended loadings to be used, information surely of interest to the maintenance engineer.

This book is written with the engineer in mind, and as cooling towers are rather specialized, the book will undoubtedly become a standard work of reference on this subject. It is therefore regrettable that the book lacks a bibliography which the more advanced student could turn to for further information.

E. B. Gareth Jones.

PEST CONTROL IN BANANAS

Edited by Susan D. Feakin. PANS Manual No. 1 (New Edition). PANS London, 1971. 128pp. Price £0.42

This modestly priced Manual will be of value to all persons concerned with the banana industry, and to plant pathologists and entomologists in general. It will be of particular help as introductory reading for persons about to work with bananas for the first time. The print is clear and the arrangement of headings and sub-headings is most convenient for the reader. Black and white photographs are numerous and mostly of good quality. Line drawings and colour plates are excellent. The editor is to be commended on obtaining illustrations from many parts of the 'world, and from persons actively working with bananas.

A useful glossary of banana terminology precedes a short chapter on weed control in which safety pre­cautions, to be observed when handling herbicides, are noted. A large chapter on diseases caused by fungi, bacteria and viruses follows. Each disease is dealt with under the headings Distribution, Symptom, Control. Disease Distribution Maps (Commonwealth Mycological Institute) are included. In general, accounts of individual diseases and the causal organ­isms are accurate and up•to-date. However, it is inevitable when condensing a large volume of litera­ture that certain errors or ambiguities will result. For instance, ascospores of Mycosphaerella musicola, causing banana leaf spot. (Sigatoka), do not normally 'give rise to dry weather infections' (p. 17). They are of greatest importance during rainy weather. It should also be noted that there is still no valid record of Sigatoka in Hawaii (p. 14). The account of black leaf streak disease (Mycosphaerella fijiensis) in the Pacific region is good, and the statement that 'its importance

I

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must not be overplayed because it probably could be adequately controlled in well-managed bananas' (p. 25) is perfectly true in the experience of the reviewer (e.g. in the Philipines and Hawaii).

The chapter on nematodes (probably the most important cause of loss of yield in bananas throughout the world) is excellent, and means of controlling Radoplw/us simi/is are set out in some detail.

The final chapter deals with insect pests, with special emphasis placed on the control of the banana weevil (Cosmopolites sordidus). Useful accounts of the types of damage caused by aphids, beetles, moths, thrips, mites, caterpillars, mealy bugs, and others are presented, together with methods of control, where known or practical.

It is a pity that the extent of losses caused in the past by some of the major pests and diseases are not mentioned, nor is there much information about the economics of control methods.

A bibliography of 106 references, mostly published between 1960 and 1911, a check list of diseases and insects, and an index, conclude the volume.

D. S. Meredith.

THE GENERA OF FUNGI SPORULATING IN PURE CULTURE J. A. von Arx J. Cramer, Lehre, 1970. iii + 288 pp., 136 figs. Price £6.90

Most of the fungi involved in biodeterioration sporulate readily in pure culture and a new book with a title like this one is therefore of obvious interest to workers in this field. Most mycologists will be familiar with Dr. Arx's 'Pilzkunde. Ein kurzer Abriss der Mykologie unter besonderer Beriicksichtigung der Pilze in Reinkultur' (J. Cramer, Lehre, 1967. 356 pp.) which provided keys "in German to the genera of fungi sporulating in pure culture. While this book made the identification to the rank of genus possible that of species was often difficult because the pertinent literature could not easily be found. This new book omits the detailed descriptions of orders and families seen in the earlier one and concentrates on the genera, consequently making it more valuable to those wishing to determine a particular culture.

Brief introductory chapters deal with the scope of the book, the characteristics of the major groups of fungi, and types of spores. This book is essentially concerned with microfungi (e.g. Oomycet.ales, Muco­rales, Endomycetales, Torulopsidales, Eurotiales, Sphaeriales, Pseudosphaeriales, Sphaeropsidales, Me­lanconiales, Moniliales), although some Discomycetes are included. All orders of Basidiomycetes are omitted which is perhaps unfortunate as very many have now been grown in pure culture. As it is the higher Basidio­mycetes which are the main concern of mycologists working on the biodeterioration of wood this book

175

will be less relevant to them than one might be led to suppose from its title. The genera of microfungi included are principally those maintained at the Centraalbureau voor Schimmelcultures, Baarn, of which Dr. Arx is the Director. As further genera are continually being grown in culture for the first time the author emphasises that ' ... ; . the keys and lists are not claimed to be completed'; users should bear this in mind. A main key leads to the "orders of fungi known to occur in pure culture and includes some not treated in detail in this work. Separate keys lead to the genera treated within each order. Each of the 634 genera keyed out is listed after the relevant key and the author and place of publication given. The type species, synonyms (with full citations and types), the number of species known, and references to pertinent papers dealing with identification to the rank of species are given. The inclusion of this data means that once the genus is determined the reader can go immediately to the relevant papers without recourse to other bibliographic works. Excellent clear line drawings illustrate the diagnostic characters of over 300 species and are a very valuable aid to their identifi­cation.

The keys work fairly well but some of the separa­tions may prove difficult for students unfamiliar with the group concerned. The inclusion of Anixiella Saito & Minoura in the Sphaeriales is in accord with modern concepts but one then wonders why Chaetomidium (Zopf) Sacc. (authority given as '(Fuckel) Zopf' in error) and Kernia Nieuwland were retained in the Eurotiales. The inclusion of details of conidium forma­tion in the Sphaeropsidales and Melanconiales is a valuable step forward but unfortunately includes some assumptive errors. The retention of Gliomastix Gueguen as a genus distinct from Acremonium Link ex Fr. (with which it is united by Dr. W. Gams) will be welcomed by many mycologists, and the union of Xeromyces Fraser with Monascus Tieghem appears to be well founded. Specialists in various groups will doubtless question some of the generic concepts adopted in this work, such as the phicing of both the ostiolate genus Stratton/a Cif. and the cleistocarpic genus Tripterospora Cain. as synonyms of Zopfiella Wint., and the union of Blennoria Fr. and Ceuthospora Fr. Dr. Arx may well have good grounds for this type of revision but one wonders if they should have been presented here without arguments to support them. Two new genera, Thamnostylum Arx & Upadhay (Mucorales, Thamnidiaceae) and Achaetomie/la Arx. (Ascomycetes, Sphaeriales), and one new species are described, and eighteen new combinations introduced.

There are, unfortunately, some errors in the biblio­graphic citation of genera and one ~uspects that Dr. Arx was unable to check many of them in the original. The species stated to be the types are generally correct but include some slips (e.g. Actinonema rosae"(Lib.) Fr. cannot be the type of Actinonema Fr. as it was not one of the original species and the name has not been conserved in a _later sense). The general layout and quality of production is good but the binding in paperback is rather poor for a work intended for the laboratory, and many users will probably find re­binding necessary within a few years.

This book should, however, be judged in comparison with the other works in English currently available. None attempts to cover such a wide field and with its lucid drawings and helpful bibliography this book will prove a valuable aid to all concerned with the identifi­cation of cultures of microfungi. Dr. Arx is to be congratulated on yet another useful publication which has attempted, and in large measure succeeded, to provide a lucid key to the genera of microfungi sporulating in pure culture.

D. L. Hawksworth.

ECOLOGY OF REFUSE TIPS

A. Darlington Heinemann Educational Books Ltd., London, 1969. 138 pp. £1.25

This low cost handbook in the Scholarship Series in Biology is a collection of ecological data concerning almost every systematic group of organisms found on refuse tips in Britain. It appeals to the naturalist by providing a guide to those macro- and micro­organisms encountered on local and household refuse tips with some explanations of the environmental pressures that influence their growth. It will help students to understand ecology as well as stimulate investigations of this unique and usually accessible habitat. It is a source book for teachers organising inexpensive field courses to tips. This book pioneers refuse ecology.

The book begins by outlining some of the problems of refuse disposal. It might have been more informative if detail were given on variations employed in disposal practice as well as listing some refuse constituents and their proportions found. This leads to a discussion on edaphic conditions that have been determined and is followed by a chapter on colonising species with extensive lists for higher plants and adequate ones for other groupings. The next two chapters deal with dispersal and dependence on water with some experi­ments described to confirm observed features. The rest of the book is devoted to specialised organisms found and the domestic refuse tip with its own par­ticular conditions. The text is filled with descriptions of ecological curiosities that maintain the reader's interest. At the end of most chapters there is a reference list containing books that are helpful for tracing further information, and the end of the book has a useful index.

The book could probably have benefited from more photographs and less imprecisely scaled drawings and some ecology of those organisms within the refuse actively degrading the substrate. This quibble does not detract anything from the merit of the book with its theme of refuse tips being an easily accessible and rich natural habitat.

R. F. Sharp.

176

CHEMICALS CONTROLliNG INSECT BEHAVIOUR Edited by M. Beroza Academic Press, N.Y. & London, 1970. 170 pp. Price £4.65

Those concerned with the effect of broad spectrum insecticides on non-target organisms in the environ­ment, will be encouraged by the wealth of information on insect pheromones reviewed in this volume: it directs the attention of the chemist, biochemist and entomologist to the ultimate goal of effective manage­ment of insect populations by non"insecticidal means.

In six papers, involving 16 authors, the reader is given an up to date account of present knowledge on communication in reproductive processes, defence mechanisms and other aspects of insect attractants and repellents.

These papers, originally presented at a Symposium of the American Chemical Society, Minneapolis, 1969, emphasise the subtleties of insect communication compared with the relative crudity of man's present attempts at pest elimination. Almost without exception, each author draws upon the "pollution of the environ­ment", the "disturbance of ecological balance" and the "rapid development of resistance to insecticides" as a background against which to review such subjects as: chemical communication in the finding of mates, food and egg laying sites, the co•ordinated behaviour patterns of social insects, and the use of lures in combination with insecticides to minimise chemical hazards in certain situations. This book is summed up in the Preface: "It is clear now that many insects depend on chemicals for survival ....... Clearly the key to insects control in many instances may well be the key that unlocks the structure of their secretions or of chemicals that attract or repel them".

The first three papers deal with the sex pheromones of Lepidoptera, Coleoptera and the Boll Weevil (Anthonomus grandis). From the large number of lepidopterous insects in which the presence of sex pheromones has been demonstrated, five economically important species receive special study: these include the Pink Bollworm Moth, The Cabbage Looper Moth and Fall Army Worm. The pheromones of Lepidoptera appear to be long chained esters or alcohols whilst those which have been identified for eight species of Coleoptera are more varied in struc­ture. Two papers describe the isolation, identification and synthesis of the pheromones found within these two Orders.

The paper on the sex attractant of the Boll Weevil highlights this insect as the major pest of cotton and one of the most important insects causing economic loss in the United States. The goal has been to isolate and identify the structure of the four terpenoid components of the pheromone of the male. The paper describes the synthesis of these four compounds which in certain combinations have proved as attractive to females as the natural substance. The conclusion from field tests is that these compounds may act as "aggre­gating pheromones" as well as sex pheromones, attracting males and females equally.

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The fourth and fifth papers are concerned with "communication chemicals". These review the role of pheromones in the social co-ordination and inter­action of insects within populations of Hymenoptera and Isoptera; "alarm pheromones" (excitatory sub­stances) which communicate colonial disturbances, defensive substances which play an important role in the "ever present predator-prey relationship"; and trail pheromones of ants, bees and termites. The alarm pheromones (ketones and octanones) appear to be the least specific of"chemical releasers of social behaviour' whilst 100 substances referred to in the paper on "Arthropod Defense Secretions" present a wide assemblage of compounds with differing chemical structures.

The final paper, on "Current Usage of Insect Attractants and Repellents" outlines the U.S.D.A. programme for screening large numbers of compounds against given insects, with the aim of finding perhaps weak attractants which might be improved by synthesis of related chemicals. This route to the useful attractant, is the reverse of the "isolation route" described in the earlier papers. Eight synthetic compounds are men­tioned: so far greatest advantage has been made of these in detecting infestations, or determining the extent of insect populations-information necessary for direct control or eradication programmes. Their value in the control of Mediterranean Fruit Fly, Melon Fly and Oriental Fruit Fly are cited as examples. Traps using attractant baits were deployed at U.S. ports of entry of fruits between 1958,64, forming an early warning detection system, which is said to have saved S9 million potential eradication costs.

This collection of papers points towards new insect control techniques for the future: Beroza, editor of the volume and author of the final paper, believes that, "insecticides will continue to be our mainstay in combatting insects, but attractants may reduce the amounts employed". He sums up our present position: "the action of some of these chemicals (attractants)­or more specifically their odours-is powerful enough in terms of the infinitesimal quantities needed and intensity or response, to make them valuable for combatting insect pests. In effect, we wish to utilize the survival mechanisms of insects in order to destroy or control them". The message for the layman is "hope": that for the research workers is many years of patient investigation ahead. For the latter, this book forms an excellent reference volume with extensive bibliographies to the subject matter of each paper.

P. B. Cornwell.

177

ISOLATION OF ANAEROBES Edited by D. A. Shapton and R. G. Board The Society for Applied Bacteriology Technical Series No. 5. Academic Press, London & N.Y., 1971. 270 pp. Price £4.00

This series is made up of books of great practical use in the active microbiology laboratory and the present volume is no exception. There are nineteen papers describing the isolation and cultivation of groups of anaerobic organisms, each paper being followed by a bibliography. Not surprisingly eighteen of these papers concern bacteria and a single paper deals with anaerobic protozoa associated with the highly specialised environment of the animal rumen. Gram positive anaerobic spore forming rods of the genus Clostridium have long been of concern both in the spoilage of foods and, more dramatically, in several types of food poisoning. The isolation and enumeration of members of this group is of great importance and about one third of the book is concerned with them.

The decomposition of cellulose is not only of major importance in the cycling of carbon through the biosphere but it is also a practical problem in the spoilage of specific commodities enjoyed by man. In this latter context cellulose decomposing fungi are of the greatest concern but there may be circumstances in which bacteria need also to be considered. The methodology described for the isolation of anaerobic cellulolytic bacteria both from the soil and from the animal rumen could be of interest to those working on the deterioration of cellulosic materials.

Among the gram negative anaerobes, species of Zymomonas may be associated with the spoilage of beer and cider and a short paper describes the detec­tion of Zymomonas anaerobia in fermented beverages.

Sulphate reducing bacteria are of considerable interest, not only in the anaerobic corrosion of metals such as iron, but also because they may be isolated from complex microbial populations associated with the deterioration of commodities such as industrial cutting oils. The isolation and examination of Desul­fovibrio and Desulfomacu/atum are described, the former in considerable detail, in a paper which helps to remove some of the reputation that sulphate reducing bacteria have "of being difficult to grow and enumerate".

Indeed the whole book could help to overcome the difficulties which many of us assume are inherent in the study of the anaerobic component of the microflora in which we are interested.

M. 0. Moss.

INDEX

VOLUME 7, 1971

AUTHOR INDEX

Barnes, T. G. 7(3):105 Lavers, G. M. 7(3) :129 Savory, J. G. Bieniada, J. 7(3):109 Schipper, A. L. Bomar, M. T. 7(1): 25 Mills, J. 7(3):105 Sheridan, J. E. Bravery, A. F. 7(3):129 Mulder, J. L. 7(2): 69 Shields, J. K.

7(4):169 Stewart, C. S.

David, J. 7(1): 3 Nelson, J. 7(4):161 Stranks, D. W. ..

Desai, R. L. 7(1): 11 Drummond, D. C. 7(2): 73

Parbery, D. G. 7(1): 5 Upsher, F. J.

Eggins, H. 0. W ... 7(3):105 Parker, M. S. 7(2): 47

Eltringham, S. K ... 7(2): 61 Petersson, B. 7(3):115 Vilnina, G. L.

Eriksson, K.-E. 7(3):115 Pugh, G. J. F. 7(1): 35

Fudalej, P. S. 7(4):155 7(1): 37 7(2): 69 Walsh, J. H.

Grant, C. .. 7(4):169 Wastie, R. L.

Hueck, H. J. 7(2): 81 Ridley, J. E. 7(2): 89 Williams, B. W.

Hughes, R. L. 7(2): 87 Rogers, M. R. 7(1): 15 Williams, J. I.

Rossmoore, H. W. '7(2): 55 Kaplan, A. M. 7(1): 15 7(4):147

Kestelman, V. N. 7(3): 99 Rytych, B. J. 7(4):155 Zyska, B. J.

SUBJECT INDEX

Alga Scytonema stuposum on fabrics

Algae versus man Amorphotheca resinae: physical factors influencing growth in culture Antimicrobial compounds: stability in the soil com­plex Basidiomycetes: wood preservatives: evaluation .. British Pest Control Association Carbon dioxide evolution technique Celltilolytic activity: rapid test for .• Cladosporium resinae: selective isolation Coolant breakdown: methylene blue reduction Cosmetics preparation: screening of preservatives .. Cotton yam: attack: technique Cutting fluids: procedure for preservation .. Environmental pollution Epicoccum nigrum Fabrics: blue green alga Scytonem stuposum Fermentative activities of microorganisms: influ· ence of polymers Fungi colonizing hardwood chips: effect of near ultraviolet light Fungi of importance in biodeterioration Fungicides for protecting baled rubber G/iomastix murorum .. Hardwood chips: effect of near ultraviolet light on fungi Limnoria: distribution of burrows .• Methylene blue reduction: coolant breakdown

7(3) :113 7(2): 89

7(1): 5

7(1): 25 7(4):169 7(1): 3 7(4):163 7(3):109 7(4):161 7(4):147 7(2): 47 7(4):163 7(2): 55 7(2): 81 7(2): 69 7(3):109

7(3): 99

7(1): 11 7(1): 35 7(3):121 7(1): 37

7(1): 11 7(2): 61 7(4):147

178

Nylon: effect of Penici/liumjanthinellum Paper industry: biodegradation Parachute nylon: effects of Penicillium janthinellum Paraffin oil-carbon black: influence of Pseudomonas sp. Penicil/iumjanthine/lum: effects on parachute nylon Phannaceutical preparations: screening of preServa­tives PJasticiser-treated cotton yarn: attack: technique .. Polymers: influence on the fermentative and other activities of microorganisms .. Preservatives for pharmaceutical and cosmetic pre­parations: screening .. Pseudomonas sp.: influence on paraffin oil-carbon black system .. Rodents and biodeterioration Rubber: fungicides .. Scytonema stuposum on fabrics Softwood, decayed: strength properties Softwood timber, newly felled: biodeterioration problems Soil complex: stability of antimicrobial compounds Stereum sanguinolentum: xylanase .. Talaromyces emersonii Tensile strength test: wood preservative evaluation Ultraviolet light: effect on fungi Vulcanizates: microbial deterioration Wood preservative: evaluation Xylanase from Stereum sanguinolentum: purifica· cation and characterization

7(2): 95 7(3):121 7(4):161 7(1): 11 7(4):163 7(3):109

7(3):113

7(3): 99

7(4):163 7(3):121 7(2): 55 7(1): 37

7(4):155

7(1): 15 7(2): 87 7(1): 15

7(4):155 7(1): 15

7(2): 47 7(4):163

7(3): 99

7(2): 47

7(4):155 7(2): 73 7(3):121 7(3):113 7(3):129

7(2): 91 7(1): 25 7(3):115 7(3):105 7(4):169 7(1): 11 7(4):155 7(4):169

7(3):115

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