BIA-Report 3/96 Workshop Microorganisms · PDF filecio del trabajo», un temo tratado con creciente interes por parte de las institu

BIA-Report 3/96 Workshop "Microorganisms" (Mikroorganismen)

A Arbetslivsinstitutet

HVBG Hauptverband der gewerblichen Berufsgenossenschaften

Bearbeitet von:

Redaktion,

Herausgeber,

Satz und Layout:

Druck,

ISBN, ISSN,

Christoph Deininger

HVBG, Berufsgenossenschaftliches Institut für Arbeitssicherheit - BIA, Sankt Augustin

Göran Blomquist

Arbetslivsinstitutet, Umea, Schweden

Ute Warkalla HVBG, Berufsgenossenschaftliches Institut für Arbeitssicherheit - BIA

Hauptverband der gewerblichen Berufsgenossenschaften (HVBG) Alte Heerstraße I II, 53754 Sankt Augustin Tel., 0 22 41 I 2 31 - 0 I Fax, 0 22 41 I 2 31 - 3 33

- April 1996 -

HVBG, Abteilung Öffentlichkeitsarbeit

kj-druck, ßonn

3-68383-405-X 0173-0387

Kurzfassung

Fragen zu Gefährdungen durch Mikroorganismen om Arbeitsplatz wurden in den letzten Jahren, nicht zuletzt wegen des lnkrafttretens der EU-Richtlinie 90/679/EWG "Schutz der Arbeitnehmer gegen Gefährdung durch biolagisehe Arbeitsstalle bei der Arbeit" in zunehmendem Maße von den nationalen Arbeitsschutzinstitutionen bearbeitet. Im Rahmen der notwendigen Umsetzung dieser Richtlinie in nationales Recht sind die Mitgliedsstaaten angehalten, adäquate Vorschriftenregelungen zu erlassen.

ln diesem Zusammenhang veranstaltete das Berufsgenossenschaftliche Institut für Arbeitssicherheit - BIA im März 1995 in Zusammenarbeit mit dem schwedischen Arbetslivsinstitutet einen internationalen Workshop zum Thema "Mikroorganismen", dessen Beiträge nunmehr

der hier vorliegende BIA-Report zusammenfaßt. Ziel des Workshops mit Experten aus acht europäischen Ländern war es, neben den grundsätzlich möglichen Risiken durch Exposition gegenüber biologischen Agenzien bei der Arbeit auch den Stand der Technik bei der Probenahme und Analyse von Bakterien und Pilzen zu erörtern. Ein wichtiger Punkt

war es zudem, die Arbeit der neuen und inzwischen konstituierten europäischen

Normungsgruppe WG 5 "Messung biologischer Arbeitsstolle" im CEN/TC 137 zu diskutieren. Nicht zuletzt wurde durch die Teilnehmer des Workshops die Eimeichung eines europäischen Projektvorschlages bei der EU beschlossen, der die Entwicklung - z.T. neuerstandardisierter Meßmethoden für Mikroorganismen und Endotoxin am Arbeitsplatz auf europäischer Ebene voranbringen soll.

Abstract

Problems concernlng hazards from microorganisms in the workplace have been dealt with to an increasing extent in recent years by the national Institutions concerning protection at werk, not least due to the EU directive 90/679/ EEC "Protection of Workers from Risks Related to Exposure to Biological Agents at Werk", which has recently come into force. The Member States are required to establish suitable regulations within the framework of the required implementation of this Directive into their own national law to provide workers with protection against hazards to their health and safety arising from the handling of biological agents in the course of their work.

ln this context the Berufsgenossenschaftliches Institute for Occupotional Sofety, the BIA, held an international workshop in March 1995 on the topic of "Micro·

organisms" in coniunction with the Arbetslivsinstitutet, whose proceedings have now been summarised in this BIA report. The goal of the Workshop, which was attended by experts from eight countries, was to discuss the state of the art in sampling and analysing bacteria and fungi, in addition to the risks that are likely to be encountered through exposure to biological agents at work. A further major point was to discuss the work of the new Europeon standards group WG5 "Measurement of Biological Agents" in CEN/TC 137 that has now been constituted. And, not least, it was decided by the participants in the workshop to submit o proposal for a Europeon project to the EU aimed at promoting the development at Europeon Ievei of standardised measuring methods, some of them new, for microorganisms and endotoxins in the work place.

Resume

Des problemes relatifs aux dangers 8manant des micro-organismes au poste de trava\1 ont ete de plus en plus trait8s par \es institutions nationales de protection du travail, en particulier 0 cause de I' entree en vigueur de Ia directive 90/679/CEE «Protection des salaries contre les dangers dus a des substances de trovail biologiques pendant leur activite professionnelle» de Ia CE. Dans le cadre de l'int8gration n8cessaire de cette directive dans le droit national, \es Etats membres sont tenus de promulguer des reglements odequots, ofin de proleger les solariSs contre les dangers qu'ils courent en matiere de sante et de securite lors de Ia monipulotion d' agents biologiques o leur poste de trovoil.

Dans ce contexte, l'institut des Berufsgenossenschaften de Ia SEkurite du trovail (BIA) o orgonise en mors 1995 - en coop8ration avec l'institut suedois Arbetslivsinstitutet - un atelier international qui avait pour theme «Les microorganismes)), dont les contributions

sont maintenont r8unies dans le pr8sent ropport du BIA. L'objectif de l'atelier, qui a rossemble des experts de 8 poys europ8ens, 8tait d'8voquer non seulement les risques fondamentalement courus suite 0 I' exposition 0 des agents biologiques au poste de travail, mais egalement l'etat de Ia technique en motiere de prelevements d' echantillons et d'anolyses des bacteries et des champignons. La discussion sur le travail du nouveau groupe de normalisation europeen WG 5 «Mesure des substances de travail biologiques)), entre-temps constitue, au sein du CEN!TEC 137 etoit egalement un point important. Les participants a l'atelier ont en particulier decide de deposer oupres de Ia CE une proposition de projet europ8en, qui a pour objet de faire progresser le developpement o l'echelle europeenne de methodes de mesure standardisees - en partie nouvelles - des microorganismes et des endotoxines aux postes de travail.

Resumen

Los problemas respecto a los riesgos existentes en el puesto de trabajo a causa de microorganismos fueron en los Ultimos afios, tambien como consecuencia de Ia puesta en vigor de Ia directiva de Ia UE 90/679/CEE «protecci6n de los trabajadores de los riesgos emergentes de los materiales biol6gicos en el ejercicio del trabajo», un temo tratado con creciente interes por parte de las instituciones nacionales para Ia protecci6n laboral. En el marco de Ia transposici6n de esta directiva o las legislaciones nacionales, los Estados-miembro deben decretar reglamentaciones adeevadas para proteger a los trabajadores de perjuicios para Ia salud y Ia seguridod relacionados con Ia manipulaci6n de agentes biol6gicos en el puesto de trabajo.

En el marco de esta tem6tica, el lnstituto de las Berufsgenossenschaften para Ia Seguridad laboral (BIA), en colaboraci6n con el instituto sueco Arbetslivsinstitutet organiz6 en Marzo de 1995

un drculo internacional de trabajo sobre el tema «microorganismos», cuyas aportaciones est6n resumidas en el presente informe BIA. EI objetivo del grupo de trabajo compuesto por expertos de 8 pa(ses europeos era el intercombio de experiencias en torno a todos los riesgos posibles emergentes de Ia exposici6n por porte de los trabajadores a los ogentes biol6gicos, asf como tambiEm en lo concerniente al estado de la tecnica para Ia toma de muestras y para el an6lisis de bacterias y hongos. Otro aspecto importante era tambien Ia discusi6n en torno ol trabajo de los nueve grupos europeos constitufdos de normalrzad6n WG 5 «medici6n de las sustancias biol6gicas» en el CEN/TC 137. los participantes del drculo de trabajo resolvieren adem6s presentar a Ia UE una propuesta de proyecto tendiente a lograr un avance a nivel europeo en el desarrollo de los metodos est6ndares - en parte nuevos - de medici6n de microorganismos y endotoxina en los puestos de trabajo.

Contents

poge

I nlroduction I I

Different Environments Carrying the Risk of Disease Caused by Exposure to Fungoi Spores - Swedish Experiences 17 Göron Blomquist, National Institute of Occupationai Health, Umea, Sweden

Microorganisms and their Products in Occupational Environments 29 John Lacey, IACR - Rothamsted, Harpenden, United Kingdom

Evaluation of Methods for Quantitative Assessment of Microorganisms in Highly Cantominated Werking Environments 43 Wijnand Eduard, National Institute of Occupational Health, Oslo, Norway

Methods for Analysis of Microbial Sampies from the Work Environment 55 B. Crook, Microbiology Section; Biomedical Seiences Group, Health and Safety Laboratory, Sheffield, United Kingdom

Microbiological Sampling in Finland 67 Marjut Kotimaa, Kuopio Regional Institute of Occupational Health, Kuopio, Finland

Measurement of Microorganisms in the Werk Environment in Fronce 75 J.F. Fabries, Institut National de Recherche et de Securite (INRS), Vandceuvre, France

Contents

Measurements of Microorganisms in the Werk Environment in Denmark Birgitte Herbert Nielsen, Department ofT oxicology and Biology, National Institute of Occupational Health, Copenhagen, Denmark

Sampling of Biooerosols in lndoor Environments in Finland Aino Nevalainen, National Public Health Institute, Division of Environmental Heolth, Kuopio, Finland

The Development of Standardization Procedures for the Assessment of Bioaerosols in the Workplace W.D. Griffiths, and I.W. Stewart, AEA Technology, Harwell, United Kingdom

Harmonised Methods for Measuring Bioaerosols ot the Workploce in Germany Christoph Deininger, Berufsgenossenschaftliches Institute for Occupational Safety, Sankt Augustin, Germany

poge

81

91

97

110

Inhaltsverzeichnis

Seite

Einleitung 14

Erkrankungsrisiko durch Pilzsporen an verschiedenen Arbeitsplätzen Schwedische Erfahrungen 18 Göran Blomquist, Nationales Institut für Gesundheitsschutz am Arbeitsplatz, Umea, Schweden

Mikroorganismen und ihre Produkte in der Arbeitsumgebung 30 John Lacey, IACR - Rothamsted, Harpenden, Großbritannien

Bewertung der Verfahren zur quantitativen Erfassung von Mikroorganismen an hochkontaminierten Arbeitsplätzen 44 Wijnand Eduard, Nationales Institut für Gesundheitsschutz am Arbeitsplatz, Oslo, Norwegen

Verfahren zur Analyse von am Arbeitsplatz entnommenen, mikrobiellen Proben 56 B. Crook, Abteilung für Mikrobiologie, Arbeitsgruppe Biomedizin, Labor für Gesundheit und Sicherheit, Sheffield, Großbritannien

Mikrobiologische Probenohme in Finnland 68 Marjut Kotimaa, Regionalinstitut für Gesundheit am Arbeitsplatz, Kuopio, Finnland

Messung von Mikroorganismen am Arbeitsplatz in Frankreich 76 J.F. Fabries, Nationales Institut für Forschung und Sicherheit (INRS), Vandceuvre, Frankreich

Inhaltsverzeichnis

Seite

Messung von Mikroorganismen am Arbeitsplatz in Dänemark 82 Birgitte Herber! Nielsen, Abteilung für Toxikologie und Biologie, Nationales Institut für Gesundheit am Arbeitsplatz, Kopenhagen, Dänemark

Probenohme von Bioaerosolen in Innenräumen am Beispiel Finnlands Aino Nevalainen, Nationales Öffentliches Institut für Gesundheit, Abteilung Umweltgesundheit, Kuopio, Finnland

Die Entwicklung standardisierter Verfahren zur Erfassung von Bioaerosolen am Arbeitsplatz W.D. Griffiths und I.W. Stewart, AEA Technologie, Harwell, Großbritannien

Standardisierte Verfahren zur Messung von Bioaerosolen

am Arbeitsplatz in Deutschland Christoph Deininger, Berufsgenossenschaftliches Institut für Arbeitssicherheil - BIA, Sankt Augustin, Deutschland

92

98

112

lntroduction

ln addition to physical, mechanical and chemical influences at the workplace, workers can also be exposed to biological agents. This exposure to microorganisms (bacterio, moulds/ yeasts, viruses) and their constituents, as weil as to human endoparasites and cell cultures, can occur in the most varied of working environments.

ln addition to those areas in which there has been an early awareness of the potential risks from biological agents facing workers, particularly with regard to possible infection, and where the health and safety authorities have dealt with the problern at an early stage (e.g. in the medical professions, laboratories, abattoirs, tropical diseoses during periods spent abroad), this subject is becoming increasingly important in other areas of werk that also come to involve the unintentionol handling of biological agents. These include 11 classic" examples such as agriculture, the timber industry, the metal processing industry (cool"1ng lubr"1cants), wholesale storage, sewage installations and areas in which circulating water systems are used in the working processes. However, the growing significance of the subject of "biologica\ agents" is also, not least, a result of the fact that over recent years, as new waste monagement and disposal strotegies have been pursued, a number of new areas has emerged

such as bio-composting, and the sorting and recycling of valuable materials.

ln EU directive 90/679/EEC the Europeon Commission has addressed the subject of biological agents. The aim of this directive is to protect workers from hazards caused by the materials to which they can be exposed at work, and also to ovoid these risks in the first place. Accordingly, in future, the type, extent and durotion of the workers' exposure must be determined for every activity where exposure to biological agents can occur, so that oll risks to the health and safety of the workers can be assessed and appropriate measures set out. The most importont criterion for assessing the risks is the classification of the biological agents according to the Ievei of risk involved. Also taken into account are the allergising ond toxic properties of microorganisms, since these can have significant effects, especially whenever high concentrations of airborne germs arise in the event of unintentional use. Excessive concentrotions can cause diseoses that obstruct the respiratory tract, extrinsic-allergic alveolitides ond 11 organic dust toxic syndrome".

This BIA report summarises the contributions of an international "Microorgonisms" workshop held by the BIA in March 1995. The aim of the workshop,

II

lntroduction

attended by I I scientists from 8 Europeon countries, was to discuss the current Ievei of knowledge, the occurrence and the measurement of microorganisms in the air at the workplace. ln addition to the fundamental danger posed by microorganisms, particular emphasis was also laid during the discussions on techniques for sampling and analysing bacteria and fungi. The problems that can arise in this area were also discussed.

This discussion, bringing tagether experts at a Europeon Ievei, was held following the suggestion from the BIA that, against the background of EU directive 90/679/ EEC, there should be some form of Europeon agreement and Europeon harmonisation with regard to meosuring biological agents. T o help achieve this, the BIA has been able to draw on the experience obtained at a national Ievei from the working group "Measuring Methods, Measuring Strategy'' (now Project Group 4 "Workplace Assessment" as part of the 11 ßiological Agents" committee under the ouspices of the Federal Ministry of Labour and Social Affairs). This working group is chaired by the BIA and its task is to standardise methods of measuring microorganisms at the workplace, os

weil as to determine appropriate measuring strategy and parameters for different oreas of work. ln a wider con-

12

text, CEN!TC 137 "Assessment of Exposure at the Workplace" resolved to set up a WG5 "Measurement of Biological Agents". An important aspect of the workshop was therefore to discuss the possible practical procedures in the Europeon standardisation committee and to acquire members for this committee in the form of interested Europeon experts. ln August 1995, the standardisation group started its work at the constituent meeting in Stockholm. lts aim is also to provide Europe with a uniform measuring procedure for assessing the microbiological contamination of workplaces.

ln addition, the workshop participants resolved to initiate a Europeon project in the 4'' EU framework programme for Research and T echnological Development (RTD) within the field of Standards, Measurement and Testing (SMT), III "Measurements related to the Needs of Society''. ln addition to the development of new and/or improved detection methods for microorganisms at the workplace, the aim of the draft project "Bioaerosols - Development of Standardised Measurements of Methods for Microorganisms and Endotoxin - lnterLaboratory Comparison", submitted to the EU in November 1995, was also to harmonise the established standardised measuring procedures by way of carrying out Europeon inter-laboratory tests with standard contaminated filter samp-

les. The project, designed in 3 phases, is also intended to provide particular specialist support for the work of the Europeon standards committee CEN/ TC 137WG5.

The contributions from the 11 Microorganisms" workshop, jointly organised and implemented by NIOH, Sweden ond BIA, Germony will be pr"tnted in

English. This is to ensure the Europewide distribution and acceptance of the present BIA report, the necessity of which can be seen from the current situation and the Europeon dimension of the subject of "biological agents". The numerous enquiries already made from other Europeon countries, outside the EU, are evidence of the interest in this report.

13

Different Environments Carrying the Risk of

Disease Caused by Exposure to Fungoi Spores

Swedish Experiences

Göran Blomquist, National Institute of Occupational Health,

Umea, Sweden

Abstract

Airborne biological particles in the work environment which may cause allergic symptoms and other diseases ore octinomycetes, algae, animol dander, bacteria, fungal propagules, mites and their faeces, pellen, spores, viruses and yeasts. T o study these diseases measurements that accurately describe the bioaerosol are necessary. Sampling of biological particles differs from other types of sampl·mg in that the partdes of biological origin are often viable, and that the sampling method should not affect the viability of the particle. The choice of method for sampling and analysis depends on many factors, such as environment, agent, Ievei of airborne microorganisms and analysis. When sampling microorganisms, the sampling time is a factor of crucial importance because the bioaerosol varies with time depending on different factors. This is also discussed in the article.

The different sampling methods thot have been used ore slit somplers, coscode impoctors (Andersen sompler) and filters. The disadvantage of the slit sampler is that they yield no infarmation on the size distribution of the bioaerosol, this is obtained by using the Anderson sampler. The methods used for analysis of the samples are discussed in the paper.

Measurements of different microorganisms have been performed in a variety of work environments in Sweden, such os wood-trimming deportments in sowmills; farms; pigsties; wood chip handling; citrus fruit handling; peat bogs; pototo storage; greenhouses; offices; museum ond waste sorting. These results are discussed in the paper. The highest Ieveis of airborne microorgonisms are usuolly found in the form environment, where Ieveis of airborne microorganisms higher thon 1 0 8/m3 have been registered.

17

Different Environments Carrying the Risk of Disease Caused by Exposure to Fungoi Spores

Swedish Experiences

Göran Blamquist, National Institute of Occupational Health,

Umea, Sweden

lntroduction

Airborne biological portides in the work environment that may cause allergic symptoms and other diseoses are pellen, mites and their faeces, animal dander, viruses, fungal propagules, spores, bacteria, yeasts, actinomycetes end algae. The nature of the partide that causes problems for the worker is strongly dependent on the work environment. Thus, it has been shown that airborne fungal spores and bacteria can cause allergic alveolitis (AA) or Organic Dust T oxic Syndrome (ODTS) in farms and in the wood trimming departments of sawmills, and during handling of wood fuel chips. [I, 2, 3]1n other cases airborne bacteria are mainly responsible for causing the disease, as for example in waste-water cleaning plants [4]. ln addition to the partides, secondary metabolites formed by the microorganism or parts of the cell wall, such as endetaxins and glucans, are often suspected of causing problems for the worker. This complex situation puts greot demands an sampling to identify the actual exposure.

T o study these environments, measurements thot accurately describe the bioaerosol are necessary. Sampling of biological portides differs from other types of sampling in that the portides of biologicol origin are often viable, and

that the sampling method should not af

fect the viability of the partide. The choice of method for sampling and analysis depends on many factors, such as env·1ronment, agent, Ievei of airborne microorganisms and analysis [5]. This poper will mainly discuss meosurements of fungol spores performed in different work environments in Sweden.

Sampling criteria

ln many work environments the process in question creotes o high Ievei of airborne microorganisms, while in other environments mouldy materiols are handled, possibly causing problems for the worker.

Meosurements of airborne microorganisms are of the utmost importonce in investigating o diseose. Another reason for performing measurements is thot the effect of action taken to diminish the concentration of airborne microorganisms can be controlled. A third reoson pertains to new processes or techniques thot emit microorganisms or involve an uncontrolled growth of microorganisms.

Before measurements are performed, it is important that underlying reasons should be considered carefully. ln the wood-trimming departments of sawmills or when wood fuel chips are handled,

19



Swedish Experiences

extrinsic allergic alveolitis or ODTS has been found to be caused by airborne fungal spores. High concentrations of bacteria in sewage-cleaning plonts can also cause health problems and in farms high Ieveis of airborne fungal spores ond bacteria or mites ore known to

cause diseases.

When sampling microorganisms, the choice of sampling time is o foctor of crucial importance. The bioaerosol varies with time depending on several factors. Outdoor airborne microorganisms vary, depending on the season. The highest Ieveis are usually registered in lote summer when the fungi sporulate; concentrations up to some 1 0,000 spores per cubic metre may occur. At an indoor workplace, the variation may be quite different. The concentration of airborne fungal spores emitted into the air in a potato storehause reoches its peak during winter. This is caused by the fact that the activities are highest du ring the winter seoson. When growth is suspected in humidifier or ventilation systems, the measurements should be performed when the ventilation system or humidifier is switched on. ln investigating a disease, the times when the highest concentrations may be suspected should be chosen for making observations. Such measurements are called "warst case" measurements. By inspection of the workplace it is often

20

possible to decide when the warst case usually arises. ln farms the warst case occurs when straw, hay or grain are handled [6]. ln other cases it may be difficult to decide when the warst case occurs, in such cases a series of measurements must be performed during the day to identify the warst case. T able I shows the variation of airborne fungal spores at a heating plant using wood chips as fuel. As the table shows, the concentration varies drastically between different days.

Toble 1: Variation of oirborne fungol propogules ot a heoting plant using wood fuel chips as fuel. The measurements were performed when the chips were dumped from lorries at the heating plant. Six somples were collected an each occasion.

Date Before dumping CFU/m3

20/ 9 106 - 107

21/ 9 104

27/ 9 103 - 10'

29/ 9 104 - 106

1/10 lOS- 107

13/10 IO'

2/11 1 o3 - IO'

15/11 106 - 107

22/ 2 I 03 - IO'

24/ 2 104 - I 05

T able 2, Concentration of airborne fungal propagules during autumn and winter. The spores were collected on polycarbonate filters and analysed by cultivation (malt extract agar) or by acridine orange staining and microscopy. At least six samples were takenon each occasion.

Plant Autumn Winter CFU Total CFU

A 103 -3·106 9·106 2·104 - 6·105

B 2·106 - 3·106 2·107 6·103 - 8·104

c 8 ·I 05 - 107 9·107 104 -2·105

D 2·107 - 4· 107 4· I 07 - 6·1 07 8·103 104

E 6·106 - I 07 2·107 -5· 107 2· 103 - 2·106

F 2·107 - 3·108 5·10' -3· 108 2· 103 - 104

ßecause of aerosol-creating processes outside the workplace, the concentration might show extreme variation during the day. The choice of sampling method consequently depends on the environment studied and of the fact that the microorganisms are viable. The sampling method chosen should not affect the viability of the microorganisms. On the other hand, when impactors such as slit samp\ers and Andersen samplers are used, it should be borne in mind that they are easily overloaded. [8] Therefore, different sampling techniques are usual/y used when sampling is performed in environments with low or high concentrations of airborne microorganisms.

Sampling

A wide range of microorganisms occur in the work environment. The organisms may differ widely in size, as bacteria with a diameter of 0.5- I 0 11m and spores with diameters between l .um and I 00 iJ.m. Sampling of microorganisms in such an environment puts large demands on the sampling methods to enable a representative sample describing the microbial flora to be obtained. Sometimes it is important that the sampling method should not affect the viability of the microorganisms, as is the case when qualitative analysis is performed. ln other cases, measurement of only the total number of microorganisms is of

21



Swedish Experiences

interest, for example, when the risk of allergic alveolitis is investigated. The main principles for sampling microorganisms ore based on either impaction or filtration. The impaction may eilher be performed on a gel, tape or a liquid film. The filtration may be performed using gelatine filters, polycarbonate filters or cellulose acetote - cellulose nitrate filters.

lmpactors

The most commonly used techniques for sampling microorganisms on gels by impaction enteil the use of slit samplers or cascade impactors, such as the Andersen sampler. ln 1941 Bauedillion described a slit sampler that could be used for sampling and enumeration of bacteria [9]. Slit samplers have since then been used routinely for sampling airborne bacteria, fungal spores and

pollens.

The slit samplers are very useful at low Ieveis of airborne microorganisms and have therefore found application in hospitals and pharmaceuticol companies. Outdoor registration of pollens is

routinely performed to help people suffering from allergy. Lately, much interest has been focused on the problems caused by microorgonisms in the indoor environment. Slit samplers and cascade

22

impactors have been used in severa! studies, as in the office sickness project in northern Sweden [I 0]. ln these environments the spore Ieveis rarely exceed I 0 4 CFU/m3 air. ln the form environment or when wood fuel chips are handled, much higher concentrations of airborne micro-organisms are present. Concentrations between 1 0 7/m 3 and more than I 09/m 3 occur frequently [II]. ln these cases the slit samplers are rapidly overloaded and cannot be used. T o ovoid the problern of overloading, especially when fungal propagules are collected, air sampling can be performed on special collection gels, which, after sampling, are either homogenised or melted in the laboratory, and then stepwise diluted and an aliquot spread on cultivation gels. With this technique slit samplers have been used even at concentrations of 1 08/m 3 and higher [ 12]. A Iimitation of the slit sampler is that lang sampling Iimes cannot be used because of water lass from the gel, which increases the distance between the gel and the slit, affecting the sampling efficiency. Prolongation of the sampling time can be effected by using an agar gel containing glycerol and water, instead of water.

Slit samplers provide no information about the size distribution of the portides collected. Accordingly, cascode impactors, such as Andersen samp-

lers [ 13] or a multistage liquid impinger, can be used. One impinger that has found great use in heavily contaminated environments is May's impinger [14]. The Andersen sampler is widely used in indoor as weil as industrial environments.

Filters

Cellulose acetote filters, gelatine filters and polycarbonate filters have been used for sampling microorganisms in different environments. During the post decade, polycarbonate filters have been used for sampling microorganisms in different work environments [15, 16]. They can easily be dispatched to occupational hygienists for sampling and returned to the Iabaratory for analysis. Eduard et al showed that the method of transportation of the samples did not affect sample analysis [16]. According to the CAMNEA-technique, the microorganisms on the filter are first suspended in peptone-water solution and then analysed by cultivation and epifluorescence microscopy [I 7].

Eduard et al showed that the analysis of microorgonisms sompled on Nuclepore filters is dependent on the microscopic method used [16]. He found that, by electron microscopy or light microscopy, up to twice as many microorganisms

were discovered as cornpared with the CAMNEA-technique. Cultivation also gave a lower value since the non-viable microorgonisms are not registered.

The fungal spores in an aerosol consist of single spores and aggregates of two or more spores. The fungal aerosol was created by blowing air over a cultivation plate containing Penicillium commune cultivoted for 20 days. The aerosol was collected on polycarbonate filters and analysed eilher by scanning electron microscopy (SEM) or by epifluorescence microscopy (EP). The result showed, that when the aerosol was suspended in water or a peptonewater solution, the aggregates in the aerosol break down into single spores or smaller aggregates (table 3, see poge 24). When analysed by SEM, araund 30 o/o of the fungal particles are composed of aggregates of three spores or more, while only araund 20 % are detected with EP.

Environments

Measurements of different microorganisms have been performed in a variety of work environments in Sweden. ln wood-trimming deportments of sowmills, the Ievei of airborne funga\ spores varied between I 03 - I 05 spores1m3 du ring normal work. The wood was arti-

23



Swedish Experiences

T able 3, Analysis of fungol Spores sampled with polycarbonate filter by SEM and epifluorescence microscopy (EP).

Methad n/m3

SEM 3, I ·I 08

SEM 3.2·108

EP 2.2·108

EP 2.3·1 08

EP 2.3· 108

EP 1.9·108

ficiolly dried and sometimes became mouldy du ring that process. Under such conditions, Ieveis up to 1 0 8/m3 have been registered, using the CAMNEAtechnique. The mostfrequent fungal species were Aspergillus fumigatus and Rhizopus rhizopodiformis.

A work area that developed rapidly under the eighties was wood chip handling. The wood chips were used as a substitute for oil at heating plants. Workplaces where AA was reported were found in different regions of Sweden. The highest Ieveis of airborne microorganisms were usually registered during the autumn season with Ieveis of airborne fungol spores up to l 0 9/m 3

.

When fresh chips were handled, the Ieveis of oirborne microorganisms usually varied between I 0 4 and 106 Other

24

I

36 35 38 36 33 42

o/o of spores in aggregate of

2-3 > 3 spores

33 31 33 32 45 17 41 23 42 25 39 19

types of organic materials that that have found use as fuel for heating include peat and pellets made of bark and chaff. Measurement of stored pellets showed considerable fungal growth when the pellets were stored under humid conditions. At a peat bog the Ieveis of airborne fungol spores were up to 1 05/m 3 outside the tractor cabin. Because of the filter in the air in Iet, the Ieveis were about 1 02 lower inside the cabin. At these Ieveis no cases of ODTS or AA were registered in Sweden.

High Ieveis of airborne fungal Spores were also measured when mouldy books were handled at a museum. The fungal flora was mainly composed of Aspergillus versicolor. The concentration of viable spores was I 06 per m3 air while the total number was 108 .

A woman working with the mouldy books suffered from symptoms described as ODTS. [ 18] These high concentrations of airborne fungal spores were even found in a fruit-sorting department of a large ware-hause for vegetables and fruits. [ 19] A man

Table 4,

engaged in sorting mouldy citrus fruits was exposed to 7 · 1 08 spores per m3 . The dominafing species was Penicillium italicum. Although subjected to this high exposure, the worker did not suffer from ODTS or AA.

Airborne, fungol spores in different work environments

Farms

Pig houses Wood chip handling

Citrus fruit handling

Peat bog

Potato storage

Greenhauses

Offices

Museum

Waste sorting

ln other types of storage, such as potato storage, the workers are exposed to high amounts of fungal spores without suffering from any disease. ln Sweden potatoes are stored indoors at a temperature that prevents them from freezing during winter. The highest spare Ievels are registered during the winter months, when the

Numberlm3 AA.

>JOB Yes

>108 Yes

104 - 106 No 108 No?

103 - 105 No 103 -106 No 103 - 105 No

> 103 No ]Q8 Yes

103 - 105 No

potatoes are dry and when the potatoes that have been damaged in harvesting have started to mould. The Ieveis at that time vary between I 04 CFU/m3 and I 0 6 CFU/m3 Lately, manual waste sorting has been used in Sweden to diminish hausehold waste. So far 1he Ieveis registered have not exceeded I 05/m3 No cases of AA or

25



Swedish Experiences

ODTS have so far been reported in Sweden.

ln the non-industriol indoor environment, the Ieveis of airborne fungal spores are much lower. A questionnaire was sent out to about 6,000 workers in about 160 buildings in northern Sweden. Fifteen workplaces with a high prevalence of Siek Building Syndrome and 15 workplaces with a low prevalence were chosen from the replies to the questionnaire. The result showed that the Ieveis of colony-forming units per m3 varied between I 0 and 300 per m3

. The registered values did not differ from outdoor measurements performed at the same time.

Conclusions

The choice of sampling method to be used is dependent on the environment studied. Thus, in the indoor environment, where very low concentrations of airborne spores and bacteria are usually suspected, a sampler that collects the microorganisms an a gel or in a liquid is preferred. Sampling with the use of filters demands lang sampling times, which may affect the viability of bacteria in particular. ln the industrial environment, where high concentrations of airborne microorganisms are suspected, usually I 0 - I 06 Iimes higher,

26

sampling of microorganisms with the use of filters as collection media has been applied. The onalytical method used depends on the problem studied. Determination of the total number of microorganisms is recommended when a disease is suspected, while cultivation must be undertaken when species analysis is performed.

References

[I J Rylonder, R., Lung diseases coused by organic dusts in the form environment. Am. J. lndust. Med. I 0, 1986, 221 - 227

[2] Ko/modin-Hedmon, 8., 8/omquist, G., and Lölgren, F., Chipped wood os o source of mould exposure. Europ. J. of Respir. Dis. suppl. No. 154, Val. 71, 1987, 44- 51

[3] Be/in, l., Clinical and immunological data on wood trimmer's disease in Sweden. J. Respir. Dis. (Suppl.) I 07, I 980, I 69 - I 7 5

[4] Outkiewicz, J., Joblonski, l., ond 0/enchock, S.A., Am. J. lnd. Med 14, 1988, 605

[5] 8/omquisl, G., Sampling of Biological Particles. Analyst vol. 119, 1994, 53- 56

[6]lundgren, R., Bierrner, L., Rosenhall, L., and Blomquist, G., Work with mouldy straw causes often allergic

alveolitis among farmers. ln, Swedish, läkartidningen 85A2, 1988, 3495- 3463

[7] Bovallius, A., Bucht, B., Roffey, R., and Aniis, P.: Three year investigotion of the natural airborne bacterial flora of four localities in Sweden. Appl. Environm. Microbiol., May 1978, 847- 852

[8] Blomquist, G., Palmgren, U., and Ström, G., lmproved techniques for sompling airborne fungal portides in highly contaminated enviromnents.

Scand. J. Work Environ. Health I 0, 1984, 253- 258

[9] Bourdillion, R.B., lidwe/1, O.M., and Thomas, J.C., A slit-sompler for collecting and counting airborne bacteria. J. Hyg. 41, 1941, 97 - 224

[10] Blomquist, G., and Andersson, B., ln, Proceedings of the workshop on health. lmplications of fungi, in Baarn nov. 1992, Editor Samsan R.

[I I] Malmberg, P., Rask Andersen, A., Polmgren, U., Höglund, S., KolmodinHedmon, B., Stalenhielm G., Exposure to microorganisms, febrile and airvvayobstructive symptoms, immune status

and lung function in Swedish farmers.

Scand J. Work Environ. Health II, 1985, 287 - 293

[I 2] Blomquist, G., Strom, G., and Strömqvist, L.H., Sampling of high concentrations of airborne fungi. Scand. J. Work Environ. Health 10, 1984, 109- 113

[13] Andersen, A.A., New sompler for collection, sizing ond enumeration of

viable airborne partie/es. J. Bacteriol. 76, 1958, 471 - 484

[14] May, K.R., Multistage liquid impinger. Bact. rev. 30,3, 1966, 559- 570

[ 15] Blomquist, G., Palmgren, U., and Ström, G., Methodological aspects of measurement of exposure to mould.

Europ. J. of Repir. Dis. Suppl. No. 154, vol. 71, 1987,29-36

[16] Wiinand, E., Lacey, J., Karisson, K., Palmgren, U., Strom, G., and 8/omquist, G., Evaluation of Methods for Enumerating Microorganisms in Filter Sampies from Highly Cantaminoted occupational Environments. Am. lnd. Hyg. Assoc. J. 51 ,8, 1990, 427- 436

[I 7] Palmgren, U., Strom, G., Blomquist, G., and Malmberg, P., Collection of airborne Microorganisms on Nudeopore filters, Estimation and Analysis -CAMNEA method. J. Appl. Bact. 61, 1986, 401 - 406

27



Swedish Experiences

[ 18] Kolmodin-Hedmon, B., Blom-quisl, G., ond Sikström, E., Mould exposure in museum personnel. lnt. Arch. Occup. Environ. Heallh 57, 1986, 321 - 323

28

[19] Strom, G., ond Blomquisl, G., Airborne spores from mouldy citrus fruit - A potential occupational hazord. Ann. Occ. Hyg. 30,4, 1986, 455- 460

Microorganisms and their Products

in Occupational Environments

John lacey, IACR - Rothamsted, Harpenden, Un.lted K"mgdom

Abstract

Microorganisms occur in many work

places, where they can be released from many different sources into the a·lr.

Depending on their specific properties, the exposure circumstances and the personal disposition of the workers, microorganisms can trigger various illnesses. Certain viruses, bacteria and fungi, occurring as pothagenie and/or opportunistic pothagenie species, can cause more or less specific infections.

ln addition, it is possible that through their allergising and toxic properties primarily non-infectious germs can make

the persons exposed ill. ln particular, this pothagenie effect of microorganisms also arises as a result of the quantitative effects of the various components that make up one microorganism. Examples of diseases transmitted in this way are obstructive respiratory tract illnesses and extrinsic allergic alveolitides (e.g. mould fungi, actinomycetes) as weil as "ergonie dust toxic syndrome" for which

endotoxins oct os the co-triggering agent. ln addition, fungi, which cantain mycotoxins, can have toxic effects.

Different microorgonisms and their constituents and products are described on the basis of their detection, by way of an exomple, ot various workplaces. Descriptions are also provided of the illnesses they cause. Viruses, bacteria (Chlamydia, Gram-negative bacteria, legionellae, Gram-positive cocci, bacilli, corynebacteria, mycobacteria, including actinomycetes) and filament fungi (ascomycetes, basidiomycetes) are mentioned. The occurrence of protozoa (amoebae), insects and mites at the workplace is also dealt with. Discussion of the possible health risks caused by endotoxins, glucanes, enzymes, proteins and other allergenic and toxic constituents of microorganisms, plants and animals forms the concluding section and rounds off the now very wide spectrum of oll the possible biological Iadars that have a potential effect on the worker.

29



John Lacey, IACR - Rothamsted, Harpenden, United Kingdom

lntroduction

Airborne microorganisms of many different types may occur on occupational environments and present hazards to the health of workers. They come from many different sources and their composition depends on the source material and, often, the way in which this has been stored. Depending on their composition, the immunological reactivity of the exposed workforce and the circumstances of exposure, they may cause a range of occupational diseases, through infection and also through noninfectious and often immunological mechanisms.

lnfection may be caused by viruses, bacteria and fungi. Viruses and bacteria are often primary pathogens and usually originate from animal sources. Fungi are more often opportunistic pothogens originoting from plant Sources where they grow os saprophytes. lnfection may be aided by immunodeficiency, resulting from underlying diseose or therapeutic treatments, or perhops resulting from mycotoxins in the dust.

Non-infectious occupational diseases, resulting from the ollergenic ond/or immunotoxic properfies of airborne biological agents, have been recognised in many diHerent situotions. The dassie examples are byssinosis and farmer's

lung, although similar occupotional lung diseases were described by Ramazzini as early as 1713. Endotoxins from Gram-negative bacteria have been implicated in byssinosis and airborne spores of thermophilic actinomycetes and, sometimes, fungi in farmer's lung. T ypical concentration ranges for some components of airborne dust on farms and related environments are shown in Table I (see poge 32). Recently, the development of biotechnological processes, utilising microorgonisms to produce usefvl pharmacevtical prodvcts, enzymes and food substitutes, has created new environments where occupational exposure to microorganisms can occur.

This article will describe the ran_ge of microorganisms implicated in occupationol lung diseases. References are not given but will be found in Lacey and Dutkiewicz ( 1994).

Microorganisms in occupational enviranment

Viruses

Two groups of viruses, referred to, respectively, as anthropogenic and zoonotic, can be implicated in occupotional infections. Anthropogenie viruses are carried in droplet aerosols produced

31



Table I, Concentrations of some microorgonisms and their products in some occupational environments (after Lacey and Dutkiewicz, 1994)

Agent Unit of Cow shed' measurement

Total micro-

organisms cells m- 3 -lQ-6 0.4 ~ 30.5

Viable (v) or total (t) fungi cfLI m-3.1Q-3 1.0- 7.2 ,,,

Viable (v) or total (t) bacteria dc m-3.10-3 43.9 ~ 281 ,,,

Aflatoxin !-lg/m3 N/D

Endotoxin ~g/m 3 0.001 ~ o.oJ? H

Plant (pj/ani-mal (a) ollergen t~glm 3 0.04 ~ 9.5 jo)

from infected people during coughing or sneezing and creote potential hazards for medical staff, dentists, Iabaratory personnel, social care workers ond teochers. They include rubella, Influenza, mumps, herpes simplex and respiratory-syncytial viruses, adenoviruses, reoviruses and cytomegalovirus. Zoonotic viruses primarily cause disease in animals but can also infect farmers, poultry processing workers and others. They may be dispersed in dust, e.g., from poultny infected with Newcastle disease virus, or in droplet oerosols, e.g., from cattle ond pigs infected with foot and mouth disease or vesicular stomatitis viruses.

32

Harvesting grain Grainstore Sugar beet foctory

0.49~213 20.2 ~ 2860 N/D

3.5~210jtl 2.4 ~ 45 ,,, 0.8 ~ 2.3 (,)

0.08 ~ 37.8 (I) 20.2 ~ 12401,1 82~203(,)

up to 1.6 0.0~0.107 N/0

N/0 up to 54.9 0.003 ~ 0.03

N/D N/0 3.5 (p)

Bacteria and actinomycetes

Some spedes of bacteria cavse infection but the greatest hazard is usually cavsed through allergenic and/or immunotoxic effects. The organisms most frequently implicated are Gram-negative bacteria and actinomycetes, often present in large numbers from plants or animals. Beeterio in work environments include:

Rickettsiae ond chlamydiae

The zoonotic species, Coxie//a burnetii, from sheep's wool end excreta, can

couse Q fever and Chlomydio psitfoci, in dust derived from the excreta of ducks, chickens, turkeys, geese, pigeons, parrots and other birds, can cause ornithosis.

Gram-negative bacteria

Bruceffa suis, the cause of brucellosis in man and swine, can be spread in oerosols through sloughterhouses while tularaemia bacilli (Francisella tularensis) occur in the air of sugar beet factories in Centrd end Eastern Europe, when roots ore washed ond in dust from grain, hay, straw and sugar beets, both as a result of contamination by infected rodents. legionella is probably the most important infectious non-zoonotic Gram-negative bacterium but potentially infectious Salmonella spp. can occur in aerosols produced from sewage. Other Gramnegative bocteria may cause opportunistic infections in immunocompromised people and/or in some specific environmental conditions, e.g. Acinetobocter calcoaceticus infection of faundry workers exposed to respirable metallic dust.

A wide range of Gram-negative bocteria of plant origin present potential respirotory hozards os sources of endotoxin and allergens. The best known is the

epiphytic Enferobacter agglomerans (syn. Pantoea agglomerans, Erwinia herbicola). This species occurs on a range af plants and plant products, especially on cereal grains and cotton bracts, and is characterised by chromogenic, yellow, facultatively anaerobic, fermentative rods with peritr\chous flagella and it produces a strong endotoxin and is a cause of allergic alveolitis. Other Gram-negative bacteria common in organ·lc dusts include species of Pseudomonas, Klebsiella, Alcali-genes ond Acinetobacter. Cytophago aflerginoe, Pseudomonas, including Ps. pseudoolcaligenes and Ps. tesferonilalcaligenes, Flavobaclerium and Aeromonas hydrophile may be present in aerosols produced by the holding tanks for cutting oils on metalworking machines, humidifiers end sewage treatment plants. Concentrations close to metalworking mochines may reoch I 0 3 to I 08 bacteria m-3 air.

Gram-positive cocci

Staphylococci and, less often, streptococci form a large proportion of the bocteria in bioaerosols in animol breeding and processing facilities. However, the role of these cocci in cousing respiratory disorders in exposed workers is still uncertain.

33



Spore-forming bacilli

Bacillus species are often numerous in organic dusts but they have seldom been implicated in respiratory disease. However, aeroso(s of B. subtifis and B. licheniformis were formed during bathroom remodelling, and B. subtilis was implicated in allergic alvealitis.

Corynebocteria

Like cocci, corynebacteria form a large part of the bioaerosol in animal breeding and processing facilities and they may also occur in /arge numbers in dusts of plant arigin. Arthrabacter spp. and Brevibacterium Jinens have been implicated in allergic alveolitis in Polish farmers.

Mycobacteria

lnfection with tuberde bacilli (Mycobacterium tuberculosis) is, perhaps, an increasing hazord for clinicians and workers in medical diagnostic laboratories. Acquisition is usually by aerosols generated at the bench, du ring the manipulation of cultures or specimens or at post-mortem examinations, during the removal of organs or the use of highspeed saws.

34

Actinomycetes

Actinomycetes are Gram-positive, filamentaus bocteria. Many species have spores about l mm diameter which easily become airborne and which penetrate deeply into the lung on inhalation. Some actinomycetes, e.g., Nocardia asteroides, can cause infection, especially in immunodeficient patients. Other species are implicated in allergic alveolitis, e.g. Saccharopolyspora rectivirgula (Synonyms: Faenia rec

tivirgulo, Micropolyspora faeni), Thermoactinomyces vulgoris, T. thalpophilus and Saccharomonospora viridis. All these species are characteristic components of the microflora of hays that have been baled weiter than about 35 % water content and which have

heated to 50 to 65 °C, giving rise to up to l 0 10 spores m- 3 air. Thermoactinemyces sacchari occurs in bales of sugar cane bagasse, is present in fewer samples but is more often abundant and although Thermooctinomyces thalpophi/us is present in most samples, it is never found in such large numbers as T. sacchari which seems to be the more important cause of bagassosis. Thermomonospora spp., especially T. fusca, T. curvata and T. chromogena, become abundant during composting for both mushroom production and the disposal of municipal wastes in which temperatures of 55 to 60 °C occur. Streplo-

myces spp. are also important in biooerasals originating from soil, hay and other plant materials. Streptomyces albus, S. olivaceus and S. thermohygroscopicus have oll been implicated in the etiology of allergic alveolitis.

Fungi

Filamentaus fungi

Fungi are ubiquitous in the environment, causing diseases of plants and animals, colonising plant surfaces as saprophytes and decomposing organic matter. Many species produce abundant spores which easily dispersed in the air. Many fungi are weil known as allergens but some species can cause infection and occupational disease. Large numbers of fungal spores are present in the dust from both the cutter bar and the rear of combine harvesters when harvesting; cereal crops can contain up to about 2.0 · 108

spores m-3 air, while drivers can be exposed to about 10 % of this concentration. Cladosporium accounts for up to 75 % of the spores, Alternaria for up to 25 % and Verticillium lecanii for up to 10 %. No other spore type accounts for more than about 5 % of the total. Farm workers affected by the dust gave positive skin tests or yielded precipitins in gel diffusion tests to extracts of V. lecanii which has spores

about 3 · I mm. Spores of some pathogens of wheat, such as rusts (Puccinia graminis) and smuts (Ustilago and Tilletia spp.), which cannot be isolated in culture have caused rhinitis, asthma and coniunctivitis in farmers, millers, granary workers and others.

Fungoi spores are often abundant in stored hay and cereal grains, tagether with actinomycetes if the storage conditions are favourable, and many have been implicated in asthma and allergic alveolitis. Same species, particularly Eurotium spp. (Aspergillus glaucus group) and Wallemia sebi, are xerophilic and require media with low water activities for their isolation, such as DG 18. The predominant Aspergillus spp. generally give a good indication of the previous storage conditions and A. fumigatus is offen common in hay baled at about 35 % water content which has heated to 50 oc or above. Eurotium rubrum, a cause of farmer's lung in Finland is most abundant at water activities of about 0.85 Ow.

Other fungi implicated in allergic alveolitis include Penicillium glabrum (syn. Penicil/ium frequentans) in suberosis; Cryptostroma corticale in maple bark disease; Aspergillus clavatus in malt worker's lung; Rhizopus microsporus vor. rhizopodiformis in respiratory disease of wood trimmers in Norway; Penicillium

35

Microorganisms and their Products in Occupational Environments

spp. also in wood dust and in cheese washer's disease; Aureobasidium pul/ulons and Graphium spp. in sequoiosis, although both species are unusual in producing slimy spores that might not be expected to easily become airborne; Eurotium sp. from campest implicated in mushroom worker's disease in Japan; and Wo/lemia sebi, a xerophile from dried fruit and flour in bakeries, from coffee beans, from hay and in indoor environments.

Edible fungi (Basidiomycetes)

Spores of the commonly cultivated Pleurotus ostreotus (oyster mushroom), Lentinus edodes and Pholiota name

ko are highly allergenic and can cause allergic alveol"1tis and asthma in exposed persons. However, none of these species will sporulate in culture although their spores may be counted by light or scanning electron microscopy on spare trap slides. Symptoms of respiratory allergy have also been described in mushroom growers and processors of dried mushroom soups exposed to spores and mycelium of Agaricus bisporus and other mushroom species (Lentinus edodes, Boletus edu/is).

36

Protozoa

Amoebae of the genera Acanthamoeba ond Noeglerio ore found in a wide ronge of oquatic habitats, including industrial humidification systems. They have been implicoted in infection ond humidHier fever in workers inholing the oerosol from contominated woter but confirmotion is required. These protozoo moy also serve os vectors of Legione/lo bacteria, trapping them within phagocytic vocuoles ond so proteefing from destruction in chlorinated woter.

lnsects

Airborne portides of insect origin, e.g., poisonous hairs, body frogments, excreto, moy couse asthma, rhinitis, conjunctivitis ond dermatitis. lnsects ore used widely in loborotories for reseorch purposes and allergy to them or to their products has been identified in Iaboretory workers. Allergy has also been observed in foresters, silk producers, granary workers, workers of food industry and farmers. Gypsy moths (Lymantria dispar), Douglas fir tussock moths (Orgyia pseudotsugata), silkworms (Bombyx mori), locusts (Locusto migratoria), cockrooches (Periplaneta, Blatte/la), grain weevils (Sitophilus

granarius), mealworms (T enebrio), larvae of Chironomus, and cochineal insects (Coccus cactus) are among the strengest sensitizers. Seekeepers and people processing honey may become allergic to honeybee-body dust and hive particles. The strengest hause fly allergens were found in settled dust containing faecal material while locust allergens were most potent in the peritrophic membrane. Allergens to both insects were detected in air samples.

Mites

Hause dust mites (Dermatophagoides spp.) areweil recognised as potent allergens but much less attention has been paid to the role of storage mites in occupational allergy. Large populations of these mites can develop in stored hay and grain, often when these are too dry for fungal colonisation. However, there are also close interactions between mite populations and fungi. Mite respiration can release water and cause heating, making conditions more suitable for fungal invasion while some storoge mites are mycophagous and can feed and complete their life cycles only an fungi. The most numerous species found in hay, grain, flour and sometimes in houses include Acarus siro, A. farris, Lepidoglyphus destructor, Glycyphagus

domesticus, Tyrophagus putrescentiae, T arsonemus sp., Tydeus interruptus and Cheyletus eruditus. ßy analogy with hause dust mite allergy, sensitivity is probably associated with Inhalation of the faecal pellets.

Microbial products in occupational environments

Endetoxins and glucons

Endetoxins

Endetoxins are highmolecularweight, heatstable lipopolysaccharides (LPS), consisting of a characteristic Iipid component, Iipid A, covalently bound to a heteropolysaccharide. They occur in the outer membrane of the cell walls of Gramnegative bacteria as heteropolymers with profeins and phospholipids, and can easily be released in large quantities into ergonie dusts in the form of discoid portides (microvesicles) 30 lo 50 nm in diameter with a characteristic tripletracked membrane. The quantities of endetaxins in different ergonie dusts and in their source materials (groin, cotton, herbs), as determined using o specffic assay, the Limulus amoebocyte \ysate test ranged from I 0 2 to 106 ng g- 1

• Concentrations of airborne endotoxin reported from dif-

37



ferent types of farms and industrial settings in different countries ranged from l 0 1 ta I 0 5 ng m-3 Mast determinations

exceeded a suggested th reshold Ievei af 200 ng m-3 10.2119 m-3). The greatest concentrations, 103 to 105 ng m-3, were faund when grain and other vegetable materials were being handled.

Glucans

Glucans are camponents of the cell walls of fungi and may also be secreted by Iew bacteria, e.g. A/ca/igenes faecalis. Glucans from fungi consist of a chain d glucopyranose rings, united primarily by I I ~ 3)-ß-D-polyglucoside linkages. I I ~3)-ß-D-glucans can stimulate the reticuloendothelial system and cause a variety of biological effects, primarily through an activation of macrophages. They cause a delayed effect appearing 3 to 7 days alter exposure and can also cause cell sensitization.

Recently, Rylander et al. have suggested that I I ~3)-ß-D-glucans, released from the cell walls of fungi or bacteria into airborne ergonie dusts, may cause ehrenie symptoms on inhalation, in

cluding ehrenie byssinosis or sick hause syndrome lbuilding-related disease). Concentrations of glucans in the air of cotton cardrooms were within the range

38

of 280 to 4330 ng m-3 Although concentrations in the air of sick office buildings were much smaller 10.06 to 0.55 ng m-3), there was still a significant correlation betvveen concentrations of I I ~ 3)-ß-D-glucan and the occurrence of respiratory symptoms in exposed workers. This could indicate a possible role for I I ~ 3)-ß-D-glucans in the etiology of chronic work-related diseases caused by the inhalation of the airborne ergonie dusts.

Mycotoxins

Mycotoxins are low-molecular-weight toxic secondary metabolites of fungi produced during their growth an growing and stored crops and foods. Among the most important are aflatoxins produced by Aspergillus flovus and A. porasiticus, ochratoxin A produced by Penicil/ium verrucosum and Aspergillus alufaceus (A. ochroceus) and the trichothecenes and fumonisins produced by Fusarium spp. Mycotoxins characteristic of the species have been reported in the spores of Fusarium graminearum, F. sporotrichioides, F. moniliforme, Stachybotrys otro lsyn. Stachybotrys chartorum), Penicillium expansum, P. brevicompactum, Aspergillus versico/or and A. flovus!porasiticus. Spores of A. f/avus and A. parosilicus, which are often airborne when

affected crops are handled, may contain up to 84 to 200 119 aflatoxin g- 1

• There is circumstantial evidence, from their presence in settled dust deposits, that other mycotoxins can occur in air. For instance, secalenie acid D may occur in dust deposits in maize elevators and macrocyclic trichothecenes, including satratoxin H, in aerosolised spores of Stachybotrys afra isoloted from mouldy buildings. However, so far mycotoxins have been found in occupational cerosals in only small quantities olthough heavy exposures to the spores of toxigenic species may sometimes occur.

There is streng evidence that aflatoxins and some other mycotoxins, e.g., secalenie acid D, moy be airborne in /arge concentrations in some work environments, as when processing peanuts and maize, and may be sufficient to cause toxic or carcinogenic effects.

Volotile metabolites produced by fungi couse musty odours in mouldy buildings and may cause o range of symptoms on inhalation. The predominant volotile produced by a ronge of fungi is 1-octen-3-ol but ethyl acetote, ethanol, butonol and other short-choin alcohols ond aldehydes con also be produced. The eorthy odour alten reported is due to 2-octen-1-ol and geosmin, the Ietter a product also of some actinomycetes.

Enzymes and cell products

Enzymes are essential for the metabolism and growth of microorganisms and can be detected in their spores. lt is therefore not surprising that antibodies against enzymes from the spores of 5. rectivirgula have been demonstrated in farmer' s lung patients. However, many enzymes are now produced industrially from microorganisms tagether with antibiolies and other microbial products, some from genetically engineered species, and aerosols may form as a consequence of leaks in fermentation systems or when contoinment systems fail. Proteolytic enzymes of Bacillus subtilis (subtilisins) can be released into the air of factories during the production of "biological" washing pawders and couse pulmonary disorders in exposed workers. Göthe et al. (1972) found a meon concentrations in two detergent factories, respectively, of 17.5 ng subtilisins m-3 air (activity equivalent to 0.7 glycine units [GU] m-3) and 130.0 ng m-3 (5.4 GU m-3). The greater concentration exceeded the Ihreshold Ievei of 24 ng m-3 ( 1.0 GU m-3). There is little other data on amounts in the air but sensitisation has been reported an a number of occasions. Pharmaceutical and Iaboretory workers may also be exposed to aerosols of powdered enzymes of mammals such as pepsin ond trypsin.

39



Allergenie and toxic substances of plant origin

Apart from their content of microorganisms, airborne dusfs from crushed or pulverized plant materials may contain plant materials that may be allergenic.

These may include pollens, especially of grasses and, in some countries, ragweed (Ambrosia spp.); dusts from tea, coffee, vanilla, soybean, castor beon, rice, herbs, buckwheat; the tuberaus root of devil 's tongue (Amorphophollus koniac cultivated in Japan, the source of "Moiko" dust); powdered plant tissues used as drugs, e.g., psyllium, ipecacuanha; plant proteases (papain from Carica papaya, bromelain from Ananas comosus) ond, perhaps, tannins. Plant allergens may also cause allergic alveolitis, as in stipatosis caused by low molecular weight allergens in esparto gross (Stipo lenocissimaJ in Spoin. Wood dust, particularly from deciduous trees, such os oak and beech, appears to be linked to the occurrence of adenocarcinoma of the nasal sinuses and, perhops, also to lung cancer and Hodgkin's disease. The incidence of the adenocarcfnoma is ab out l 000 tim es greater in woodworkers than in other people. Other wood dusts, chiefly exotic Woods and from western red cedar (Thuio plicota), may also couse dermatitis, rhinitis, conjunctivitis and asthma. T. pJicata cantoins plicatic acid, o !ow

40

molecular weight allergen. Pine resin cantoins colophony (rosin), a solid substance used as a flux in solder or as an additive to glues, which can cause occupational asthma in electronics workers exposed to soldering fumes.

Protein aerosols

Proteins are essential components of oll cells and may form aerosols whenever cells are broken. Thus, proteins from cereal grains occur in airborne flour dust during milling ond baking, from sugor beet in the slicing area of sugar factories and from scampi in sea food processing plants. Scampi ore shelled using water jets which create fine aerosols containing up to 8.5 ,ug Scampi antigen m-3 air, as determined by large volume elecirostatic sampling and radioallergosorbent test (RAST) inhibition ossoy. Other allergies have been recorded to proteins from marine animals. For instance, hypersensitive lung disease may be coused in exposed workers by ollergenic portides from prawn (Nephrops norvegicus) ond snow crob (Chionoecetes opilio) during the production of sea food. Proteins of vertebrate animals can also give rise to asthma and allergic alveolitis, e.g., from fish meal and frogs; from epithelium, feathers and droppings of poultry; from egg allergen in egg-

L

processing plants; and from oerosols containing epithelium, hair, urine, faeces, milk and saliva ol cattle and other form animals.

Conclusion

Many different types of microorganisms can be found in the air of occupational environments presenting hazards of infection, mucous membrane irritation, immediate allergy and allergic alveolitis, toxic alveolitis ond, perhaps, carcinogenicity. Some types are hazardous in low concentrations while others require much more intense exposure to produce

symptoms. Some bioaerosols are composed of viable microorganisms, others ol non-viable particles. Consequently, different hazards require different sampling strategies and different assay methods if representative samp\es are to be obtained, microorganisms are to be quantilied and their clinical signilicance established.

References

Lacey, J., and Outkiewicz, J., Bioaerosols and occupational lung disease. Journal ol Aerosol Science 25, 1994, 1371- 1404

41

Evaluation of Methods for Quantitative Assessment of Microorganisms in Highly Cantominated Warking Environments

Wijnand Eduard, National Institute of Occupational Health, Oslo, Norway

Abstract

Exposure to microorganisms can be measured by different methods. T raditionally, mainly viable methods have been used that detect microorganisms which are able to grow in culture, but also a non-viable method based on light microscopy. More recently, nonviable methods have been developed that are based on counting of microorganisms by microscopic techniques or detection of microbial markers by chemical, bio-chemical and immunochemical methods. These methods may asess different microbial agents. At present, it is not clear which agents should be assessed, but viable methods are probably not satisfactory because

exposure to non-viable microbial agents may cause similar effects as exposure to viable agents. Relations between different methods must therefore be taken into account when resu\ts obtained by different methods are compared.

ln this presentation, viable and nonviable microscopic methods for measurement of airborne microorganism Ievei and comparative studies of these methods are reviewed and evaluated for exposure assessment purposes in the working environment. The paper focuses

on exposure assessment of non-infectious microbial agents in epidemiological studies and the potential for compliance testing.

43

Evaluation of Methods for Quantitative

Assessment of Microorganisms in Highly

Cantominated Warking Environments

Wijnand Eduard, National Institute of Occupational Health, Oslo, Norway

lntroduction

Exposure to microorganisms is common among workers handling biological materials, e.g. farmers, workers exposed to contaminated humidifiers and workers handling malt, cork, wood, waste and waste water (Lacey and Crook, 1988). Exposure to microorganisms may cause health effects, especially from the respiratory system. The viability of microorganisms is probably not essential for development of these effects (Malmberg, 1991).

Exposure Ieveis to airborne microorganisms have been assessed by different methods that are likely to yield different estimates. A main distindien is between viable methods which detect microorganisms that are able to grow in culture and non-viable methods which estimate the sum of viable and nonviable microorganisms. Recently, new non-viable methods have been developed. Microorganisms may be counted by fluorescence microscopy (Palmgren er al., 1986) and scanning electron microscopy (Eduard et al., 1988). Markers of microorganisms may be determined by chemical (Sonesson et al., 1990), bio-chemical (Obayashi, 1990) and immuno-chemical methods (Topping et al., 1985; Campbell et al., 1989). As the latter methods are in their infancy for assessment of microorgan-

isms, only the methods using culture and microscopic methods are considered in the following.

ln this presentation, viable and microscopic methods for measurement of airborne microorganism Ievei and comparative studies of these methods are reviewed and evaluated for exposure assessment purposes in the working environment. The paper focuses on exposure assessment of non-infectious microbial agents in epidemiological studies and potential for compliance testing.

Agents

Microorganisms systematically belang to different classes such as virus, bacteria, fungi, algae and protozoa. Among these, fungi and bacteria have been studied most frequently as risk factors of diseases and other health defects in the working environment. Two important groups of bacteria have been recognized: Gram-negative 1l bacteria that contain endetaxins in their cell wolls and

Gram-positive actinomycetes that have a similar life cycle as moulds and may produce large amounts of spores that

I) Bacteria can be devided by their abilitytobe stained by the Gram stoin that also devide bacteria by their cell wall structure

45




easily become airborne. Fungoi and bacterial Spores are therefore important components in microbiol aerosols. The size of spores from different species varies from 0.5 to I .5 11m for actinomycetes to 2 to I 0 11m for moulds. Spores may also be present in aggregates of up to hundreds of spores, especially spores from actinomycetes (Karlsson and Malmberg, 1989). Vegetative bacterial cells may dominate in some working environments. Single bacterial cells have small aerodynamic size similar to bacterial spores, but bacteria may be present in aggregates with larger particles. The microbial aerosol may therefore contain respirable portides as weil as tracheabronchial and extra-thoracic particles.

There is some evidence that some species have a greater potency than other species to induce health defects but further data about dose-response relationships are needed to clarify this point (Thurston et al. 1979; Baselee et al. 1983; l'ogelmark et al. 1991). The viability of moulds and actinomycetes is probably of less importance in the work environment. Aerosolized extracts of moulds and actinomycetes are also used in provocation tests of patients with extrinsic allergic alveolitis and may cause fever attacks and pulmonary reactions (Pepys and Jenkins, 1965; Parkes, 1982). However, it cannot be ruled out

46

that viable microorganisms may induce a strenger response if, after deposition in the lung, they produce antigens that are not present in dead microorganisms.

lnstead of measuring viable or nonviable microorganisms, exposure to particular toxic or allergenic constituents, metabolites and chemical markers can also be determined. Examples are the measurement of endotoxins, components of membranes of Gram-negative bacteria, (I ~3)-ß-D-glucons, components of cell walls of many moulds, allergens, mycotoxins and 3-hydroxyfatty acids as markers for endotoxins/Gram-negative bacteria. Measuring constituents and metabolites can have advantages compared to counting microorganisms as these agents may be present in the environment in measurable form even if whole microorganisms cannot be recognized (a), they are alten stable and can be measured with Ionger sampling times (b), and many constituents and metabolites are of direct etiologic relevance (c). T able I g·1ves an overview of microbial agents that have been measured.

Methods for measurement of airborne microorganisms

The measurement of microorganisms in air involves aspiration of the bioaerosol,

Table I' Microbial agents

Agent Fungi Bacteria

Gram+ Gram-

Microbial portides

- spores + + 1+1 - vegetative cells + + - viable aggregates + + +

Constituents and metabolites

- antigens and allergens + + + e.g. glucans

- taxins mycotoxins endetaxins

- chemical markers

separation of the portides from the air stream and analysis of the collected particles. Many ·Instruments have been specifically designed for analysis by culture, except that filter samples can be analysed by culture, microscopic methods, gas chromatography mass spectrometry of chemica\ markers, immuno-chemical and biochemicol detection of constituents and metabolites, e.g. antigens and toxins.

The evaluated methods have been summarized under analytical method, sampling method 1 and assessed agent in table 2 {see poge 48). Fora description of viable methods and light microscopy see Gregony (1973), for scanning elec-

+ + +

tron microscopy Eduard et al. ( 1988) and Eduard ( 1993), and for fluorescence m·,croscopy Palmgren et al. (1986).

Systematic errors

Several sources of sampling errors have been described. Aspiraf1on errors may be present for oll methods since none confirms the inhalable convention for meosurement of airborne portides ot the workplace (CEN, 1993a). lnhaloble aerosol sampling is possible for collection on filters but this has not been described for microorgonisms. Microorganisms must also be separated from the

47

Evaluation of Methods for Quantitative Assessment of Microorganisms in Highly

Cantaminoted Working Environments

T able 2, Evaluated methods

Analytical method • Sampling method Agent

Culture impactorll, centrifugal sampler viable aggregates Jmpingerll, filter, cyclone, liquid scrubber dispersed viable

oggregates

Microscopy

-light filter, impoctor2l spores

- fluorescence filter spores, vegetative cells

- scanning electron filter spores

II Both single stoge and multi-stage instruments have been described.

2) Multi-stage instrument

collected air. This eHiciency is high for most instruments except the centrifugal sampler (RCS) and the SAS sampler. Personal sampling is a necessity for exposure assessment in the working environment. This is Straightforward for filter sampling. An impactor and an impinger have also been described for personal sampling, but oll other instruments can only be used in the stationary state. Strain during sampling may kill or weoken microorganisms and reduce their viability. This is an important error in filter sampling of vegetative cells, but spores may be collected with high eHiciency.

Airborne aggregates of microorganisms that are collected in liquid or are resuspended before culture, may dispersex

48

and yield a higher colony count than directly cultured samples. lf this difference between estimated agents is not taken into account, disruption of aggregates may introduce a positive bias.

Preparation Iosses are weil known from viable analysis using dilution series and similar errors hove been found for fluorescence microscopy. Growth of microorganisms in culture may be inhibited or enhonced by the presence of other species. An issue that has not been assessed is the recognition of microorganisms, which is a possible source of error for microscopic methods.

The systematic errors of various methods have mainly been assessed by comparison of the methods in assumed homo-

geous Iobaratory ond workplace atmospheres. Labaratory studies are usually carried out with monodisperse aerosols of o single organism by which errors from disruption of aggregates in liquid Suspension are avoided. ln lield studies the bioaerosol may contain many different organisms perhops in large aggregates. These studies have been summorized in figures l to 4. For more details see Eduard (1993).

Studies o/ viable methods have been summarized by comparison with the 6-stage Andersen sampler. This sampler has generally shawn the highest yields among samplers that collect microorganisms direct\y on nutrient p\ates, ligure I . The relative yield ol the lollowing methods was low: the centrifugal

' • arllt1meoc mean i I-range [

sampler, the SAS sampler, a personal impoctor and in tests with vegetative bacterial cells lilter samplers. Results from field studies were similar to Iabaratory studies.

Methods that collect microorganisms in liquid, or redisperse them alter collection, are also compared with the 6-stage Andersen sampler, figure 2 (see poge 50). ln Iabaratory studies, these methods have lower yields than the Andersen sampler, although the relative yield of the all-glass impinger with impoction distance 30 mm (AGI-30) is relotively high. Yields in field studies are much higher than the Andersen sampler, however, as may be expected as a consequence of disruption of aggregates.

laboratory swdies field studies

1mpactor Andcrsen sampler 2s Andersen sampler ls personal slit sampter SAS

' '

centrifugal samplcr lllter

• f--

0

• ~

• - -% 1000 %

Relauve yield 100 figure 1 :

Viable methods using direct culture compared with the 6-stage Andersen sampler

49




I ~ ~;,:",;etc m .. an 1 15,10

hodc1. ~s~ory

L--'----~ l~'_'c.,__ field studics -f/1------~~-

imptnger all-g/ass 4mm all-glass 30mm personal multistage

resuspended filter

• --~---270

• ________ ", Figure 2: Viable methods using dilution plating compared with the 6-stage Andersen sampler

slit ~ampler cyclone

Different viable methods have also been compared with microscopic methods in field studies, Figure 3. These studies show large differences between methods, where non-viable methods may estimote Ieveis that are one or more orders of a magnitude higher than

viable methods.

Figure 3: Viable methods compared with microscopic methods

e anthmet1c mean -range

Andersen sampler 6st

Andcrsen samplcr Ist

filter

50

0

field sturlies

% 100

Relative yield

• '----_j~-1::============~/:J,~w: 0 % 100 !00 % 200

Relative yteld

Differences have also been observed between non-viable methods, Figure 4. Fluorescence microscopy showed lower yields than scanning electron microscopy, even though bacterial cells were not observed with the scanning electron microscope.

Random errors

Probably the largest sampling error arises from the day-to-day variability of exposure Ieveis. Exposure measurements ollen follow a log-normal distribution. Distributions of 8 h time-weighted average microorganism Ieveis have been reported with geometric standard deviations (GSD) of 4 to 6, which is considerably higher than usually found for exposure to other pollutants in the working environment. ln consequence

L

Karlsson & Malmbcrg, 1989

Eduard et al.. 1990

FM

FM

LM D 0 100

Rdative yicld Figure 4:

FM= tluoresccnce microscopy LM = llght microscopy Fluorescence and light microscopy compared with scanning electro microscopy

o subset ofsamples with >50% fungi (Eduard, 1993)

the random error of a single measurement is high. For exomple, if the geometric meon is 4 · I 05 m-3 ond the GSD is 5, then the 95 % confidence intervol of one measurement is l ,6 · l 04

to I 07 m-3

Rendom errors from the onolyticol procedure, the precision, have been studied for vioble methods in Iabaratory studies ond in field studies, Toble 3.

Only two studies hove adressed the precision of microscopic methods

Table 3, Analytical precision of viable methods

Studies

Lobaratory

Field

(Käpylä ond Penttinen, 1981; Eduord ond Aalen, 1988). These studies show thot the precision follows the voriobility expected for random counts (Poisson distributed) il the oggregote size is occounted for. A relative precision of 10 % moy then be achieved if 200 to 500 microorganisms are counted, depending on the oggregote size distribution (Eduord ond Aalen, 1988). The microscopic methods have the potential to conflrm the performance requirement for compliance testing,

Precision, CV

arlthmetic mean range

16% 6to3lo/o

23% 9 to 51%

51

Evaluation of Methods for Quantitative Assessment of Microorganisms in Highly

Cantaminoted Working Environments

whereas this seems difficult with vioble methods (CEN 1993b).

Potential for compliance testing

The results from the reviewed studies indicate the following aspects of methods for measurement of microorganisms that are important for future compliance testing by comparison with occupational exposure Iimits:

D The accuracy of sampling in microscopic methods, and non-vioble methods in general, is better than in non-viable methods because personal samples can be collected on lilters and inhaloble sampling seems possible. Sampling times can be varied over a large range, and usualfy permit sampling over 8 hours, which is not possible with vioble methods.

0 The onalytical precision of microscopic methods is sufficient to meet performance requirements for compliance testing. The variability of vioble methods is probobly too high.

D Non-viable ogents moy cause health effects and should be assessed. This is not possible with viable methods

Jdentificotion of species has not been discussed here. lt is not clear whether

52

species identification will be necessary, but this will be required if the health relevance of different species is studied in epidemiological studies of working populations. Viable methods hove the greatest potential for the identification of species. However, the non-viable have some potential for classification of species and moy also be necessary for identificotion of certain spedes thot are not able to grow in culture.

References

Boseler, M. W., Fogelmork, B., and Burre//, R., Differential toxicity of inholed Gram-negative bacteria. lnfect. Immun.

40, 1983, 133- 138

Campbe/1, A.R., Swanson, M.C., Fernondez-Coldas, E., Reed, C.E., May, J.J, and Pratt, D.S., Aeroallergens in dairy barns near Cooperstown, New York and Rochester, Minnesota. Am Rev. Respir. Dis. 140, 1989, 317- 320

Comite Europeen de Normalisation (CEN 1993a), Workplace otmospheres. Size fractions definition procedures for measurement of oirborne portides (EN481 ). ßrussels, Belgium, CEN

Comite Europeen de Normalisation (CEN 1993b), Workploce otmospheres. General requirements for the perfor-

mance of procedures for meosurement of chemical agents [CENfTC1371 WG2/N137). Brussels, Belgium, CEN

Eduard, W., Sandven, P., Johansen, B. V., and Bruun R.), ldentificotion and quontification of mou\d spores by scanning electron microscopy (SEM), Analysis of filter samples collected in Norwegian saw mills. Ann. Occup. Hyg. (Suppl 1) 32, 1988, 447 - 455

Eduard, W., and Aalen, 0., The effect of oggregotion on the counting predsion of mould spores on filters. Ann. Occup. Hyg. 32, 1988, 471 - 479

Eduord, W., Assessment of mould Spore exposure ond re\ations to symptoms in wood trimmers. Ph.D. diss., University of Wageningen, the Netherlands, 1993

Fogelmark, 8., Lacey, J., and Rylan-der, R.: Experimental allergic alveolitis ofter exposure to different microorganisms. lnt. J. Exp. Path 72, 1991, 387- 395

Gregory, P.H., Microbiology of the Atmosphere. 2'd ed. Aylesbury, leonard Hili, 1973

Käpylä, M., and Penttinen, A, An evoluation of the microscopic counting methods of the tape in Hirst-Burkard pollen and spare trap. Grane 20, 1981, 131 - 141

Kar/sson, K., and Mo/mberg, P., Characterization of exposure to moulds ond actinomycetes in agriculturol dusts by scanning electron microscopy, f\uorescence microscopy and the culture method. Scand. J. Work Environ. Health 15, 1989, 353- 359

lacey, J., and Crook, 8., Review. Fungoi and actinomycete spores os pollutants of the workplace and occupational allergens. Ann. Occup. Hyg. 32, 1988' 51 5 - 533

Malmberg, P.: Microorganisms. Criterio documents from the Nordic expert group 1991. Arbete och Hälse 50, 1991, 39- 69

Obayashi, L A new endotoxin-specific assay. Adv. Exp. Med. Bio\. 256, 1990, 215- 222

Pa/mgren, U ., Ström, G., 8/omquisl, G., and Malmberg, P., Collection of airborne microorgonisms on Nuclepore filters: estimotion and onolysis -CAMNEA. Method. J. Bacteriol 41, 1986, 401 - 406

Parkes, W. L Occupational lung disorders. 2'd ed. Butterworths, london, 1982

Pepys, J., and Jenkins, P.A., Precipitin (F.l.H.) test in farmer's lung. Thorax 20, 1965,21-35

53




Sonesson, A., Larsson, A., Schüfz, A., Hagmar, L., and Hol/berg, T., Camparisan of the Iimuius amebocyte lysate lest and chromatographymass spectrometry for meosuring lipopolysaccharides in airborne dust from poultry processing industries. Appl. Environ. Microbiol. 56, 1990, 1271 - 1278

Thurslon, J.R., Cysewski, S.J., Richard, J.L., Exposure of rabbits to

54

spores of Aspergillus fumigatus or Penicillium sp.• Survival of fungi and microscopic changes in the respiratory and gastraintestinal tracts. Am. J. Vet. Res. 40, 1979, 1443 - 1449

Topping, M.D., Scarisbrick, D.A., Luczynska, C.M., Clarke, E.C., Seaton, A., Clinical and immunological reactions to to Aspergillus niger among workers at a biotechnology plant. Br:t. J. lnd. Med. 42, 1985, 312-318

Methods for Analysis of Microbial Sampies from the Work Environment

B. Crook, Microbiology Section; Biomedicol Seiences Group, Health and Safety Laboratory, Sheffield, United Kingdom

Abstract

There is a need to sample for microorganisms in the work environment, either to ensure product quality or to assess the risk to the workforce from exposure to microorganisms. The Ietter consideration must take into account the possibility of infection, ollergy or toxicologicol effect upon the exposed worker. in many instances, the raute af expasure for the worker is via the airborne raute. A number of sampling devices are available far measuring oirborne biological materials (bioaerosols). the performonce of which have been reviewed previously.

Assuming that a suitable bioaerosol collection method has been used, there are a range af options from which to choose for analysing the collected material. T raditionally, analysis has been by culturing the microbiol content of the material, but this does not necessari\y provide full information, because it will only account for those organisms capoble of growing on Iabaratory media, wi\1 underestimate ce\\s that are difficult to culture ond will not account for ce\\ components such os endotoxins.

Any of these may also be important when assessing the risk of bioaerosols to the exposed worker. Therefore, to ougment information received from culturing, alternatives shovld also be considered. These alternatives include microscopic ana\ysis, immunodetection, meosurements of cell metabolism or cell constituents and molecular techniques. Methods are ovailable for oll of these, with molecular techniques oHering perhops the most sensitive and specific meons, although adoptation moy be needed to make some of the techniques fit with bioaerosol collection.

Consideration must a\so be given to ways in which samples are collected and subsequently handled prior to onalysis, especially where biologicol material may be liable to changes ·,n its properlies (reduction in viability or multiplication of cell numbers) between collection ond onolysis. This paper considers individually the steps token in sample collection, transport, storage and analys·ls, and documents the criteria for success. Based on this informotion, recommendations are made for future progress in the analysis of bioaerosols.

55


ß. Crook, Microbiology Section; ßiomedical Seiences Group, Health and Safety Laboratory, Sheffield, United Kingdom

Why is there a need to sample microorganisms in the workplace?

The presence of microorganisms in the work environment may compromise the quality of work material or the health of the exposed worker. A more obvious example af the former is food quality, where there is a clear need to monitor for potential contamination. Less opparent is the problern that may be caused by biological contamination of electronic components. For example, fungal spores settling on microprocessors end magnetic media can cause performance problems (McCain and Mirocha, 1994}, hence the need for weil-monitared clean room conditions during manufacture.

The health of workers may be compromised by exposure to microorganisms via ingestion, ingress through cuts, punctures, abrasions or the mucous membranes, but the most likely form of exposure in the general workplace is through inhalation. This can result in infection (e.g. legionellosis and some dust-borne zoonoses such as chlamydiosis), toxicosis (e.g. inhalation fever from endotoxin} and allergic lung disease (Crook and Olenchock, 1995}. ßoth inhalation fever (organic dust toxic syndrome} and a!Jergic Jung disease arise from exposure to large concentrotions of oirborne biological material (bioaerosols}, often

ossocioted with work in agriculture (Crook, 1994}. Respiratory sensitisation presents a serious burden to industry. for example in the UK some 3,500 new cases of work-reloted respiratory disease are recorded each year, with the associated industrial cost of loss of productivity and the social cost of lass of livelihood. Almost 40 % of these cases are occupational asthma, as outlined in o recent report from an ongoing survey (Sallie et al, 1994} and approximately I 2 to I 5 o/o of these may be attributed to exposure to materleis of organic origin. Consequently, there is a need to be able to measure workplace exposure to bioaerosols to enable a risk assessment to be made end, if appropriate, to initiate prevention or control measures.

Sampling and Analysis of Workplace Bioaerosols

Several methods are available to measure bioaerosols, ronging from those designed specifically for recovery of airborne biological material, to those adapted from methods to collect airborne dust for gravimetric analysis (Crook, 1995a; 1995b}. following collection, the methods for analysis are those used for m·,crobiological assay in other applications. The complete pro-

57


cess, from choice of the location and time of sampling, through to final data analysis and sample archiving, can be subdivided into six distinct activities, as listed in fable I . Each activity has Iimitations to its complete success which, if not overcome by taking actions such as those suggested, could by multiplication of their effects greatly reduce the ac· curacy of the analysis. However, the remedies to some of these limitations have yet to be fully researched.

Culturing Methods

The conventional method for counting and characterising ony microorganism is by culturing. ln some samples, biological material is deposited directly onto agor media (e.g. Andersen impactors and RCS centrifugal samplers), for others the material is collected into liquid (e.g. impingers and Aeroiet cyclone samplers) which can be diluted and used to inoculate agar media, while for filtration samples the dry deposit generally is resuspended in liquid, then diluted as obove. ln this woy, a range of general purpese and selective ogar media can be inoculated, for incubation at suitoble temperatures for a sufficient period to allow growth, then the resulting colonles counted and the numbers expressed as colony-forming units (cfu)/m3 oir sompled.

58

The conventionol culturing method is the best, ond cheapest, method for characterising the microorganisms in a somple, but at the same time it makes o number of ossumptions, as listed below:

D the collected microorganism is vioble. Non~viable microorganisms represent no infectious hazord, but if the microorgonism is o potential ollergen it will remoin a hazard even when dead if the allergenic protein or carbohydrate moiety remoins in o configurotion recognisable by the human immune system. Similarly, toxic components of cells, e.g., endotoxin, will remain active even if the cell from which they have been secreted is dead. lndeed, more endotoxin may be released as Gram-negative bacterial cell walls break down alter death

D the collected microorganism is culturable. Some microorgonisms ore known to enter a metabolic state referred to as non-culturable but viable (NCBV; Co/weil et al, 1985). This is

especially important for certain pathogens as they can escape detection by culturing but if they enter a suitable host they can "revive '' to cause infection.

D the collected microorgonism is readily culturable in a laborotory medium. While not entering the classification of NCBV, certain microorganisms are particularly difficult to culture under labo-

Table I, Summary of procedures, from start to finish, for collecting and analysing bioaerosol samples, the limitations to successful sampling and ways to overcome them

Procedure Limitations Overcome by

I. Choice of sampling a} High risk werk not being done Sampling strategy; occupa-site/time/doy/seoson b) Right activity, wrang material tional hygiene observation;

c) Right activity, right material, communication with workforce amended procedure or workploce conditions

2, Choice of sampling a) lnefficient/unrepresentotive col- a) Choose, use, design, weil-method lection of aerosol in sampler characterised samplers

b) Tampering with sompler b) Communication w'1th workforce c) Physical inability to remove c) Test and design protocols for

deposit from sampler efficiently efficient removal d) Lass of viability or other pro- d) Sampier choice to minimise

perty of interest before removal stresses e) Lass of viability etc. during e) As c) and d)

removal

3. Storage and tronsport Changes in viability du ring Optimise storoge conditions of sample from site storoge; i.e., losing viability, - use of a transport medium? -to anolyser goining it (multiplicotion), differen- storoge temperoture, RH,

tial changes; resulting in timescole effects unrepresentative somple

4. Sampie preparation a) Physical lass du ring preporation a) Need to optimise protocols and anolysis (dilution ond transfer)

b) Viability lass during preporotion b) b) As a) c) Choice of analytical method c) Need to identify and optimise

methods

5, Counting and a) Counting precision/errors a) Need to optimise proteeals characterisation b) Non-representative growth, b) Choke of assay

overgrowth c) Chorocterisation errors c) Training; interlob. camparisans

6. Storage of sample o) Security of sample a) Need to optimise protocols ofter onolysis b) Repeatability of assoys - was b) Assoy portion of somple only

analysis oll or nothing? How best to store the rest?

59


ratory conditions. For example the zoonotic bacterial pathogen Leptospire hardio can take up to 16 weeks to cultivate in an artificial Iabaratory medium (Bolin et al, 1985), while some potentially allergenic thermophilic actinomycetes are also hard to grow (Lacey, 1995). Therefore a clinical diagnosis relying solely on culturing techniques has obvious limitations.

T o analyse those microbial cells which are readily cultured, samples of callected bioaerosols are usually spread onto the surface of agar plates. Pour plates and most probable number (MPN) methods of analysis are less popular. Both general purpose and selective agar media are used, the latter to aid characterisation of the collected cells, but it must be noted that some selective agents may have a detrimental effect on the subsequent cultivation of previously aerosolised microorganisms. Physical stress such as dehydration, or exposure to ultraviolet light, or airborne pollutants, may have reduced their viability (Cox, 1987) and the additional metabolic stress of selective agents moy compromise further their ability to grow. T wo alternatives exist to overcome this

problem. Firstly, the initial inoculation could be made onto a general purpese medium without selective agents added. After allowing sufficient growth to occur for total colony numbers to be recorded,

60

colony material can be transferred onto a selective medium using a suitable blotting material. Possible drawbacks to this approach are the efficiency of the transfer or the possibility that slower growing organisms moy olreody have been out-competed by Iaster growing species. A secend alternative is to use methods to enhance the recovery of stressed cells before the plating out stage. The choice of an optimal liquid collection medium for use in impingers or to resuspend deposits from the surfoce of the filter may overcome aerosolisation stress; in our studies the mineral solts solution quarter strength Ringers, with 2 o/o inositol added1 gave best recovery of aerosolised bacteria (Crook et ol, 1988). Two phase systems hove been used successfully ta separate different groups of microorganisms (Biomquist et al, 1984) and they could be used in impingers both to collect microbial cells in conditions optimised to suit as many genera os possible and also to selectively isolote genera for subsequent charocterisation. T o maximise recovery during culturing, there is a need to ensure thot conditions for growth, such as pH, incubation temperature, ond water availobility, are optimum.

Subsequent choracterisotion of cultured cells is by the standerd microbiological techniques, combining gross ond

microscopic morphology with biochemica! tests.

Alternatives to culturing

While culturing techniques are always likely to be the basis for analysis of bioaeroso\s, existing and potential alternatives also need to be considered to augment the data available from culturing. Seme of these are described below•

Microscopy

The use of light microscopy, scanning electron microscopy and epifluorescence microscopy for direct counting and partial characterisation of collected bioaerosols is weil established (Palmgren et al, 1986; Eduard et al, 1990). The techniques offer the advantage of enumeration of cells irrespective of their ability to grow, although further work is required to select the best fluorochromes for staining and counting accuracy and also to standardise protocols so that automated image analysis can be used to overcome the labour intensiveness of the procedure. Greater specificity of analysis could also be achieved by the use of immuno-linked fluorochromes.

lmmunodetection

An alternative to microscopy for counting and characterising fluorescent - or immunofluorescent- tagged cells is flow cytometry. T agged cells in suspension are possed through a constricted chonnel which forces them to flow in single file post a Iaser light source. Measurement of light scottering allows cells to be counted and their size, shape ond fluorescent property recorded, thereby allowing discrimination. The technique has been used for some microbiologicol analyses (Nelson, 1993) ond potentially could be used to anolyse cells collected from biooerosols. Microorganisms can be detected sensitively and specifically by ELISA-based immunoassoys bosed on monoclonol or polyclonal antibodies ond many tests are available commercially. However, many record only the presence or absence of cells and work may be needed to establish a correlotion with ce\1 numbers.

Cell metobolism

Products of microbial cell metabolism, such os adenosine triphosphate (ATP) can be measured using the enzyme complex luciferin-luciferase. There are limitations to this method which would need to be oddressed. For example, ATP Ieveis fall rapidly os cells lose their

61


viabi!ity, so such a measurement would be of actively metabolising cells only, also there are diHerences in the quantity of ATP present in different cell types, depending on their size and metabolic activity, so it may be difficult to relate data to cell numbers.

Cell constituents

Where Gram-negative bacteria form a significant proportion of the bioaerosols, measurement of endotoxin concentrations is dinically relevant due to their possible role in ODTS (doPico, 1986) and endotoxin assay is a widely used alternative to enumeration by culturing. However, at present the relationship between cell number and endotoxin Ieveis is not clearly defined and may differ in different sample types. The assay, based on the Limulus amoebocyte lysate test, is highly sensitive and has improved in quality in recent years. Further work needed to improve detection of endotoxin in bioaerosols includes optimisation of the collection method and the assay. Same researchers favour collection by filtration onto cellulose acetote membranes (Gordon et al, 1992), while others prefer teflon membranes (Reynolds and Milton, 1993). lt is also necessary to compare the two types of assay currently available, end-point ond

62

kinetic, if an optimised procedure is to be set up.

D Fungoi I - 3 ß glucans may also contribute to respiratory symptoms in ODTS following worker exposure to lorge contributions of contaminated dust (Rylander 1992). Methods to measure their concentration in biooerosol samples have yet to be elucidated.

D As an alternative to counting growing colonies, recent studies have suggested detecting a chemical marker of the presence of contaminants. For example, muramic acid has been used to quantify the presence of bacterial peptidoglycan in dust samples, with analysis by gas chromatography-mass spectrometny (Fox and Rosario, 1994).

Molecu/ar techniques

Molecular biological methods offer the possibility to detect the presence of specific microorganisms in very small numbers in a sample even in the presence of large quantities of other organic material. Gene probes are used to recognise nucleotide sequences of the target organism and polymerase chain reaction (PCR) can be used to amplify the signal to a detectable Ievei (Pickup, 1991). lt is possible to apply these techniques to bioaerosol sampling to

detect microorganisms either at species or genus Ievei, although more werk is needed to make the techniques quantifiable.

Future progress on bioaerosol analysis

As described above, a range of analytical techniques exist which may be applied to bioaerosol measurement. At present, there is no consensus in Europe on sampling and analytical methods, although work done by the Nordic Research Group (Nordisk Minsterrad, 1988) has provided a basis for establishing a common approach. ln USA, the Bioaerosols Committee set up by the American Congress of Government lndustrial Hygienists (ACGIH) is providing a similar function. There is a diverse range of environments where bioaerosol samples need to be taken. These include indoor and outdoor environments at high and low concentrations, to recover fungi, bacteria, actinomycetes and viruses. lndoor environments include domestic premises, offices, agriculture (animal confinement and food storage}, manufacturing industry, biotechnology and clean room work, while out of doors the environments may include measuring emissions from waste water treatment plants or monitaring

dispersal from the site of application of genetically modified organisms. lt is most likely therefore that no single sampling method or analytical procedure will be appropriate for oll applications.

As a starting point for detailed characterisation, filtration is probably best because of its simplicity, although dehydration effects on collected cells may result in loss of viability of bacterial vegetative cells (less so for spare forming bacteria and fungi). The choice of filter membrane and filter holder is likely to be important. For example, the plastic monitor cassette used in the CAMNEA method (Palmgren et al, 1986) may be subject to collection Iosses because of electrostatic effects, whereas samplers such as the lOM head have been designed to conform to ISO standards for collection of inhalable portides (Mark and Vincent, 1986). lf the assays used are optimised for analysis of samples callected on filters they are likely also to be applicable to analysis of bioaero-sol samples collected by other means. For simplicity, culturing, counting and characterisation by conventional means should be the basic method, but this will need to be augmented by total and differential counting by microscopy, endotoxin assays, and techniques under development such as mo\ecular analysis.

63


Consideration will need to be given to the way that samples are treated following collection and prior to analysis to ensure maximum survival. A recent study campared the performance of identical analytical procedures in two different laboratories, one analysing bioaerosol samples shortly alter collection and one after three days transportation an ice (Thorne et al, 1994). The study concluded that samples could be stored reliably prior to analysis with some restrictions, but the report highlighted the following points,

0 ln some of the impinger collection media tested bacterial numbers increased significantly in summer but remained unchanged in winter. Therefore more work is needed to develop an optimum collection/recovery medium.

D Although not addressed by the study, the results suggested that a liquid transport medium, similar to those used to maintain viability of microorganisms on clinical swabs, could possibly be developed to maintain viability without multiplication of microorganisms.

D The study did not record any data far samples stored for more than three days. Information is needed an the stability of samples stored for extended periods.

64

D The microorganisms collected were characterised only as total colonyforming units in groups defined by incubation temperature and isolation medium. No data was recorded for possible differences in the balance of different genera depending on the season, a factor which must be taken into consideration as it may alter their ability to survive and recover.

The best way to establish practical, representative and consistent results is by a thorough interragation, by repeated sampling, of the methods to be used. The procedures established should then be examined further by inter-laboratory comparisons to establish the margins of acceptable error.

References

B/omquist, G., Strom, G., Soderstrom, B., Separation of fungal propagules by partition in aqueous polymer two-phase systems. Appl. Env. Microbiol. 47, 1984, 1316- 1318

Bolin, C.A., Zuerner, R.L., Trueba, G., Comparison of three techniques to detect Leptospire interragans serovor hardjo type hardjobovis in bovine urine. Amer. J. Vet. Res. 50, 1989, 1001 I 003

Co/weil, R.R., Braton, P.R., Grimes, D.J., Roszak, D.R., Hugo, S.A., Palmer, L.H., Viabie but non cuiturobie Vibrio choierae and reiated pathogens in the environment; implications for the release of genetically engineered microorganisms. Biotechnoiogy 3, 1985, 817 - 820

Cox, C.S., The Aerobioiogicai Pathway of Microorganisms. Wiley lnterscience, Chichester, 1987

Crook, B., Aerobioiogical investigation of occupational respiratory allergy in agriculture in the UK. Grana 33, 1994, 81 - 84

Crook, B.' Chapter 8 - inertial samplers: biological perspectives. ln: Wathes, C.M., and Cox, C.S. (eds.), Bioaerosois, o Handbook of Sampiers and Sampling. 1995, lewis Publ., USA, 241 - 262

Crook, B., Chapter 9- Non-inertiai samplers: biological perspectives. in: Wathes, C.M., and Cox, C.S. (eds.), Bioaerosols, a Hondbook of Sampiers and Sampiing. 1995, lewis Publ., USA, 263- 278

Crook, B., Higgins, S., Lacey, J., Methods for sampling airborne microorganisms ot solid waste disposo\ sites. in, Houghton, D.R., Smith, R.N., Eggins, H.O.W. (eds.), Biodeterioration 7. Proceedings 71

h International Biodeteriora-

tion Symposium. London, Elsevier, 1988, 791 - 797

Crook, B., 0/enchock, S.A., Chap-ter 18 - industrioi Workpioces. in, Wathes, C.M., and Cox, C.S. (eds.), Bioaerosols, o Hondbook of Sampiers ond Sompling. 1995, Lewis Publ., USA, 527-542

doPico, G.A.: Report on diseases. Amer. J. indust. Med. 10, 1986, 261 - 265

Eduard, W., Lacey, J., Karlsson, K., Pa/mgren, U., Strom, G., 8/omquist, G., Evaluation of methods for enumerating microorganisms in filter samples from highiy contominoted occupationai environments. Amer. lnd. Hyg. Assoc. J. 51' 1990, 427- 436

Fox, A., Rosario, R.M. L Quantificotion of muramic acid, a marker for bocterial peptidogiycan, in dust coiiected from hospital and hame air conditioning filters using gas chromatography-mass spectrometry. indoor Air 4, 1994, 239- 247

Gordon, T., Ga/danes, K., Bras-seau, L.: Camparisan of sampling media for endotoxin-containing aerosols. Appi. Occup. Environ. Hyg. 7, 1992, 472 - 477

Lacey, L Sompiing and assoy of biooerasals ln: Aerosols, their Generation,

65

Methods for Analysis of Microbial Sampies

from the Work Environment

Behoviour and Applications. Proceedings 9th Aerosol Society Conference. Bristol, The Aerosol Society, 1995, 97- I 02

Mark, 0., Vincent, J.H., A new personal sampler for oirborne total dust in workplaces. Ann. Occup. Hyg. 30, 1986, 89- I 02

McCain, J. W., Mirocha, C. J., Screening computer diskettes and other magnetic media for susceptibility to fungal colonisation. International Biodeterioration and Biodegradation 33, 1994, 255 - 268

Nelson, 0., Flow cytometry in bacteriology. Rev. Med. Microbiol. 4, 1993, 215-221

Nordisk Minsterrad: Harmonisation of sampling and analysis of mould spores. Nordic Council of Ministers T echnical Report from Project 170.21-2.29, 1988

Palmgren, U., Strom, G., 8/omquist, G., Malmberg, P., Collection of airborne microorganisms on Nuclepore filters - estimation and analysis - CAMNEA

66

method. J. Appl. Bacteriol. 61, 1986, 401 - 406

Pickup, R. W., Development of molecular methods for the detection of specific bacteria in the environment. J. Gen. Microbiol. I 37, 1991, I 009 - I 019

Reynolds, S.J., Mi/ton, O.K., Camparisan of methods for analysis of airborne endotoxin. Appl. Occup. Environ. Hyg. 8, 1993, 761-767

Rylander, R., Persson, K., Goto, H., Yuasa, K., Tanaka, S., Airbornebeta 1-3 glucon may be related to symptoms in sick buildings. lndoor Environ. 1, 1992, 263 - 267

Sallie, 8.A., Ross, O.J., Meredith, S.K., McOonald, J.C., SWORD '93, Surveillance of work-related and occupational respiratory diseose in the UK. Occup. Med. 44, 1994, 177- 182

Thorne, P.S., lange, J.l., 8/oebaum, P., Ku/Iman, G.L Bioaerosol sampling in field studies, can samples be mail expressed? Amer. lnd. Hyg. Assoc. J. 55, 1994, 1072- 1079

Microbiological Sampling in Finland

Marjut Kof1maa, Kuopio Regional Institute of Occupational Health,

Kuopio, Finland

Abstract

Although Initial microbiological investigotions in Finland concentrated on agricultural questions, for some years now, oll possible working environments have been the subject of microbiological studies. Whereas other sampling and analysis methods, such as collection on filters, were used relatively rarely, the Andersen cascade impactor was employed in a large number of studies to examine air contaminafton. Certain working environments stand out due to the high Ievei of mould and actinomycetes contaminating the air, while in other areas a concentrafton of bacteria predominates.

The microbiology section of the Kuopio Institute of Occupational Health (FlOH) investigates exposure to microorganisms at the workplace and the ensuing consequences for health. On the basis of the workplace ana\ysis, work can begin on the planning of microbiological measurements. ln order to determine the source of the organism emission, material and/ or water samples as weil as surface samples can be Iaken. For studies of

the air the 6-step Andersen impactor is used. The choice of incubation temperature and nutrient medium for the culture depends on the aim of the microbiological analysis and will be described in more detail below. For personal sampling, filtration collectors are used in conjunction with polycarbonate filters, which also allow measurements to be Iaken in highly contaminated areas. Easy-to-use culturing methods in the analysis of microorganisms have the advontage of also making it possible to identify individual species. On the other hand, methods whereby microorganisms are counted directly provide higher figures than would be possible by culturing on nutrient media. The most suitable method for a study relating to health problems at the workplace must be carefully selected in accordance with the aim of the analysis. Modern ·lmmuno-chemical and electron microscopic analysis methods are still to be developed further. To detect antibodies in the blood of persans exposed, the FlOH has currently developed six antigen sets for various occupational areas.

67


Marjut Kotimaa, Kuopio Regional Institute of Occupational Health,

Kuopio, Finland

Introduttion

Microbiologicol samplings were first corried out in agricultural settings, e.g. in cow sheds and in hay barns in the early '80s in Finland. Sedimentation plates were used to deted airborne microbes. loter, an six-stage Andersen impoctor was used in oll kinds of occupational environments. Other sampling and analysing methods such as CAMNEA and filter collection with SEM (scanning electron microscopy) onolysis have been used in research projects more thon in routine service.

By Andersen impactor, it has been possible to find both quantitative and qualitative differences in different fields of occupation. Agriculture is characterised by high-level exposure to airborne microbes arising from various materials used on farms. ln oddition to mesophilic fungi ond bacteria, thermotolerant fungi and thermophilic actinomycetes are often found. Wood chip handling may cause o similar type of exposure os is found on farms. The saw mill industry differs from the above-mentioned environments, because there is usuolly only one source of moid, kiln-dried timber. Jt creates the exposure in "dry work . phases" in saw mills. Very high exposure to thermotolerant fungi have been measured in wood trimming departments.

Certain work environments are characterised by the occurrence of high amounts of bacteria in addition to minor amounts of funga! spores. Examples of this kind of environment are waste water treatment plants, metal industry, textile industry and printing offices. The microbial exposure in the biotechnical industry is typically to the microbial strain used in the process.

Du ring the last few yeors, waterdamaged buildings have gained Iot of publicity, and people have become aware of the possible health effects of quite low-level, but Iang-term exposure to fungol spores in these environments. Today, the Microbiology Unit at the Kuopio Regional Institute of Occupational Health (FlOH) has two main octivities: to characterise the exposure to microbes in work environment, and the biological monitaring of the workers' exposure to microbes. The demand for field service and for biological monitaring has increased about three-fold during the last year.

Steps in microbiological sampling

Gelhering the background informalion

\n oll work environments, it is essential to gother os much background informotion as possible to measure the right

69


impurities at the right moment, and to use correct methods for analysis.

Background information should include oll the possible sources of microbes in the work process, transmission routes of mkrobes from the source to the worker, the mechanism of exposure, perceived health effects of the workers, ventilation technique of the work place, ambient air temperature and relative humidity, and the main constructional features of the building itself, e.g. the history of pipe leaks or other water-damage and maisture problems.

On the basis of these data, a smart guess is made about the quantity and the quality of the microbial problem, and the sampling strategy can be

planned.

Sampling strategy

T o recognise the source of microbes, bulk samples are taken from the materia\s used in work environments, samples are also taken from suspected contaminated liquids, end wipe samples are taken from suspected contaminated surfaces into buffer solution. These samples are diluted and plated out onto proper cultivation media, which are then incubated at proper temperatures.

70

A six-stage Andersen impactor is used in air sampling. Because the ana!ysis of collected microbes is done after cultivation, there is need for the selection of proper media, too. The selection of media is based on the information about the materials handled in the work environment. The combination of media generally used is malt-extract-roseBengal-medium (incubation at 20 "C), dichloran-glycerol-medium (incubation at 20 "C) for fungi, and tryptone-glucoseyeast-extract-medium (incubation at 20 "C) for mesophilic bacteria. ln the environments where there is a probable source of thermotolerant fungi or thermophilic octinomycetes, correspondlng media (dichloran-glycerol-medium incubation at 40 "C ond halfstrength nutrient-agar incubation at 55 °C) are used. (n waste water treatment p!ants or on certain types of production in agriculture (pigsties, swineries, hatcher\es etc.) Gram-negative bacteria ore expected to be found. for their detection, eosin-methyl-blue-medium incubotion at 37 "C is used. in biotechnology plants, highly selective medium for the production organism ond incubation at the optimum temperature is used.

The Andersen impactor con only be used for environmental sampling, and not as a persona! sampler. H a personal air sample from the workers breathing zone is needed, collection onto the

Nuclepore filter is performed, and analysis is made using CAMNEA or SEM.

With the Andersen impactor it is possible to collect for only short periods (some minutes), in very highly contaminated environments samp\ing time has to be within seconds to ovoid the risk of overloading the sampling plates. By taking many successive air samples, the work period can be covered representatively, but this increases the number of plates to be analysed. Some of the problems of Andersen sampling can be overcome by CAMNEA, which is a very useful method in occupational environments with high concentrations of airborne microbes. The detection Iimit of CAMNEA is quite high, thus it cannot be succesfully used in environments with only slightly elevated microbe concentrations.

Problems in detecting microbes

When vioble methods are used, we always face the problem that the result is an underestimate of the total number of microbes in the air. The percentage of viable microflora varies araund 1 to I 0 % of oll microbes. Some of the fungal spores (e.g. Serpula lacrymons) are those not capable of growing on artificial media, thus their detection must

be based on other methods, e.g. microscopic counting or immunochemistry. Slowly growing species (e.g. Stachybotrys atra) are easily overgrown by fast-growing fungal species, and thus other thon cultivation methods ore preferable. Some species have speciol requirements for growing and their detection needs speciol, selective media.

Although the viable methods' disadvantage is thot the number of microbes obtoined os o result is an underestimate, the species con be identified eosily compored to microscopic counting methods, which give more reliable result for the total number of microbes.

Relevance of sampling and analysing methods to the existing health problems

Most common and best-known workrelated health consequences caused by the exposure to microorganisms are infections, allergic rhinitis, allergic alveolitis, asthma, ergonie dust toxic syndrome (ODTS), chronic bronchitis and combination of unspecific symptoms. These health consequences are caused either by intoct orgonisms or by the"1r "natural" chemical components (endotoxins or glucons) or secondary metabolites (mycotoxins).

71

Microbiological Sampling 1n Finland

lf the problern deals with occupational infection, viable methods with a highly selective medium for the detection of the particular pathogen have to be used.

As to allergens, it is difficult to decide whether viable methods with slightly selective media or microscopic counting methods should be used. Even nonviable microbes or microbederived portides are immunogenic. By viable methods, good specificity of the diagnosis may be achieved because of the better identification of the microfiora. The enumeration of nonviable microbes by microscopic counting methods gives the estimate of "total microbial Ioad". ßy SEM even the identification of some fungi is possible. The combination of the two main analytical methods would be ideal.

lmmunochemical methods are not in wide-spread use in the anolysis of airborne microbes. The problern in these methods (ELISA-inhibition, RAST-inhibition) is that every microbe needs its own method. Highly cross-reactive microbes, e.g. some plant pothagenie field fungi, are exceptions. lmmunochemical methods could be saved for those microbes which are not detected by other anolyticol methods. Their odvantage is that they are highly specific and can detect small amounts of microbespecific profein in an air sample (some

72

nanograms/ml of sample suspension). The detection Iimit may, however, be quite high when the profein amount is converted to the number of spores (approx. I 0 4 spores/ml of sample suspension).

Biological monitaring of the exposure to microbes

Microbe-specific antibodies (immunglobulin class G) in workers' sera serve as a tool in biological monitoring. Enzyme-linked immunosorbent assay (ELISA) is sensitive enough to detect even small antibody concentrations and can also be used in follow-up studies to indicate the difference in exposure. Antibody determination can be used to evaluate the amount of Iang-term exposure and to motivate the worker to find ways of decreasing the exposure. Microbial antigens used in the assays are selected according to the exposure data. For practical use, only eight typical microbial antigens are selected to represent each work environment. At the moment, our Iabaratory has antigen sets for six different environments:

1.) forms,

2.) wood chip work

3.) saw mills

4.) greenhouses

5.) printing offices, and

6.) water damoged buildings

Combining the data and its interpretation

ln the ideal situation, oll the data obtained in different phases of the process support each other. ln practice, the interpretation of the results is quite a complicated task.

ln industrial environments, the source of airborne microbes is usually the material used in the work (wood, grain etc.). lf the concentrations of airborne microbes is high, and the workers have warkrelated symptoms suggesting a microbial background, and the workers have antibodies to the microbes found in the air, action should be taken to lower the exposure by proper hygienic measures. ln some cases, the material itself used in work the environment is not the source (at the moment microbes are collected at the workplace), but if the source is the settled dust, then cleaning routines

should be changed to remove the dust from the collecting surfaces.

Usually, the comparison of the microflora of material/liquid, surface and air samples is made. lf the antibodies of exposed people have been determined, the comparison of the antibodies that are elevated with the microflora of whatever sample in that environment is done. lf the results of microbiological analyses and aerological findings are similar, the conclusion is that those people are exposed to microbes in that particular environment. A waterdamaged building is a very complicated environment where serological findings are often positive but it is quite an effort to find "matching" microbes from the building. Often this requires removing of the carpet, wall or ceiling to get material samples from e.g. insulation materials which then may show mold growth. The concentrations of airborne microbes in water-damoged buildings is seldom high, thus the diagnosis of a moldy house is based moinly on the presence of indicotor orgonisms. At least in these coses, viable methods are preferred, because of the better identification of fungal genero.

73

Measurement of Microorganisms in the Work Environment in France

J.F. Fabries, Institut National de Recherche et de Securite (INRS), Vandceuvre, France

Abstract

After having recalled the main problems raised by sampling microorganisms dispersed into the air at workplace, the guidelines of a study conducted by INRS and beginning in 1996 are presented. This study will be achieved in collaboration with external laboratories having a good proctice in environmental microbiology. Five main directions should be followed, choice of

some sampling and analysis methods for which reliable data are already availoble, and measvrement campaigns at some workplaces; extensive criti-cal analysis of the capabilities affered by the most commonly used methods; assessment of the physical characteristics of samplers; impact of sampling parameters an the biological activity of collected microorganisms; and finally optimisation of the sampling methods.

75

Measurement of Microorganisms 1n the

Work Environment in France

J.f. fabries, Institut National de Recherche et de Securite (INRS), Vandceuvre, France

The Europeon Directive 90/679/CEE of 26 November 1990 concerning the biological hazards at the workplace requires exposure assessment of workers to biological agents, including microorganisms (bacteria, fungi, viruses, ... ). This directive has been translated into french law by the order 94-352 of 4 May 1994.

Exposure assessment to microorganisms dispersed into air, making bioaerosols, is an important challenge for the future, and prevention is now encouraged through the Directive. Biological hazards effectively constitute a new field of investigation, parallel to physical and chemical hazards.

This theme has been retained by INRS for the next years. A seminar was organised by the institute in June 1993, followed by an instruction period obout what could be done by the Social Security Institution in that field, in order to answer the numerous questions that are periodically raised by inspectors and occupational physicians. Finally it was recently decided to start a research activity in bioaerosols measurement within our institute. lt could be followed by epidemiological studies in some particular sectors where biological hazards are known to be relatively high, and other tasks including the preparation of

information and training services (database, seminars, courses, ... ).

Bioaerosol sampling

The assessment of biological hazards due to airborne microorganisms requires the availability of well-adapted sampling methods. Many techniques have been proposed, but oll the users are confronted with technical problems that could easily alter the significance of the results obtained.

As for non-biological particles, a sampling device is characterised by its sampling efficiency, which expresses the probability for an airborne particle to be effectively collected onto the terminal substrate. This physical parameter reflects aerodynamic phenomena related to particle behaviour, in the vicinity of the sampling orifice or through the sampler itself. Sampling efficiency measures the contribuf1on of several factors in the ospiration ond collection processes. Aspiration efficiency is generather factors moy alter meosurements, like sompling time. For exomple, too lang sampling Iimes may affect viability, or alter the quality of the subsequent analysis. \n cantrast too short sompling times may affect accuracy or significance of the results.

77

Measurement of Microorganisms 1n

the Work Environment in France

Besides these sampling problems, other difficulties may arise from transportation of the samples, analysis and interpretation of the data.

Considering oll these facts, bioaerosol sampling appears to be a very difficult task, with no absolutely reliable methods validated for every situation that can be encountered in occupational hygiene. Several institutes in charge of health prevention at the workplace have now taken this new dimension into account, and INRS has decided to focus on the metrological aspects for the next few years.

INRS study

This study will start in 1996. lt will be carried out by the Aerosol Measurement Laboratory, located in the research centre of Vandoeuvre (Nancy district). As the study will require two complementary skills, an external microbiology Iabaratory (or perhops more) will be associated with the research programme. The investigation is expected to pursue five moin opproaches, detailed below,

1.) ln o first step some sampling techniques will be specifically selected, and the corresponding equipment purchased. For example the Bioaerosol Sampling

78

Subcommittee of ACGIH [I] gave some indication which are the useful choices of sampling device for indoor environments. Other more recent recommendations [2, 3] can be taken into account, although many samplers are not still completely characterised in terms of sampling efficiency or microorganism integrity during sampling. With these techniques and auxilliary analysis methods including colony counting, germ primary identification by means of biochemical screening assays, and direct observation and counting (classical or epifluorescence microscopy), some measurements will be performed at various workplaces in order to develop procedures adapted to each situation. The aim of this preliminary part is to provide the institutional users with sampling protocols and assay methods as soon as possible, and to start measurements in the field of bioaerosol hazards ot the workplace, in the event of registered comploints or clinical ob

servations.

2.) An extensive critical analysis of the capabilities of oll the most commonly used methods will be carried out, on the bosis of a Iiterature review.

3.) The third part of the study will be

devoted to the physical aspects of aerosol sampling of biooerosol samplers. Although some recent studies yielded

again in the the knowledge of samplers' behaviour [ 4, 9], it would be of some interest to proceed to additional measurements of aspiration and collection efficiencies for some samplers. Relevant parameters would be examined: particle aerodynamic diameter, external wind velocity, orientation of the sampler to the wind, internal parameters, ... etc. Our new wind tunnel facility [I 0] could be used for this investigation.

4.) The impact of sampling parameters on microorganism viability will be examined. This problern is more acute in the case of bacteria than with fungal spores, which are generally much more resistent to stress. Same microorganisms were recommended for this type of investigation [I I, 12]. Some authors [ 13], for example used Pseudomonas fluorescens as the test microorganism for studying the potential effect of dessication. This part will preferebly be performed using existing test chambers availoble in our institute, after possible modification.

5.) Finally oll the methods that will have been retained for their best performance will be tested in the field, and the sampling and assay procedures will be optimised.

Conclusion

The work environment in France raises the same questions obout the hozords due to biooerosols os in other countries. As no orgonised measurement progromme hos ever been performed for the ossessment of biooerosol chorocteristics ot the workplace, except in buildings [14], this important item will be studied extensively from 1996. lt is highly desirable that some standardised methods could emerge os soon os possible from collaborative work at the Europeon Ievei, in order to prepore criteria with regard to air quality.

References

Burge, H.A., Chatigny, M.A., Fee-ley, J., Kreiss, K., Morey, P., Otten, J., Peterson, K.: American Conference of Governmental lndustrial Hygienists (ACGIH) Bioaerosols - Guidelines for assessment ond sompling of saprophytic bioaerosols in the indoor environment. Appl. lnd. Hyg., 2, 1987, RIO-RI6

Grilliths, W.D., DeCosemo, G.A.L., The ossessment of biooerosols : o criticol review. J. Aerosol Sei. 25 (8), 1994, 1425- 1458

79

Measurement of Microorganisms 1n

the Work Environment in France

Macher, J.M., Willeke, K., Performance criteria for bioaerosol samplers. J. Aerosol Sei. 23 (suppl. 1), 1992, 647- 650

Nevalainen, A., Pasfuszka, J., Liebhaber, F., Willeke, K., Performance of bioaerosol samplers : collection characteristics and sampler design considerations. Atm. Env. 26A (4), 1992, 531 - 540

Macher, J.M., First, M. W., Reuter centrifugal air sampler : meosurement of effective oirflow rate and collection efficiency. Appl. Envir. Microbiol. 45, 1983, 1960- 1962

Fängmark, 1., Wikström, L.E., Henningson, E.W., Collection efficiency of a personal sampler for microbiological aerosols. Am. lnd. Hyg. Assoc. J. 52 I I 2), I 991 ' 516-520

Henningson, E. W., Ahlberg, M.S., Evaluation of microbiological aerosol samplers , a review. J. Aerosol Sei. 25 (8), 1994, 1459- 1492

Uplon, S.l., Mark, 0., Douglass, E.J., Hall, D.J., Griffiths, WO., A wind tunnel evaluation of the physical sampling efficiencies of three bioaerosol samplers. J. Aerosol Sei. 25 (8), 1994, 1493 - 1501

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Grinshpun, S.A., Chang, C.-W., Nevalainen, A., Willeke, K., lnlet choracteristics of bioaerosol samplers. J. Aerosol Sei. 25 (8), 1994, 1503-1522

Wifschger, 0., Wrobel, R., Fabries, J.F., Görner, P., Renoux, A: A new experimental facility for studying oerosol samplers. J. Aerosol Sei. 25 (suppl. I), 1994, 333- 334

Grilfilhs, W.O., Report an Bioaerosols Workshop- Europeon Aerosols Conference, Oxford 1992. J. Aerosol Sei. 24 (7), 1993, 973-976

Jensen, P.A., Todd, W.F., Davis, G.N., Scarpino, P.V., Evaluation of eight bioaerosol samplers challenged with aerosols of free bacteria. Am. lnd. Hyg. Assoc. J. 53 (10), 1992, 660- 667

Thompson, M. W., Oonnelly, J., Grinshpun, S.A., Juozaifis, A., Willeke, K.,

Method and lest system far evaluation of bioaerosol samplers. J. Aerosol Sei. 25 (8), 1994, 1579- 1593

Squinazi, F.: Microbiologic air contamination and building-ossociated illness. Aerobiologia 6, 1990, 45- 50

Measurements of Microorganisms in

the Work Environment in Denmark

Birgitte Herbeet Nielsen, Deportment of Toxicology and Biology, National Institute of Occupational Health, Copenhagen, Denmark

Abstract

The Institute of Occupational Health in Denmark consider meosurements of microorgonisms based on personal sompling techniques os the most valid estimate of the exposure Ievei. Full shift exposure is performed ofter the generol principles in determination of oerosols. Routine meosurements are executed on two membrane filters:

I.) Total particulate matter is collected on 25 mm 8.0 l'm cellulose nilrate filters using closed-face field monitors operating at I . 9 Um in with a inlet velocity at 1.25 m/s. After grovimetric determinotion of total dust the filters are extracted and the suspension is further analysed for endotoxin. The endotoxin assay is corried out by the kinetic-chromogenic version of the Limulus Amoebocyte Lysate test.

2.) Microorganisms ore measured according to the principles of the CAMNEA- method. Sampling is corried out on 25 mm 0.4 l'm polycarbonate filters placed in a closed- foce filter

cossettes with o pump colibrated to an airflow of I .0 Umin. After extraction the suspension is onalysed by culturing and by microscopy. Vioble counts ore performed in order to enumerote the colony forming units of live groups of culturoble microorgonisms: mesophilic bacterio, actinomycetes ond fungi (oll 25 oq plus thermophilic fungi (45 oq and actinomycetes (55 oq. Se Ieeted isolates are occotionolly identified by dossie microbiologicol procedures.

Total counts ore carried out by epifluorescence microscopy. Acridinorange is used as fluorochrome and the dyed microbial cells ore occumuloted on dark 0.4 l'm filters. The number of viable and non-viable microorganisms are counted in forty randomly chosen fields at 1250 times magnification grouping into bocteriol rods, bacterial spheres, yeast cells and fungi spores.

Basically oll analysis are quantitative methods used in order to estimate the exposure Ievei in the breath"mg zone for people employed in different work environments.

81



Birgitte Herbert Nielsen, Department of T oxicology and Biology, National Institute of Occupational Health, Copenhagen, Denmark

The Institute of Occupational Health in Denmark has only worked intensively with measurements of microorganisms since 1989, but the institute has more than forty years of experience in measuring dust as aerosols in different werk environments. Measurements of aerosols in generd have focused on personal sampling techniques os the most valid estimate of exposure Ievei. Today, the moin approach for measurements of microorganisms is based on quantitative methods ond personal sompling is performed on filters alter the same principles as the general determination of aerosols.

Sampling techniques

Total aerosols ore collected du ring Iu II work periods on membrane filters for subsequent analysis in the laboratory.

The routine sampling techniques are:

D T otol dust collection on 25 mm 8.0 pm cellulose nilrate filters (Sartorius) using closed-face lield monitors (Millipore) with o 5.6 mm inlet and operated at 1.9 Umin (1.25 m/s inlet velocity).

0 Bioaeroso! cdlection according to the CAMNEA-method [I] on 25 mm 0.4 pm polycarbonate filters placed in a closedface filter cassettes (Nuclepore) with

a pump calibrated to an airflow of 1.0 Umin.

ln some investigations, the fraction of respiroble dust is collected using a modified Higgins and Dewell cyclone connected to the same equipment as for collection ol total dust [2].

Currenl Analyses

The routine analyses of the two different types of membrane filters are

I. o) Total dust

b) Endetoxin

2. a) Total counts ol bacteria and fungi by microscopy

b) Viable counts of culturable bacteria and fungi

Analysis of dust and endotoxin

Total dust

The moss of total particulote matter, total dust, is determined gravimetrically ot a 60 pg Iimit of detection. Pre- and post-weighings of filters are made in a climate chamber kept at a constant temperature and humidity. T o correct for any hygroscopic effects on filters, three blank filters are kept as a standard.

83



Endetoxin

Endetoxin assay is performed on the collected mass of the dust filters. The filters are extracted in I 0.0 ml sterile, non-pyrogenic water by orbital shaking at 250 rpm for 15 min ot room temperature. The extraction fluid is stored at -20 oc until analysis by a chromogenic Limulus Amoebocyte L ysate ( LAL) assoy.

The analysis is performed in duplicate at 37 oC by the kinetic-chromagenic

version of the LAL assay (Kinetic-QCL kit from Bio Whittaker) using an automated microtiter reader (ThermoMax, Molecular Devices) connected to a computer with software for data analyses (SoftMax, Molecular Devices). The endotoxin reaction curve is measured by recording the absorbance at 405 nm every 30 s. during a 60 min. period. The reaction time for the absorbonce to increase 0.200 units is an estimate of the endotoxin concentration. Reference endotoxin E. coli 055,85 is used as standard. The concentration is given in endotoxin units per cbm air (EU/m3

)

which can be converted into nanogrammes per cbm. The Iimit of detection is below 0.5 EU/m3

The kinetic approach has been used at the Danish institute since 1994. Previously 1 the endotoxin ossoy was bosed on the principle of the end-point method

84

applied to chromogenic LAL-assay (Coatest Endotaxin kit from Chromogenix, Kabi Pharmacia). Today the kinetic assay is considered to be o more occurate and sensitive method for quantificotion of airborne endotoxin. A further development of the kinetic assay makes it possible to determine interference in the analysis, e.g. Inhibition or enhancement from other sub

stances in the sample [3].

Analysis ol bacteria and lungi

Before analysing the microorganisms, the polycarbonate filters are extracted in the filter cassettes by adding 5.0 ml 0.05% Tween 80 solution and shoking for 15 min. at room temperature. Sampies for viable counts are immediately used for plating (see poge 84) while the rest is kept at -80 oc for later analysis by fluorescence microscapy (see poge 84). The fallawing counting techniques are performed according to the CAMNEA-method described by Palmgren et al. [I].

Total counts by fluorescence microscopy

Counting by epi-fluorescence microscopy is carried out by staining 1 ml extraction fluid with 0.3 ml 0.0 I %

acridine orange in acetote buffer (bioMerieux) for 30 s. and filtered through a dark 0.4 11m polycarbonate filter (Nuclepore). The number of microbial cells in forty randomly chosen fields is counted by epi-fluorescence microscopy at 1250 times magnification, grouping into bacterial rods, bacterial spheres, yeast cells and fungus spores. The concentration of m·lcroorganisms is given in total counts per cbm air (counts/m 3). The lowest countable concentration of microorganisms is approximately 3 · 1 03 counts per sample. Dependent an the sampled air volume, this corresponds to a Iimit of detection from 104 to 106 counts1m3

air.

Using this method, both viable and nonviable microorganisms are enumerated. At present, the analysis is based on a manual counting technique, which is rather time-consuming. The Iimit of

Microorganisms Media

detection is rather high compared to other microbial analyse.

Viable counts by cultivation

Culturable bacteria and fungi are quantified by inoculation of suitable dilutions of the extraction fluid fram the filters an plates with selective medio for mojor groups of bacteria, actinomycetes and fungi. After incubation, the colony forming units (du) are counted and the concentration is calculated as cfu/m3 oir. The minimum detectable concentration of colony {orming units is 50 cfu/filter, which, depending on the volume of the oir somple, corresponds to a Iimit of detection between 102 and 103 cfu/m3

At present, the following media, incubation temperature 1 oq and incubation time (days) are used as the basic parameters for vioble counts of oir samples,

'C Days

Mesophilic bacteria Nutrient agar1l 25 7 Mesophilic actinomycetes 10% Nutrient agarll Mesophilic fungi DG 18 ogar2l Thermophilie fungi3l DG 18 agar2l Thermophilie actinomycetes Nutrient agar 1l

l

ll Nutnent agar (Oxo1d) w1th act1d1one (cydohex1m1de) 50 mg/l to prevent fungal growth 21 DG 18 agar (Oxo1d) D1chloran Glycerol ogar - the med1a mh1b1tS fast grawmg fung1 31 A;.perg1Uus fum1gatus ore determ1ned oller 3 days o{ mcubat1on

25 7 25 7 45 3 -7 55 7

85

Measurements of Microorganisms in the Work Environment in Denmark

On special occasions or for examinotions of other types of samples, e.g. liquids or solid materiols, highly selective media moy be used for detection of specific genus or species. ldentification of selected isolates is performed by classicol microbiological principles.

Discussion of current and future methods

Sampling lechnique

Air contaminants, including aerosols, are to be expected in o spatially nonuniform distribution at workplaces in general. Therefore, personal sampling is used to abtein a valid estimate of the exposure Ievei in the breathing zone for people employed in different workplace environments [ 4].

Due to evaluating the hazards of the breathing atmosphere, different working groups in standordisation, ISO and ACGIH, have recommended that the collection of "total dust" be replaced by "inhalable dust" measurements. This new concept recognises the fact that only aerosols which pose o potential risk are those that enter the body through the nose or mouth during breathing. At present, the Donish institute is testing a personal lOM- sampler for collection of inhalable dust, but this approach has

86

not yet been implemented as a routine method, while the sampler has not been fully validated. lt has ta be emphasized that the IOM-sampler may cause a considerable increase in the Iimit of detection of gravimetric analysis [5].

Membrane lilters

Cellulose-nitrate filters of pore size 8.0 11m have been used for dust measurements at the Danish institute for many years. When the endotoxin analysis was implemented it was obvious that the filter quality and the extraction procedure had some influence on the results. These problems have previously been described by Olenchock el al. [6] and recently, Douwes et al. [7] have examined the influence of various dust sampling and extraction methods on the measurement of airborne endotoxin. Both studies emphasize the requirement for standardization of filters used for bioaerosols and standards of reference for further preparation and analyses of the samples.

Total caunts by epi-lluorescence microscopy

Oirect detection of microorganisms using microscopy is a dassie way of determining microscopic particles, i.e. by coun-

ting the individual microorganism cells. Especially in aerosols where the survival of microorganisms is compromised by a number of different factors [8], this direct method for quantification of viable and non-viable microorganisms is of great importance. ln different work environments, non-viable microorganisms in bioaerosols may cause, e.g., allergic diseases or toxic reactions [9].

As mentioned above, the manual counting technique is time-consuming and has a high detection Iimit. T o improve the method, the use of automatic image processing would be of great advantage. But this technique is only suitable where it is possible to differentiate between microbial cells and particles of non-microbial origin.

Viable counts by cultivation

Viable counts of microorganisms by counting colony forming units after plating and incubation is also a dassie method first described by Koch in the last century. This technique is still the most common method in quantitative analyses of microorganisms in food and water. Most of the knowledge of airborne exposures to microorganisms is based on measurements of viable counts, although only a minor fraction of the total number of microorganisms can

be detected by cultivation. Hence, to enable comparison with previous studies, it may be necessary to include some measurements of viable counts. Furthermore, the method is the most common way to cultivate specific isolates of microorganisms for identification.

Future methods for analysis of bioaerosols

Endotoxin, or LPS from the cell wall of Gram-negative bacterio, is a welldefined biological agent which has been implicated in a number of occupationa\ diseoses in the work environment. lnhaled endotoxin causes inflammatory responses by activoting macrophages or epithelial cells from the lungs so mediotors causing the inflammotory reactions are released [I 0]. ln fungi, the cells have a major structural wo II component, ß- 1 ,3-D-glucan, which consists of polysaccharides. Glucans are also biological active and able to induce inflammatory responses in humans [1 0] so an ossay to determine glucons will be considered in the future.

Biooerosols often contain a number of different microorganisms, and numerous other components of non-microbial origin may also contribute to possible biological eHects. Therefore, a general and

87



simple dose-response relationship of microorganisms is difficult to apply in toxicological risk assessments of bioaerosol exposure. To improve this, it would be of great importance to develop methods to determinate the "total biological effects" of bioaerosols or in other complex samples. For this purpose, the Danish institute has started to develop an in vitro assay for the quantification of the inflommatory potential of bioaerosols. The measurements consist of ELISA analysis of interleukins, IL-6 and IL-8, secreted from human lung epithelial cells after stimulation with extracts of bioaerosols. Hopefully, this ossoy will turn out to be a useful Supplement to the traditionol microbial characterisotion of bioaerosol exposure.

References

[I] Polmgren, U., Ström, G., Blomquist, G., Molmberg, P., Collection of airborne microorganisms an Nuclepore filters, estimation and analysis -CAMNEA method. J. Appl. Bocteriol. 61, 1986, 40 I - 406

[2] Higgins, R.l., Dewe/1, P., A gravimetric size-selective personal dust sampler. ln, lnhaled portides and vapors. Edited by C.N. Davies, London, Pergarnon Press, 1967

88

[3] Hollander, A., Heederik, D., Versfoot, P., Douwes, )_, Inhibition and enhancement in the analysis of airborne endotoxin Ieveis in various occupational environments. Ann. lnd. Hyg. Assoc. J. 54 I I I), 1993, 647 - 653

[4] Cohen, B.S., Horfey, N.H., Lippmonn, M.: Bias in the air sampling techniques used to measure inhalation exposure. Ann. lnd. Hyg. Assoc. J. 45 (3), 1984, 187- 192

[5] Scheeper, B., Kromhoul, H., Bo-leii, J.S.M., Wood-dust exposure during wood-working processes. Ann. Occ. Hyg. 39 (2), 1995, 141- 154

[6] 0/enchock, S.A, Lewis, D.M., Mull, J. C., Effects of different extraction protocols on endotoxin analysis of airborne grain dust. Scand.J. Work Environ. Health 15, 1989, 430- 435

[7] Douwes, J., Versloat, P., Hol/an-der, A., Heederik, D., Doekes, G., lnfluence of various dust sampling and extraction methods on the measurement of airborne endotoxin. Appl. Environ. Microbiol. 61 (5), 1995, 1763 - 1769

[8] Grifliths, W.D., DeCosemo, G.A.L., The assessment of bioaerosols - a criticol review. J. Aerosol Sei. 25 (8), 1994, 1425- 1458

[9]locey, J., Dutkiewicz, J., Biooerosols and occupational lung disease. (review) J. Aerosol. Sei. 25 (8), 1994, 1371- 1404

[I 0] Rylander, L Symptoms ond mechanisms - inflammation of the lung. Ann. J. lnd. Med. 25, 1994, 19- 23

89

Sampling of Bioaerosols 1n lndoor Environments in Finland

Aino Nevalainen, National Public Health Institute, Division of Environmental Health, Kuopio, Finland

Abstract

There are two porameters affecting the quality of microbiological indoor air dah the physical collection efficiency of the chosen sampling device and its biological collection behaviour. Detection of microorganisms by culture growth is one analytical approachi

besides, there are other analytical methods which do not require the use of viable material. Dependent on the expected germ concentration, it is possible to use impaction or fi\tration methods which can be completed by additional information collected on the premises (e.g. inspection, surface samplesl.

91

Sampling of Bioaerosols in lndoor Environments in Finland

Aino Nevalainen, National Public Health Institute, Division of Environmental Health, Kuopio, finland

Criteria in selecting a sampler for indoor air sampling - physical properfies -

ln sampling bioaerosol particles, the total efficiency of the method is a complex phenomenon that includes the physical properlies of the sampling device and the biological properlies of the analysis phase (Nevalainen et al., 1993). The biological properlies of the portides indude many variables, such as survival or alteration of the organisms during sampling. There is no method able to reveal everything about the whole bioaerosol. One must decide beforehand what the targets of the sampling are and then select the sampling method, focusing on that particular " of bioaerosol.

The physical collection efficiency of the sampler is influenced by the geometry and other physical characteristics of the sampling device, which determine the inlet efficiency. ln practice, it is mainly the cut-off size (d50 ) of the sampler that the investigator will consider. The cut-off size is the diameter at which half the portides pass through the inlet (Willeke and Baron, 1993). ln indoor oir sampling, where bacteria and bacterial and fungal spores are the main interest, the cut-off of the sampler should be dose to I /.lffi.

Biological efliciency

Biological efficiency comprises many aspects. Where viable organisms are to be collected, it is essential to maintain their viability during the sampling process which may \ndude abrupt changes in pressure, desiccation and other effects. Once collected, the viable organism must be detected. This can be done by cultivating it and detecting the colony formed. ln this case only the colanies of those organisms develop for which the nutritional, temperature and other growth requirements are met. However, only I to I 0 % of the environmental organisms are culturable in Iabaratory conditions. The rest may be dead cells or "viable but not culturable". While most of the health effects are also caused by these particles, the total amount of cells or spores should be detected by direct counting of the partides. Other possibilities for detection are immunochemical methods (presence of an allergen) and those based on molecular biology, e.g., PCR (polymer chain reaction). The analysis may also be based on the detection of the total amount ot micro-bial material, e.g., endotoxins of Gram-negative bacteria or the ergosterol of fungi.

93

Sampling of Bioaerosols 1n lndoor

Environments in Finland

Other important characteristics

Most bioaerosol sampling is carried out in practical situations, in workplaces, homes or schools, for the purpose of hygienic monitaring of the indoor air. Therefore, there are limitations on the sampler regarding its size, noise and collection time. The sampler must be portable, and its noise Ievei should not disturb the activities in the facilities to be

monitored. Sampling Iimes depend on the concentration Ievei. Again, sampling for more than o few hours is seldom possible, and much shorter tim es are preferred.

Creating a logical strategy for the monitaring purposes also includes selecting reference material for the bioaerosol types and the environments to be sampled. For bioaerosols, there are no TLV values and few guidelines for the sampling have been published. Each investigator must, therefore, produce reference material for his own purposes: at least for the range of the expected concentrations and enabling the analysis of the major factors regulating them.

Strategy for indoor sampling of fungi and bacteria

0 Andersen 6-stage impactor, loaded with 2 % MEA, DG 18 (fungi) or

94

TYG medium (bacteria) is mainly used for indoor air environments with no specific sources to producing massive loading. The media cover most mesophilic and xerophilic fungi and bacteria, especially actinomycetes.

0 Filter sampling with culturing in the above-mentioned media and epifluorescence counting of total concentrations for indoor environments with expected high concentrations, e.g., during demolition or repair (Rautiala et al. 1995).

0 When the microbiological status of the building is to be assessed, surface swabbing and material samples cultured in the Iabaratory are also used and recommended, in addition to, or instead of, air sampling.

Guidelines have been set up for the normal ranges of indoor bacteria and fungal concentrations (Reponen et al., 1992).

References

Nevalainen, A., Pastuszko, J., Liebhaber, F., Willeke, K., Performance of bioaerosol samplers: collection characteristics and sampler design considerations. Atm. Env. 26A (4), 1992, 531 - 540

Nevalainen, A., Willeke, K., Liebhaber, F., Pastuszka, J., Burge, H., end Henningson, E.: Bioaerosol Sampling. Chapter 21, in, Aerasal Measurement, Princ"lples, T echniques and Applicatians. Ed. by K. Willeke and P.A. Baron, von Nastrand Reinhald, 1993, 471 - 492

Rautiala, S., Reponen, T., Hyvärinen, A., Nevalainen, A., Husman, T., Vehvilä-

inen, A., Kalliokoski, P., Exposure to air-borne microbes during the repair of mouldy buildings AIHA J (in press).

Reponen, T., Nevolainen, A., Jontu

nen, M., Pellikka, M., Kalliokoski, P., Normal range criteria for indoor air bocteria and fungal spores in a subarctic climate. lndoor Air 2, 1992, 26- 31

95

The Development of Standardization Procedures for

the Assessment of ßioaerosols in the Workplace

W.D. Griffiths, and I.W. Stewart, AEA Technalogy, Hanwell, United Kingdom

Abstract

A broad spectrum of bioaerosols is released ot the most varied of workplaces where they can result in negative consequences for the health of the workers exposed to them. Measurement of biooerosols in the workplace is therefore urgently required. Despite extensive studies and publications devoted to this subject in recent years, there are as yet no standardised instructions available for ossessing air contamination caused by the presence of germs in working environments. The performance criteria for bioaerosol collectors must therefore be defined, standard protocols drawn up and standardised measuring methods established. Measurements of the Ievei of airborne organisms with filtration collectors are mainly carried out with polycarbanate filters which, in particular, permit measurement of high concentrations of organisms. Whereas filters are ideal for the collection of robust mould fungus spores over Ionger periods of time, in the collection of sensitive microorganisms, such as bacteria and yeasts, the limitatians due to mortality rotes must be taken into consideration. Cyclone collec-

tors capture the aerosolised microorganisms in fluids. Some of the results provided by different outhors indicote a higher detection rate with germs such as Saccharomyces cervisiae that are susceptible to drying out.

Efficient methods of producing mono and polydisperse bioaerosols are described below. Polydisperse bioaerosols were produced by using simple glass atomisers and conventional nebulising devices. The ability of the microorganisms (bacteria, yeasts) to survive in the aerosols is heovily dependent on the selected test conditions. Species-specific characteristics must also be duly considered, as weil as the stress factors acting on the microorganisms. Griffiths et al. implemented a programme for developing biaaerosol standords. The bioaerosol lest chamber (BTC) developed permits the aerosolisation of various microorganism suspensions (I 06 to I 09 cells/ml) within broad temperature and air humidity ranges. The BTC chamber is suitoble for the production of standardised aerosols of bacteria, yeosts ond moulds and has already been used successfully in enzyme and antibiotic studies.

97

The Development of Standardization Procedures for the Assessment of Bioaerosols in the Workplace

W.D. Griffiths, and I.W. Stewart, AEA Technology, Harwell, United Kingdom

Health Effects

Airborne microorganisms associated with different occupations and work environments have for some time been implicated in causing occupational respiratory diseases (ßennett, 1994). Such bioaerosols can be produced from airborne portides which are biological in origin. These "particles" include bocteria, fungal spores, actinomycete, fern spores, pollen, algae and plant cells, insect and mite frogments and excreta, proteins, enzymes and antibiofies from biotechnology processes, endotoxins, and mycotoxins.

The effects of certain pothagenie bocteria, fungus and viruses hove been recorded in some environments, but the more common effects of exposure to occupotionol bioaerosols are due to mucous membrane irritation, bronchitis, allergic rhinitis, asthma, extrinsic alveolitis and organic dust toxic syndrome (Locey and Dutkiewicz, 1994).

Bioaerosols can be produced in many occupations, and are present in a variety of work environments such as farms, intensive animal houses, abattoirs, maltings, wood chip processing facilities, storage and handling facilities for organic materials, ships' holds, engineer-ing workshops, medical and dental premises, food processing plants, water

purification plants, scientific research establishments, biotechnology plants, pharmaceutical plants, and many others. The problems associated with bioaerosols are often related to production, process contamination, environmental exposure, as weil as occupational exposure (Griffiths and Sokhi, 1993).

Aerobiological Monitaring

The assessment of bioaerosols has been reviewed by Griffiths and DeCosemo ( 1994) , and they considered the main

. techniques available for sampling and

assaying airborne microorganisms. Factars which can affect the survival of airborne microorganisms and problems associated with the production of test bioaerosols were addressed.

Griffiths and DeCosemo also considered the need for aerobiological monitaring in occupational hygiene, containment of bioprocessing equipment, environmental exposures, deliberate release of microorganisms, contamination of food and pharmaceutical products, and general scientific research. They reported that despite wide application of aerobiological monitoring, no standerd protocols exist for sampling bioaerosols. Thus, six criterio were identified by the authors for the monitaring of bioaerosols. SampIers used to collect bioaerosols, and

99

The Development of Stondordizotion Procedures for

the Assessment of Biooerosols in the Workploce

assoy methods were also described, and areas of their opplication were highlighted. Henningson and Ahlberg ( 1994) reported an evaluation of numerous bioaerosol samplers and stated that there is a need for stondordizotion methods and recommended procedures for their use.

Filter Sampling

Membrane and gelatine filters (Macher and First, 1984) and polycarbonate filters (Biomquist et al, 1984a) have been used in conjunction with plastic cassette samplers for bioaerosol sampling. One of the moin disadvantages of using filters to collect airborne microorganisms is that they afford little protection to the cells, so that the I arge volumes of air passing through the filter may cause a degree of desiccation. This may affect the viability of the collected microorganisms. For exomple, Stewart ( 1995) describes experiments where aerosolised cells from liquid cultures of Soccharomyces cerevisiae were collected on polycarbonate or gelatine filters in an lOM Personal lnspirable Aerosol Sampier (PIAS) (Mark and Vincent, 1986) for periods up to 180 minutes. Under standard conditions total and

culturable counts were made and bioluminescent assays of odenylote energy charge and total adenylate con-

100

centrations were performed. S. cerevisiae cells were aerosolised at relative humidifies of 30, 40, 50, 60 and 70 %. Over this range, the results showed that the culturable fraction was highest at 70 % RH with a value of 0.21 compared with an unaerosolised value of 0.66. Generally less than I 0 % of the captured cells were culturable. The ratio of total odenylates to total count varied from 0.00 I to 0.007 compared with an unaerosolised value of 0.007. The lowest ratios were obtained with cells that had been captured an filters for up to 180 minutes at 30 % RH.

Robust moterials, such as fungol spores are little affected by aerosqlisation. Polycarbonate filters have been used extensively in Sweden with diverse types of plastic cassette filter holder to collect bioaerosols. ßlomquist et al., ( 1984b), Palmgren et al, ( 1986a and b), and Ström, ( 1986) used them to collect airborne fungol spores in highly contominated environments. Crook et ol., ( 1988a and b) used them to monitor airborne microorganisms from domestic waste composting plants and in coffee warehouses. Recent work by Birch and Griffiths ( 1993) has further demonstrated the usefulness and limitations of Nuclepore and Anopore filters (Anotec, Oxon., UK.) in assessing airborne microorgonisms. The microorganisms collected in such a fashion are eilher washed off

the filter for detection (Palmgren et al, 1986b), or detected directly on the filter using staining techniques.

Blomquist ( 1995) has reported that the sampling of bioaerosols in Sweden is carried out using polycarbonate filters (presumably contained in a plastic cassette holder). The collection efficiency of such a cassette does not match the inhalation criteria relevant to health related sampling (ISO, 1991 ), compared with alternative personal samplers such as the lOM PIAS (Mark and Vincent, 1986), and the Health and Safety Executive (HSE) 7-hole sampler (HSE, 1986). The matter of collection efficiency is fully discussed by Vincent ( 1989).

Blomquist reports that filters used to collect bioaerosols are routinely sent to d'Merent laboratories for assay. He proposes that this ' 1round-robin" exercise of analysis need not necessarily be carried out only in the country where the samples were collected. This procedure will facilitate the comparison of the analytical results of a series of samples obtained in a number of selected laboratories throughout Europe. This important process will allow the establishment of the standardisation of measurements of bioaerosols collected in the workplace environment. Forthis to come about, bioaerosols must be collected using an acceptable technique and sampl-

ing device, and analysed using an agreed methodology. ln addition to this, standard samples must be collected which accurately represent bioaerosols from a number of selected workplace environments.

Cyclone Sampling

Errington and Powell ( 1969) described two bioaerosol sampling cyclones that have liquid continuously sprayed into the sampler inlet. These sampling cyclones are constructed in Perspex or stainless steel, and have sampling rotes of 75 and 350 I min- 1

• Decker et al, ( 1969) characterised a large volume (< 1000 I min- 1

) glass sampling cyclone (Aerojet-General Liquid Scrubber Sampling Cyclone), which has been further described by Buchanan et al., (I 972). As cyclones with spray-weiters have been found to be gentle with airborne microorganisms, they help to maintain cell viability Ieveis.

The response of the Aerojet cyclone to large Windborne portides was assessed by May et al., ( 1976), who compared it to other samplers. Recent work by Upton et al., ( 1993 and 1994) and by Griffiths et al., ( 1993) has extended the range of this work to show that the collection efficiency of the sampler, Operating at a sampling rate of 500 I min- 1

101


the Assessment of Bioaerosols in the Workplace

and with speeds up to 4 m s- 1, is bet

ween 70 and I I 0 o/o for portides as large as 20 Jlm aerodynamic diameter. Penetration of the smallest lest portides (d50 ~ 1.5 11m) through the sampler was also observed. Rothwell et al., (1993), have used test aerosols of Escherichia coli, and S. cerevisiae to record similar collection efficiency as Upton et al. and show that the bioefficiency of the sampler was subjected to much variation. Recovery agents in the wetting liquid were not tested in their work, and such agents may weil improve viability retention.

Stewart et al., ( 1994) aerosolised S. cerevisiae cells and collected them in an Aeroiet General Cyclone. Using standard conditions of buffer contact and biochemical reagent incubation times, total and culturable counts were trade and biofluorescent ossays of adenylate energy charge and total adenylate concentrations were performed. S. cerevisiae cells were aerosolised at temperatures of 20 and 25 oc and

relative humidities of 30, 40, 50, 60 and 70 o/o. Over the range of temperatures and relative humidifies covered, the results showed that the metabolic activity of the microbial cells increased with increasing humidity. The ratio of culturable and total cell counts increased from 0.05 at 30 o/o RH to 0.50 at 70 o/o compared with the non-aerosolised

102

culturable fraction of 0.71. The mean ratio of total adenylates to total cell count ranged from 0.005 to 0.007 over the humidity range compared with 0.007 for non-aerosolised cells. These results show that the measurement of total adenylates can give a good estimate of total cell numbers for S. cerevisiae and the method yields results within 2 I /2 hours. The culturable fraction data also provided new insights into the oerostability of S. cerevisiae not previously observed.

Griffiths ( 1994) reported on the behaviour of bioaerosols of E. coli, S. cerevisioe and Penicil/ium exponsum spores in an Aerojet cyclone. The experi-ments were carried out under controlled environmental conditions (20 °C, 30 o/o RH) in a specially designed Bioaerosol Test chamber (Griffiths, 1993 and Williamson and Griffiths, 1993). The collected microorganisms were assayed using a total count method, following the method of Jones ( 1979), and also by o standerd culturing technique (Koch, 1981). Griffiths found that aerosolisotion extensively affected the culturability of E. ca/i and S. cere-visiae, but had little affect on P. expansum spores. Only the Ietter species of microorganism oppears to be a potential condidate for o reference material. The results of the tests indicated that culturing alone moy not be sufficient to ossess

the number of microorganisms accurately, and that other techniques, such as total count, should also be used

Bioaerosol Generation and Survival

Methods for the production of polydisperse ond monodisperse biooerosols from 1·1quid suspens·lon and from dry powder were described by Griffiths and DeCosemo (1994). The survival characteristics of microorganisms were also discussed because they con significantly affect the selection of sampler and assay method appropriate to the microorganism under consideration.

Simple glass atomisers and conventional nebulisers, such as the weil known Collison nebuliser, have been found to be effective in produöng controllable high concentrotions of polydisperse aerosols of bacteria cells and spores from liquid suspensions. The choice of liquid for suspending the m·lcroorgonisms in the generoter reseNoir has o sign·lficant effect on the oerosolised product in terms of concentration, size distribution, and viability. The quantitative loss of viability due to the process of aerosol generotion is related to many equipment porometers, ond to the species and state of the microorganism. lt is likely that the liquid (buffer or broth solution) used to moke up the stock Suspension,

the initial culturing procedure, and the residence time of the organisms in the supporting liquid prior to aerosolisotion may greatly affect the initial viability of the system. The size of the natural microorganism cell or spare con have an impact on the performance of the aerosol generator: gloss atomisers have been found to be very effective for I arger cells such os 5. cerevisiae, while nebulisers have been found (ust os effective for smaller cells, such as E. co/i and B. subti/is vor niger. The latter type of generoter concentrates \arger cell numbers in suspension and aerosolises the liquid part of the suspension.

Monodisperse aerosols of bacterial cells and spores can effectively be produced using a Spinning Disk Aerosol Generator. The oerosol concentration Ieveis produced by conventionally air-powered devices tend to be low, thus limiting their use. Higher concentrotions of conventional aerosols hove been obtained by moking use of o modified version of the generator that is electrically powered.

Dry ergonie powders, such os pellen and fungal spores, can most effectively be mode oirborne by means of oirjetting, but fluidised-bed techniques are also effective. Electrostatic charge equilibrotion by meons of ionising rodiation is recommended for such aerosols. Humi-

103


the Assessment of ßioaerosols in the Workplace

dity-related problems are frequently associated with dry powder techniques.

Griffiths and DeCosemo I 1994) and ather studies have shown that great care must be taken over the interpretation of the term "non-viable", as it may not mean that the microorganism is dead. Cax I 1989) suggested that nonviable microorganisms be considered as microorganisms that have suffered some degree of darnage and may be repaired under some selected culturing con

ditions.

Dynamic physiological phenomena, whether directly or indirectly controlled by genetic mechanisms, are involved in the survival processes of microorganisms. Specific conclusions relating to phenomena which can influence the viability of airborne microorganisms are:

D the species and strains of microorganism must be treated as separate entities; no general rules relating to the survival of microorganisms can be ap

plied,

D bacterial and fungal spores survive better than vegetative cells,

D cell mutation can make it difficult to assess whether a certain strain of microorganism will respond in a constant fashion to an applied stress over o period of time, and the survival proper-

104

ties of microorganisms are affected by the chemical properlies of the growth medium used; this introduces the possibility that storage time and conditions can influence subsequent survival,

D survival is dependent on the stresses imposed by the method used to generate the aerosol,

D the suspending medium land protective additives which may be used) in the aerosol generoter reservoir can have an effect on the viability of the microorganisms; the period of time spent suspended in this fluid lcontact time) can also affect the viability of the microorganisms,

D aerosol sampling can induce much stress and therefore greatly affect the viability of the collected microorganisms,

D collection fluids used in some samplers are usually based on simple salt solutions with protective additives which, along with the contact time and environmental conditions pertaining at the time of sampling, have an effect on the viability,

D the environment has a major influence on the viability of airborne microorganisms; the most important factors are RH and oxygen Ieveis,

D other, less important environmental factors which con cause stress are radiation, temperature, air movement, and pollutants; the most influential pollutont process is the Open Air Foctor IOAF) caused by the reactions of ozone products,

D non-lethol events and repair mechonisms have to be consideredi the degree ond permonence of damoge is dependent on species, environment, the stress which has been applied, and the ability of the microorgonisms to repair the damage.

Cell survival and the viability of biooerasals con be summarised in terms of o number of different factors and on the cumulative application of a series of different stresses. These can be applied before, during, the airborne state, and on collection and assay, and ultimate survival is dependent upon the ability of the microorganisms to repair the damage caused by oll of these stresses.

ln conclusion, Griffiths and DeCosemo I 1994) outlined recommendations and a programme of research to assess the choracteristics and behoviour of bioaerosols prior to the considerotion of the possible establishment of bioaerosal standards far test purposes.

Bioaerosol Standards

The need for standard samples has already been demonstrated by Lewis et al., I 1993) and by Griffiths and Sokhi, I 1993) in their reports relating ta the National Measurement lnfrastructure far Aerosols and Particulates in the Airborne Phase, VAM Scheme Project 14, Survey of User Needs, and The Requirements for Bioaerosol Standards. This survey was carried out mostly in the UK, but with important input from Europe and the USA, and showed that an overwhelming proportion of bioaerosol users who were questioned wanted the establishment of bioaerasol stan

dards and sampling guidelines. Many of the respondents were active in the field of occupational hygiene and workplace monitoring. The VAM survey demonstrated a need for the standardisation of bioaerosols, and for the produdien of reference microbiological material. Griffiths and Sakhi I 1993) reported on the results of the survey1

ond also outlined o programme of werk necessary to develop bioaerosol standards. Same parts of this programme were carried out during 1993 ta 1994, and the results were reported by Griffiths I 1994). This publicatian considered the results of an experimental programme to examine the validity of culture techniques in the assessment of biooerosols, with particulor reference to

105



the effects aerosolisation moy have on the viability of microorganisms.

Griffiths reported thot microorganisms could reodily be oerosolised from liquid suspensions of I 06 to I 09 cells ml- 1

using the Bioaerosol Test Chomber (8TC) developed by Griffiths ( 1993) ond characterised by Williamson ond Griffiths ( 1993). The BTC can control lest atmospheres in the temperature ronge 20 to 40 'C, and relative humidities of 20 to 80 %. The 8TC hos been extensively used to study the effects of generafing aerosols from a number of biologically octive portides including S. cerevisioe (Stewort, 1994 and Stewart et ol, 1994) and an antibiotic (Farrell et al, 1994). Biooerosols have been successfully generated for collection on filters, and into solutions by means of an Aerojet cyclone. ln addition to bacteria, yeast and fungal spores, the lest chamber has been used to produce standerd bioaerosols containing specified enzymes and antibiotics.

References

Bennetl, AM., Heolth Hazords in Biotechnology. Chapter 7, in, Hambleton, P., Melling, J., and Solusbury, T.T. (eds.), Biosafety in lndustrial Biotechnology. 1994, Chopman and Hall, London

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Birch, D.J., ond Griffiths, W.O., Personal Sampier Efficiencies. Warren Spring, Labaratory Research Paper W94009, 1993

8/omquist, G., Arbetsmiljöinstitutet, Umea, Sweden. Personal communication, 1995

Blomquist, G., Polmgren, U., ond Ström, G., lmproved techniques for sampling airborne funeal portides in highly contaminated environments. Scand. J. Environ. Health I 0, 1984a, 252- 258

8/omquist, G., Ström, G., ond Strömquist, L.H., Sompling of high concentrations of airborne fungi. Scand. J. Work Environ. Heolth I 0, 1984b, I 09- 113

Buchonon, L.M., Horstod, J. B., Phil-lips, J.C., Lofferty, E., Dohlgren, C.M., and Decker, H.M., Simple liquid scrubber for large-volume air sompling. Appl. Microbiol. 23 (6), 1972, I 140 - I 144

Cox, C.S .. ' Airborne bocteria and viruses. Sei. Pro. Oxf. 73, 1989, 469- 500

Crook, 8., Bordos, R.P., ond Locey, J., Domestic Waste composting plants as a source of airborne microorganisms. ( 1988o) Aerosols, Their Generation, ßehaviour ond Applications. Proceedings of The Aerosol Society 2'd Conference 22-24 March 1988

Crook, 8., Griffin, P., Topping, M.D., ond Locey, J., An opproisal of methods for sampling aerosols implicated as causes of work-related respiratory symptoms. I 1988b) Aerosols, Their Generation, Behaviour and Applications. Proceedings of The Aerosol Society 2cd Conference 22-24 March 1988

Decker, H.M., Buchanan, L.M., ond frisque, D.E.: Advonces in large-volume air sampling. Cantamination Control. 8, 1969, 13- 17

Errington, F.P., and Powe/1, E.O., A cyclone seporator for aerosol sampling in the field. J. Hyg. Camb. 67, 1969, 387- 399

Farre// C.D., Rowe/1, F.J., and Cumming, R.H., A Rapid, Specific ELISA for Monitaring Airborne Ceftazidime in lndustry. Proceedings of the Royal Society of Chemistry Analytical Division Meeting on Research and Development. Topics in Analytical Chemistry, University of Hertfordshire, I 8-19 July, 1994

Griffiths, W.O., Design requirements for a chamber to test airborne microorganisms and samplers. I 1993) Warren Spring Labaratory Research Paper W94014

Grifliths, W.D., Annexe C Biologieall Microbiological Aerosol Standards. Final Report , The Effect of aerosolisation

Parameters on Viability, the Validity of Culturing Techniques. I 1994) AEA Technology Report AEA-TPD-233

Griffiths, W.D., and Sokhl, R.S., An-nex C: Specification for a study on biolocgical/microbiological aerosol reference materiols (final report). ( 1993) AEA-EE-0445. Annex C of T echnical Progress Report: National Measurement lnfrastructure for Aerosols and Par

ticulates in the Gas Phase, Valid Analytical Measurement Scheme, Pro-ject 14

Grilliths, W.D., and OeCosemo, G.A.L., The assessment of bioaerosols: a critical review. J. Aerosol Sei. 25 (8), 1994,1425- 1458

Grilfiths, W.D., Upton, S.L., and Mark, 0.: An investigation into the collection efficiency and bioefficiency of a number of aerosol samplers. J. Aerosol Sei. 24 (Suppl. I), 1993, 541 - 542

Health and Safety Executive (HSE), General methods for the gravimetric determinotion of respirable and total inhalable dust. ( 1986) MDHS 14, HSE, London

Henningson, E.W., and Ahlberg, M.S., Evaluation of microbiological aeroso\ samplers: a review. J. Aerosol Sei. 25 (8), 1994, 1459- 1492

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ISO, Air quality - Parfiele size fraction definitions for health related sampling. I 1991) T echnical Report ISO!TC 146 Doc No N338. International Standards Organisation, Geneva

Jones, J.G., A guide to methods for estimating microbiol numbers and biomass in freshwater. I 1979) Freshwater Biological Association Scientific Publication N° 39

Koch, A.L., Growth Measurement. I 1981) Ch. II in Manual of Methods for General ßacteriology. Ger-hardt, P., Murray, R.G.E., Conist-low, R.N., Nester, E.W., Wood, W.A., Krieg, N.R., and Phillips, G.B. leds} American Society for Microbiology.

Lacey, J., et al: Harmonisotion of sampling and Analysis of Mould Spores. Nordic Council of Ministers, Copenhagen 1988

lacey, J., and Dutkiewicz, L Bioaerosols and occupational lung disease. J. Aerosol Sei. 25 18), 1994, 1371- 1404

lewis, G.N.J., Mitche/1, J.P., Griffiths, W.O., Mark, 0., and Sokhi, R.S., National Measurement lnfrastructure for Aerosols and Particulales in the Airborne Phase, VAM Scheme Project 14, Survey of User Needs. I 1993) AEA-EE-0442

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Macher, J.M., and First, M. W., Personal air samplers for measuring occupationol exposure to biologicol hazards. Am. lnd. Hyg. Assoc. J. 45 12), 1984, 76- 83

Mark, 0., and Vincent, J.H., A new personal sampler for airborne total dust in workplaces. Ann. occup. Hyg. 30 I 1), 1986, 89 - 102

May, K.R., Pomeroy, N.P., and Hibbs, S., Sampling techniques for I arge windbome particles. J. Aerosol Sei. 7, 1976, 55- 62

Palmgren, U., Ström, G., Malmberg, P., and Blomquisl, G., The Nuelepore filter method: A technique for enumeration of viable and non-viable airborne microorganisms. American Journal of lndustrial Medicine I 0, 1986a, 325 - 327

Palmgren, U., Ström, G., Blomquist, G., and Malmberg, P., Collection of airborne microorganisms on Nuclepore filters, estimation and analysis -CAMNEA method. J. Appl. ßact. 61, 1986b, 40 I - 406

Rothwe/1, G., Wi/liamson, P., and Grifliths, W.O., An investigation into the bioefficiency of selected aerosol samplers. I 1993) Warren Spring, Labaratory Research Paper W940 18

Stewart I. W., Wi//iomson P., and Co/. !ins, L.J.: Bioluminescent Assay of Adenylate Energy Charge and Total Adenylates in Aerosolised Saccharo· myces cerevisiae. Proceedings of the Eighth Annual Conference of the Aerosol Society, University of York, 21 · 23 March, 1994

Stewart, I. W.: Bioluminescent Assay of Adenylate Energy Charge ond Total Adenylates in Aerosolised Saccha· romyces cerev·rsiae. I 1994) AEA T ech· nology Report AEA.TPD·I65, Har· weil

Stewart, /. W.: ßioluminescent Assay of Adenylate Energy Charge and Total Adenylates in Aerosolised Saccharo· myces cerevisiae captured an Filters. I 1995) AEA T echnology Report in pre· paration

Ström, G., Qualitative and quantita· tive analysis of microorganisms. Particularly funcal spores. Methodological developments. I 1986) Umea University

Medical Dissertation, New Series N" 175

Upton, S.L., Mark, D., Douglass, E.J., and Griffiths, W.D., A w·rnd tunnel evaluation of the sampling efficiencies of some bioaerosol samplers". Pro-c. 7'h Annual Conference of The Aerosol Society 11993). From, Aerosols- Their Generation, Behaviour and Applications., 156

Upton, S.l., Mark, D., Douglass, E.J., Hall, D.J., and Griffiths, W.D., A wind tunnel evaluation of the sampling efficiencies of three bioaerosol somplers. J. Aerosol Sei. 25 18), 1994, 1493 . 1502

Vincent, J.H., Aerosol Sampling, Science and Practice. I 1989) Published by John Wiley and Sons, Chichester

Wi//iamson, P. W., and Griffiths, W.D., Characterisotion of a Biooerosol Test Chamber. I 1993) Warren Spring Labaratory Research Paper W940 15

109

Harmonised Methods for Measuring ßioaerosols

at the Workplace in Germany

Christoph Deininger, Berufsgenossenschaftliches Institute for Occupotionol Sofety, Sankt Augustin, Germony

Abstract

Proteefing workers from risks related to exposure to biological agents at work has, over recent years, become an increasingly important aspect of heolth ond sofety ot work, also finding expression in EU directive 90/679/EEC, which is devoted to this very subject. ln this BIA report, Dr locey's orticle in particular goes into more detail about the potential risks to the heolth of workers exposed to biological agents, especially microorganisms. The Berufsgenossenschaftliches Institute for Occupotionol Sofety, the BIA, Iook up this subject very eorly on ond created its own microbiology section in 1992. ln the microbiology Iobaratory subsequently instolled ot the BIA, samples collected at workplaces are analysed for their bacteria and fungi content, ollowing, by determining the germ content, an ossessment to be mode of the workploce. This ossessment deols with the possible risk orising from microorgonisms and pro-poses suitable protective measures if these prove to be necessary. Determining the Ievei of organisms present in the air at the workplace is the main focus of BIA's testing activities which ore supplemented by material sompling (e.g. from circulating water systems, air humidifiers and cooling lubriconts).

110

Project group 4 "Workploce Assessment" of the ßiologicol Agents Committee (ABAS) which was set up by the Federol Ministry of lobour ond Sociol Allairs ond which is choired by the BIA, hos the tosk of estoblishing standardised measuring procedures for biologicol ogents ot the work-place - in the initial phase bacteria (generol) ond moulds/yeosts (generol) -and developing a suitable associated meosuring strotegy thot duly tokes into account the special characteristics of the biologicol ogents. ln oddition, the working group will propose suitable microbiological meosuring parameters (sum, group and key parameters) for ossessing different types of workploce.

A first standardised measuring procedure for determining moulds/yeasts in the oir ot the workploce was developed by PG4 under the averoll control of the BIA ond put out to tender for triol in the BIA-Arbeitsmoppe. The meosuring procedure is based on sampling by collection on membrane filters (e.g. polycorbonote, cellulose ester, gelotine) ond subsequent onolyticol determination by culturing on nutrient medio (malt extroct, DG 18 ogor). A direct method is also described which is porticulorly suitoble for low concentrations of airborne organisms and orientating measurements, as is an

indirect method which also permits measurement in highly contaminated areas and long sampling times, e.g.

more than 2 hours. The results are then given in colony-forming units per m3 of air.

III

Hormonised Methods for Measuring Bioaerosols


Christoph Deininger, Berufsgenossenschaftliches Institute for Occupational Safety, Sankt Augustin, Germany

lntroduction

The EU Directive 90/679/EEC, definitively amended by the EU Directive 93/88/ EEC, aims to protect workers from safety and health hazards due to workplace exposure to biological agents; this protection includes measures to prevent such hazards. Consequently, it will be necessary in future to determine the type, extent ond duration of workers' exposure for any activity related to biological exposure. lt appears useful, against the general background of the Directive, to develop suitable methods which can be us'ed to identify biological agents at the workplace. Those responsible for safety and health protection at work should be provided with consistent methods for sampling and onalysing biological agents at the place of work.

''Measuring methods and strategies" - a task force

While transposing the above EU Directlve into national law, the Federol Ministry for Labour and Sociol Affairs set up an expert committee for biological working agents (Ausschuß für Biologische Arbeitsstoffe - ABAS), to act os a consulting body for the Ministny regarding any problem in connection with biological working agents (figure 1).

114

Figure 1: Committee for Biological Agents at Work [ABAS)

UA 1 lntended Use

UA 2 Unintended Use ---------i

PG 1 T ransposing EU-Directive 90/679/EEC

PG 2 lndustrial Medical Care Gen TSV

PG 3 Classification of Biological Agents

PG 4 Workplace Assessment _____ ___J

Part of this committee is the task force "Workplace assessment" (formerly "Measuring methods and strategies") which recently began its work. This task force, headed by the Berufsgenossenschaftliches Institute for Occupational Safety, is entrusted with the following tasks:

1. T o harmonise methods for measuring biological agents at the workplace.

2. T o determine the corresponding measuring strategy

3. To develop different measuring parameters for assessing biological agents at different workplaces.

The tosk force was convened because standardised methods for measuring microorgonisms at the place of work were lacking and measuring results and methods for biological agents (keyword, living material) had always been largely

heterogeneaus in the post. For the purpose of comporoble workploce ossessments in terms of microbiologicol exposure, hormonisotion of meosuring methods therelore appeared urgently necessory.

Instruments for samp\ing bioaerosols must meet o number of special performonce requirements, viz.:

0 The suction flow must be high enough to ensure that a sufficient quantity ol air is sucked in (which is important for little contominated working areas) and low enough to prevent microorganisms (in particular bacteria) from drying out.

0 The suction velocity must be high enough to ensure thot even sma\l particles (approx. < I 11m) are sucked in, and low enough to ovoid mechonical damage to the microorgonisms (in porticular bacteria).

0 The sampling volume must be precisely quantiliable.

0 The meosuring results must be accurate and reproducible.

0 The particle size distribution must be charocterisable.

0 Sampling must be possible in personal and/or stotionary form.

D Even extremely high contaminotion must be quantiliable.

D The instrument must be easy to handle (e.g. battery-powered, size, weight).

0 Other specHic criteria must be lullilled.

Meosuring methods for germ determination not only include

a) in-plant sampling, but also

b) storage and transportation to the laboratory,

c) sample preparation (at present mastly cultivation techniques),

d) analysis and finally

e) evaluation of measuring results.

The ossessment of atmospheric microbiological workplace exposure can generally be carried out on the basis of different parameters (figure 2, see poge 116). As in most cases complex mixtures of d·1fferent microorganisms ore present at the workplaces - particularly when contact occurs unintentionally, os e.g. in the agriculture and waste disposal industry, the consideration of so-called sum parameters (i.e. the total number ol culturable (countable) bacteria or moulds/yeasts), or group para-

115

Harmonised Methods for Measuring Bioaerosols


Figure 2: Parameters for Assessment of Microbial Workploce Load

Sum Parameters

Beeterio Moulds/Yeasts

Group Parameters

Endetoxins Enterobocteriaceae Actinomycetes

lndicator Parameters

Aspergillus fumigatus Staphylococcus aureus Escherichia coli

lnfectious Agents

Legionella pneumophila Mycobacterium tuberculosis Pseudomonas aeruginosa

meters like endotoxins and enterobacterioceae) appears useful. This is particularly advantageaus whenever ollergising and toxic effects of microorganisms are to be ossessed, the lotter depending largely on quantitative aspects (e.g. almost any mould fungus is a carrier of potential allergens). ln addition, the use of

116

sum porameters allows a simplified and thus less costly analytical determination and indicotion of total figures as, for instance, by means of epifluorescence anolysis. For speciol workplaces it is also possible to indicate key organisms, os e.g. the Aspergillus fumigotus in com~ posting plants. last but not least, even detailed problems encountered in connection with infectious germs can be tackled by investigating individual species, e.g. Legionella pneumophila and Mycobocterium tuberculosis.

The tosk force "Workplace ossessment" drew up a Iist of workplaces which require particular ottention in terms of work safety and the suitable measuring porameters for their ossessment (see figure 3). This Iist has not been completed yet, but will continuously be updated.

As o first step, consistent meosuring methods for bacteria and maulds/yeasts in the atmosphere (ond in aqueous media) are being developed. Measuring methods for viruses and microorganisms on solid surfaces will be dealt with in a second step (figure 4, see poge 118). A first investigation will focus on three basic types of procedure, viz. filtration, impoction and impingement: methods for determining bacteria (in general) and moulds/yeasts (in generoll will be developed, harmonised and made

Figure 3: PG 4 "Workplace assessment"

Workplaces Measuring Parameters Discussed

waste sorting plants, composting plants bacteria, moulds/yeasts, Gram-negative bacteria, Gram-positive bacteria, spore forming bacteria, actinomycetes, aspergilli {A. fumigatus), endetaxins

wastewater treatment plants bacteria, Gram-negative bacteria, E.coli, endetaxins

waste deposits bacteria, moulds, an(aerobic)spore forming bacteria

soil decontamination Gram-positive, Gram-negative bacteria, specific germs (e.g. pseudomonades, Nocardia spec. )

food production bacteria, moulds/yeasts, staphylococci, coliforms

biotechnology specific germs{fermentation germs, contamination germs)

health care bacteria, moulds/yeasts, specific infectious germs (f.e. legionellae, klebsiellae, mycobacteria)

air-conditioning systems, air humidifiers bacteria, moulds, actinomycetes, pseudomonades, legionellae, specific germs (f. e. biofilters), en-dotoxins

metal working (cooling lubricants) bacteria, moulds/yeasts, pseudomonades enterobacteriaceae, endetaxins

wholesale/storage moulds, actinomycetes

timber industry

II All activities in the working areas cited-above are included, f.e. cleaning, maintenance, construction works.

117



Figure 4: PG 4 "Workplace ossessment"

Parameter Measuring Procedure Stole of Affairs

o) in the air

moulds/yeasts filtrotion procedure published for testing cultivotion

bacterio filtrotion procedure in preparation cultivation

bacterio impaction procedure in preparation moulds/yeasts centrifugal sompler

bocteria Impaction procedure in preparation moulds/yeasts cascade sampler

endetaxins in preparation

staphylococci in preparotion

aerobic, onaerobic spare in preporotion beoring bacteria

pseudomonades in preparation

enterobacteriaceae in preparation

actinomycetes in preparotion

b) in fluids

f.e. bacteria moulds/yeasts pseudomonades legionellae actinomycetes endetaxins enterobacteriaceae

c) on solid surfaces

{provided for future working out)

II First of oll procedures for measuring viruses ore not worked out in thc PG 4

118

available ta the user. The compatibility af methods must of course be guaranteed. This is achieved by considering the results obtained by means af the different methads during the test phase, by assessing and campering them.

Standardised method for measuring moulds/yeasts contamination

The very first "Measuring method for determining moulds/yeasts concentrations in the workplace atmosphere" harmonised by the task force "Measuring methads and strategies" under the convenorship of the Berufsgenossenschaftliches Institut für Arbeitssicherheit (BIA) was published and released for testing in February 1995.

The measuring method embraces sampling by Separation on o membrane filter (e.g. cellulose ester, polycarbonate, gelatine) and determination by cultivation. The method enables a large concentration range of moulds/yeasts in the air to be detected, thus covering not only clean areas but also highly contaminated workplaces as they ore e.g. found in the waste disposal industry. ln the case of low moulds/yeasts concentrations in the air, the loaded filters (e.g. cellulose ester) are placed directly on a culture medium, bred and counted

(direct method). ln the presence of higher concentrations, however, the fungi units on the loaded filters (polycarbonate, gelatine) are washed off and, after preparation of a dilution series, brought onto the culture media, bred and counted (indirect method). The result is indicated in colony-forming units per m3 air (CFU/m3 air). The measuring method is split up into the following phases,

Sampling

Any filter sampling device suitable for determining health hazards at the workplace can be used for sampling, on condition that it meets the special rules of bioaerosol sampling (e.g. possibility of Sterilisation, handling). Collection characteristks should olways be specified. Particularly for personal sampling, the use of sampling devices camplying with the collection characteristic (inhalable fraction) of EN 481 "Determination of particle size distribution for measuring airborne particles" is desirable. Such an Instrument is e.g. the BIA-sampling system for total dust GSP (volume flow 3.5 1/min, filter diameter 37 mm) in combination with a personal sampling pump. Sampling includes the determination of air humidity (% R.H.) temperature ( 0 C), air flow velocity (m/s) and air flow direction at the place of measurement.

119



Different types of membrane filters are used, cellulose ester filters (pore size 0.8 ,um) for the direct method and polycarbonate (pore size 0.8 ,um) or gelatine (pore size 3 ,um) for the indirect method.

Analysis

Analysis is carried out by cultivation in an incubator at 22 °C± 1 oc and 30 oc± I oc or 25 oc± I oc for

2 to 7 days. The culture media are the following,

Malt extraet-agar (MEA) e.g.,

malt extract mycolog. peptone agar water pH 5.5±0.2

30.0 g/1 5.0 gl

15.0 g/1 1000 ml

and Dichloran-Giyceroi-(DG 18)-agar, e.g.,

peptone glucose potassium hydrogen sulphate magnesium sulphate dichloran chloramphenicol glycerol agar water pH 5.6±0.2

120

5.0 g/1 I 0.0 g/1

1.0 g/1 0.5 g/1

0.002 g/1 0.1 g/1 18 %

15.0 g/1 1000 ml

Oirect method: using a pair of tweezers the loaded filter is brought directly onto the culture medium in a Petri dish, where the gelatine filters are dissolved and the insoluble membrane filter absorbs the excess culture medium, i.e. the filter is diffused by the culture medium which thus provides the fungi with the necessary growth promoting substances. Since many fungi form bright-coloured colonies, the use of grey filters (with a grid) has turned out to be most suitable.

lndirect method: using a pair of tweezers the loaded filter is brought e.g. into I 0 ml of a 0.9% soll solution with 0.01 % Tween 80, where the gelatine filter is dissolved. The fungi units have to be washed off from the insoluble membrane filter (e.g. polycarbonate) by (heavily) agitating the solution. lf the filter is brought into a salt solution of different volume, this must be taken into account when calculating the results. The original Suspension is further diluted in decimal steps (1,1, 1,10, 1,100 etc.). Then 0. I ml of every dilution is pipetted onto ot least two agar plates of the selected culture media and spread by means of a Drigalski-spattle.

Ca Ieuiotion

Assessment is done by counting the number of moulds/yeasts colanies grown

during the incubating phase an the culture medium.

Direct method: The following formula is used to calculate the result expressed in colony-forming units per m3 air:

colonies/filter · I 000 I CfU/ m3 ~ -------

sampling volume (I)

lndirect method: The total number of colanies grown on oll solution agar plates (1,1, 1,10 etc.) are counted and the result in colony-forming units per m3 is calculated using the following formulae,

Lc c ~-----

C = weighted orithmetic mean value of oll colanies

E c = sum of oll colanies assessable for calculation (lowest ond next higher assessable) dilution

n 1 = number of plates with lowest ossessable dilution

n2 = number of plates with the next higher dilution

Then the weighted arithmetic meon value c is multiplied by the dilution factor d of the lowest assessable solution n1 (e.g. in the cose of I o- 2, d ~ 1 00) and by the factor I 00 resulting from the fact that only 0. I ml was spread on the agar plates and I ml of the original suspension of 10 ml used for preparing

the dilution series. lf a different suspension volume was used, this has to be taken into account when doing the final colculation. The volue G represents the number of fungi units contained in the sompled oir volume ond suspended in lO ml ( ~ original Suspension).

G ~ c · d · 100

Consequently, the result in colony forming units per m3 oir is calculoted as followso

G · 1000 I CfU/m3 ~ -----

Sampling volume (1\

Evaluation ol results

Only those fungi un"rts can be counted which are viable and capable of forming colonies; no statement can be mode concerning fungi units without cell division capacity. for this a (planned) detailed approach, e.g. epifluarescence analysis, is necessary.

T o ensure that a deviating value is immediately identified, the assessment of measuring results must always be corried out against the background of reference values meosured in parallel in the outside atmosphere.

A wide ronge of moulds/yeosts concentrations in the air can be detected

121



using the indirect method with separation of the fungi units from the filter and preparation of a decimal dilution series. Moreover, this methods allows sampling Iimes of ;;;:; 2 h, thus enabling the occupational mould exposure situation to be determined over a representative period of time, as in the case of chemieals (see EN 689). This includes the possibility of determining shift averages and averages for certain process phases.

lf the results obtained by means of the direct ond indirect methods are not in complete accordance, this may be due to differences in methods (e.g. dissolution of spare aggregates, Ionger incubation when the indirect method is used). Naturally, attention must be poid to this aspect when ossessing the results.

Conclusion

Standardised measuring methods for biological working agents represent the essential basis for harmonised ossessment of microbial exposure at the place of work. T o achieve this aim, it will be necessary to elaborate a measuring strategy which takes due account of the particularities microorganisms moy present (e.g. determination of measurement task, sampling time, averaging time,

122

sampling location, workplace analysis, minimum number of samples, outdoor reference measurement, physical atmosphere porameters etc.).

Meosurements for microorganisms in the workpface are carried out for different reasons. A measuring strategy should set up rules, how measurements for the detection of bioaerosols (with standardised measuring procedures) have to be done. Account must be taken of the following measurements tasks:

0 rough measurements for an approximate estimation of a worker's exposure

0 measurements for the localisation of sources emitting microorgonisms (e.g. air humidifier systems)

0 measurements for detecting the shift average concentration of microorganisms. lf there is no threshold Iimit value (related to shift duration) for microorgonisms in the workplace atmosphere, measurements should be carried out to investigate a possible correlation between employees' exposure to bioaerosols and health effects

0 meosurements for testing the efficiency of protective measures taken

0 furthermore, measurements could be done in the future to assess whether or not TLVs are observed

The practical realisation of sampling depends on the special measurement tosk quoted obove, e.g., Da I hove to sample in employees' breathing zone or

not? How lang do I have to somple? How many somples do I have to Iake?

This klnd of consistency is at the same time the prerequisite for defining Iimit values or occupational hygienic guidance concentrations whose potential role in the ossessment of biologically ex· posed workplaces is a contentious issue among experts.

References

EG-Richtlinie 90/679/EWG, Schutz der Arbeitnehmer gegen Gefährdung durch biologische Arbeitsstoffe bei der Arbeit

EG-Richtlinie 93/88/EWG, Änderung der Rl 90/679/EWG (v.a. Einstufungs. vorschläge biologischer Arbeitsstoffel

Deininger, C., Gefährdungen durch biologische Agenzien am Arbeitsplatz, BIA-Handbuch, 21. lfg.X/93. Hrsg., Berufsgenossenschaftliches Institut für Arbeitssicherheit, Erich Schmidt Verlag, Bielefeld 1993

The American Conference of Governmental lndustrial Hygienists (ACGIH), Tl V/BEl Booklet 1994 · 1995

BIA-Arbeitsmoppe Messung von Gefahr· stoffen, loseblattsommlung, 14. lfg. 11/95, Erich Schmidt Verlag,

- 9400 MeBverfohren für biologische

Agenzien (einleitender Teil mit Vor. stellung des Arbeitskreises

- 941 0 Probenohme von Bioaerosolen om Arbeitsplatz (allgemeiner praktischer und theoretischer Hintergrund)

- 9420 Verfahren zur Bestimmung der Schimmelpilz/Hefenkonzentrationen in der Luft om Arbeitsplatz ( Probenohme mit Abscheidung auf einem Membran· filter und Bestimmung durch Kultivie· · rung)

Anforderungen an sichere Arbeitsplätze in Wertstoffsortieranlagen, Niedersächsisches Sozialministerium, Juni 1994

BG Druck und Papierverarbeitung und Fachinstitut für Gebäude-Klima e. V., Wartungssicherheit für den nygienisch einwandfreien Betrieb von luftbefeuchtungsanlagen. lnformationsveranstaltung, Januar 1995

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Documents

BIA-Report 3/96 Workshop Microorganisms · PDF filecio del trabajo», un temo tratado con creciente interes por parte de las institu