SIST EN 13098:2019
(Main)Workplace exposure - Measurement of airborne microorganisms and microbial compounds - General requirements
Workplace exposure - Measurement of airborne microorganisms and microbial compounds - General requirements
This European Standard specifies general requirements for the measurement of microorganisms and microbial compounds. This European Standard provides also guidelines for the assessment of workplace exposure to airborne micro-organisms including the determination of total number and culturable number of micro-organisms and microbial compounds in the workplace atmosphere.
Exposition am Arbeitsplatz - Messung von Mikroorganismen und mikrobiellen Bestandteilen in der Luft - Allgemeine Anforderungen
Dieses Dokument legt allgemeine Anforderungen an die Messung von Mikroorganismen und mikrobiellen Bestandteilen fest.
Dieses Dokument stellt außerdem eine Anleitung für die Beurteilung der Exposition am Arbeitsplatz durch luftgetragene Mikroorganismen, einschließlich der Bestimmung der Gesamtanzahl und der anzüchtbaren Anzahl an Mikroorganismen und mikrobiellen Bestandteilen in der Arbeitsplatzatmosphäre, zur Verfügung.
Dieses Dokument gilt nicht für die Messung von Viren.
Exposition sur les lieux de travail - Mesurage de microorganismes et en suspension dans l'air - Exigences générales
Le présent document spécifie les exigences générales relatives au mesurage des micro-organismes et des composés microbiens.
Le présent document fournit également des lignes directrices pour l’évaluation de l’exposition aux micro-organismes en suspension dans l’air sur les lieux de travail, y compris la détermination du nombre total de micro-organismes et du nombre de micro-organismes cultivables et des composés microbiens dans l’atmosphère des lieux de travail.
Le présent document ne s'applique pas au mesurage des virus.
Izpostavljenost na delovnem mestu - Ugotavljanje prisotnosti mikroorganizmov v zraku in merjenje njihovih metabolitov - Splošne zahteve
Ta evropski standard določa splošne zahteve za ugotavljanje prisotnosti mikroorganizmov v zraku in merjenje njihovih metabolitov. Ta evropski standard podaja tudi smernice za oceno izpostavljenosti na delovnem mestu zaradi mikroorganizmov v zraku, vključno z določanjem skupnega števila in števila mikroorganizmov, sposobnih tvorbe kolonij, ter njihovih metabolitov v zraku na delovnem mestu.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN 13098:2019
01-oktober-2019
Nadomešča:
SIST EN 13098:2003
Izpostavljenost na delovnem mestu - Ugotavljanje prisotnosti mikroorganizmov v
zraku in merjenje njihovih metabolitov - Splošne zahteve
Workplace exposure - Measurement of airborne microorganisms and microbial
compounds - General requirements
Exposition am Arbeitsplatz - Messung von Mikroorganismen und mikrobiellen
Bestandteilen in der Luft - Allgemeine Anforderungen
Exposition sur les lieux de travail - Mesurage de microorganismes et en suspension
dans l'air - Exigences générales
Ta slovenski standard je istoveten z: EN 13098:2019
ICS:
07.100.99 Drugi standardi v zvezi z Other standards related to
mikrobiologijo microbiology
13.040.30 Kakovost zraka na delovnem Workplace atmospheres
mestu
SIST EN 13098:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN 13098:2019
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SIST EN 13098:2019
EN 13098
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2019
EUROPÄISCHE NORM
ICS 07.100.99; 13.040.30 Supersedes EN 13098:2000
English Version
Workplace exposure - Measurement of airborne
microorganisms and microbial compounds - General
requirements
Exposition sur les lieux de travail - Mesurage de Exposition am Arbeitsplatz - Messung von
microorganismes et en suspension dans l'air - luftgetragenen Mikroorganismen und mikrobiellen
Exigences générales Bestandteilen - Allgemeine Anforderungen
This European Standard was approved by CEN on 10 June 2019.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 13098:2019 E
worldwide for CEN national Members.
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SIST EN 13098:2019
EN 13098:2019 (E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Symbols and abbreviations . 9
5 Measurement of microorganisms and microbial compounds . 9
5.1 Biological agents and biological properties . 9
5.2 Aim of measurement . 10
5.3 Measurement strategy . 10
5.3.1 General . 10
5.3.2 Specification of the objectives for measurement . 10
5.3.3 Specification of the measurement task . 10
5.3.4 Collection of background information . 11
5.3.5 Sampling strategy. 11
5.4 Measurement options . 11
5.5 Uncertainty of measurement . 12
5.6 Variability of exposure level . 12
6 Sampling . 12
6.1 Principles and general requirements . 12
6.2 Sampler . 13
6.2.1 Categories . 13
6.2.2 Requirements . 13
6.3 Pumps . 13
6.4 Operator skills . 13
6.5 Transport and storage of samples . 13
6.5.1 General . 13
6.5.2 Transport . 14
6.5.3 Storage at the laboratory. 14
6.6 Sampling documentation . 14
7 Analytical method . 15
7.1 Requirements . 15
7.2 Validation . 15
7.3 Documentation . 15
7.3.1 General information . 15
7.3.2 Specific information . 16
7.4 Determination of culturable fraction. 17
7.5 Determination of direct cell count by microscopy . 17
7.5.1 General . 17
7.5.2 Epifluorescence microscopy and light microscopy . 17
7.5.3 Scanning electron microscopy . 17
7.6 Determination of microbial compounds . 17
8 Expression of results . 17
8.1 General . 17
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8.2 Cultivation methods . 17
8.3 Microscopic methods . 18
8.4 Microbial compounds. 18
9 Test report . 18
Annex A (informative) Recommendations for selection of exposure measuring procedures . 19
Annex B (informative) Sampling form example . 31
Annex C (normative) Determination of airborne microorganisms by cultivation . 33
C.1 General . 33
C.2 Requirements . 33
C.2.1 Suspension media and dilution media . 33
C.2.2 Cultivation media . 33
C.2.3 Cultivation temperature and incubation period . 33
C.2.4 Colony counts . 34
C.2.5 Identification . 34
Annex D (informative) List of generic media . 35
Annex E (informative) Formula and calculation examples for colony counting . 36
E.1 Calculation . 36
E.2 Examples . 37
Bibliography . 39
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EN 13098:2019 (E)
European foreword
This document (EN 13098:2019) has been prepared by Technical Committee CEN/TC 137 “Assessment
of workplace exposure to chemical and biological agents”, the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by March 2020, and conflicting national standards shall
be withdrawn at the latest by March 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN 13098:2000.
The major technical changes between this European Standard and the previous edition are as follows:
a) document title changed;
b) list of measurable bioaerosol compounds extended;
c) new measuring techniques added;
d) new definitions for “allergen”, “cell count of microorganisms”, “glucan”, “microbial compound”,
“mycotoxin”, and “RFc-recombinant” added;
e) existing definitions technically revised, where necessary;
f) terms and definitions already referred to in EN 1540 deleted;
g) 5.3 on “Measurement strategy” improved by providing more details;
h) Annex A updated with regard to new techniques and methods;
i) Annex B updated with new compounds that can be measured;
j) Annex C updated with regard to new counting strategies and identification methods;
k) new Annex E on “Formula and calculation examples for colony counting” added;
l) Bibliography updated and divided in informative references and other information resources;
m) whole document editorially and technically revised.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
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Introduction
Representative assessment of occupational exposure to airborne microbial organisms or compounds is
challenging. However, because of potential health consequences following exposure, it is important to
be able to evaluate and control exposure. The sampling equipment used can introduce its own critical
limitations, such as the assessment of the health-related aerosol fractions. Some sampling equipment is
capable only of measuring culturable microorganisms, while others allow the characterization of both,
the total number of microbial cells and the culturable fraction. Both preservation of samples and
analytical procedures can induce difficulties and uncertainties due to changes of microbial population
and/or unwanted interferences. However, by adhering to the principles outlined in this European
Standard for choice of sampling and analytical procedures, these uncertainties can be reduced and
controlled, allowing comparable and representative measurements to be made.
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1 Scope
This document specifies general requirements for the measurement of microorganisms and microbial
compounds.
This document provides also guidelines for the assessment of workplace exposure to airborne
microorganisms including the determination of total number and culturable number of microorganisms
and microbial compounds in the workplace atmosphere.
This document does not apply to the measurement of viruses.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 482, Workplace exposure — General requirements for the performance of procedures for the
measurement of chemical agents
EN 1540, Workplace exposure — Terminology
EN ISO 13137, Workplace atmospheres — Pumps for personal sampling of chemical and biological
agents — Requirements and test methods (ISO 13137)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 1540 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
actinomycetes
filamentous Gram-positive, aerobic or anaerobic bacteria belonging to the phylum Actinobacteria
Note 1 to entry: Filamentous actinomycetes form a branching network of thin filaments called a mycelium. Most
actinomycetes replicate by conidia-like spores which can easily be made airborne.
3.2
allergen
substance that can cause an allergic reaction in sensitized person
Note 1 to entry: Allergens from microbiological origin are usually proteins or glycoproteins derived from fungi
or bacteria.
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3.3
bacteria
large group of prokaryotic microorganisms with one chromosome in a nuclear region and which
replicate only asexually by cell division
Note 1 to entry: Different cell-wall chemistry is used for the classification of Gram-positive and Gram-negative
bacteria. Morphological criteria divide into spheres (cocci) and rods bacilli. Some species produce endospores as
survival units.
3.4
biological preservation efficiency
capability of a sampler to maintain the viability of airborne microorganisms during collection and also
to keep the microbial products intact
3.5
cell count of microorganisms
number of microorganisms determined as single organisms (or a corresponding measure)
Note 1 to entry: Both the viable and the non-viable micro-organisms are included.
3.6
colony forming unit
CFU
unit by which the culturable number (3.7) is given
Note 1 to entry: One colony forming unit can originate from one single microorganism, an aggregate of many
microorganisms or from one or many microorganisms attached to one particle.
Note 2 to entry: The number of outgrown colonies can depend on cultivation conditions.
3.7
culturable number
number of microorganisms, single cells or aggregates able to form colonies on a solid nutrient medium
3.8
elevated level
level above normal background level of microorganisms in a specified environment
3.9
endotoxin
constituent of the external membrane of Gram-negative bacteria (lipopolysaccharide), consisting of a
complex lipid, lipid A, which is covalently bound to a polysaccharide
Note 1 to entry: “Free endotoxin” is liberated after cell death and by budding from living cells. Lipid A is the
active (toxic) part and is a potent pro-inflammatory substance and can induce febrile, bronchial and other
symptoms in exposed workers. The composition and the toxicity of endotoxin differ between species.
3.10
endotoxin unit
unit standardized against the defined reference material, reference standard endotoxin
3.11
filtration
collection of particles suspended in gas or liquid by flow through a porous medium
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3.12
fungi
diverse group of eukaryotic microorganisms with membrane-bound nucleus comprising several
chromosomes
Note 1 to entry: Multiplication is mainly asexual but several groups replicate also by sexual spores. Filamentous
fungi (moulds) grow in lengthy hyphae and form compact tufts called mycelia. Asexual spores (conidia) are easily
made airborne. Yeasts are usually unicellular, of spherical shape and their cells multiply sexually or asexually by
budding.
3.13
glucan
polysaccharide molecule present in the cell walls of eukaryotes and prokaryotes including most molds,
upper fungi, yeasts, algae and certain bacteria
Note 1 to entry: Glucan is also referred as (1,3)-β-D-glucan.
3.14
impaction
collection of airborne particles accelerated through a nozzle or orifice on a surface by inertia effect
3.15
impingement
combination of impaction onto a surface and subsequent dispersion into a liquid medium
3.16
Limulus Amoebocyte Lysate
enzymes extracted from the blood cells of the horse shoe crab (Limulus polyphemus) that are activated
by endotoxin and other molecules (glucans etc.)
3.17
microbial compound
cell or cell wall component or metabolite of microbial origin
Note 1 to entry: Endotoxins, glucans, mycotoxins and enzymes are examples of microbial compounds. Microbial
DNA is also included in this definition.
3.18
microorganism
microbiological entity of any type, cellular or non-cellular, capable of replication or of transferring
genetic material, or entities that have lost these properties
Note 1 to entry: The term microorganism covers the term “biological agent” defined in EN 1540.
3.19
mycotoxin
toxic secondary metabolite produced by fungi
Note 1 to entry: One fungal species can produce many different mycotoxins, and several species can produce
the same mycotoxin.
3.20
physical sampling efficiency
capability of the sampler to collect particles with specific sizes suspended in workplace air
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3.21
RFc-recombinant
synthetic version of Factor C, an element found in the blood cells of horseshoe crabs
Note 1 to entry: Factor C is the essential biological component of bacterial endotoxin testing.
3.22
sieve sampler
multi-orifice impactor
3.23
total sampling efficiency
product of physical sampling efficiency (3.20) and biological preservation efficiency (3.4)
3.24
viable number of microorganisms
number of microorganisms having a potential for metabolic activity
Note 1 to entry: A viable microorganism is not necessarily culturable (also called “viable but not culturable”),
which means that the number of culturable microorganisms is often only part of the viable number.
4 Symbols and abbreviations
ATP adenosine triphosphate
CFU colony forming unit
CV coefficient of variation
DNA desoxyribonucleic acid
ELISA enzyme-linked immunosorbent assay
EU endotoxin unit
GSD geometric standard deviation
LAL Limulus Amoebocyte Lysate
LPS lipopolysaccharide
PCR polymerase chain reaction
SEM scanning electron microscope
5 Measurement of microorganisms and microbial compounds
5.1 Biological agents and biological properties
Bioaerosols can contain different microorganisms and/or microbial compounds originating from these.
Microorganisms can be classified in different taxonomic groups including bacteria, fungi, protozoa,
algae and viruses. These can be further classified to family, genus or species level. Immunologic
reactions (e.g. allergic) and/or toxic reactions as well as infections can result from exposure to
microorganisms and microbial compounds.
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5.2 Aim of measurement
The measurement of microorganisms and microbial compounds in the workplace air has two
objectives:
a) to assess workers' exposure, and/or
b) to assess the biological characteristics of air at different locations and/or at different times and
over different time periods.
It is essential to state the purpose of the measurement and how the results will be interpreted.
NOTE 1 Measurement tasks can be to locate sources emitting microorganisms and/or microbial compounds, to
measure a worker's exposure during work shift, to identify peaks in exposure, to test the efficiency of control
measures, or to control actions taken to diminish the exposure.
NOTE 2 It can be useful to be able to measure both, viable and non-viable bioaerosols.
5.3 Measurement strategy
5.3.1 General
The measurement strategy describes the action plan that allows for interpretation of measurement
data. The action plan shall consist of the following:
a) specification of the objectives for measurement (see 5.3.2);
b) specification of the measurement task (see 5.3.3);
c) collection of background information (see 5.3.4);
d) sampling strategy (see 5.3.5).
5.3.2 Specification of the objectives for measurement
The objectives for measurement shall be given and a strategy settled that is adapted to the objectives. It
is also to be considered that no OELs are available for the interpretation of measurement data.
5.3.3 Specification of the measurement task
Examples for measurement tasks are to
— locate the sources emitting microorganisms and/or microbial compounds,
— measure a worker’s exposure during work shift (e.g. task- or process-related),
— identify peak exposure,
— control actions taken to diminish the exposure,
— test the efficiency of control measures.
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5.3.4 Collection of background information
Background information should comprise
— the identification of potential onsite sources,
— expected biological agents, route of exposure and potentially exposed persons,
— expected concentrations or exposure levels of biological agents,
— the variability of the biological agents in space, time and particle size.
5.3.5 Sampling strategy
The sampling strategy shall specify the design of a sampling plan, which describes what should be
measured, and where, when and how measurement should be done.
The sampling plan usually includes:
— the definition of the target biological agent(s) and the corresponding analytical method(s),
— the determination of the sampling method(s) (sampling device, collection substrate, flow rate, etc.),
— the type of sampling (static sampling/personal sampling),
— the setting of sampling parameters (location, number, frequency and duration),
— requirements on sample transportation.
NOTE 1 It can be useful to know the environmental conditions (e.g. relative humidity, temperature, wind
speed), especially when extreme environmental conditions are to be expected.
NOTE 2 Passive methods (e.g. surface swabs, electrostatic dust collectors) can be applied to complement air
sampling information.
For assessment of measuring results reference samples, such as outdoor air, non-exposed workplace air
(e.g. from another office in the same workplace area) or workplace air before starting of work activity,
shall be taken, if background exposure is not known.
It is essential to verify that the sampling strategy is representative to exposure and that sampling and
analysis correspond to the assessment objectives and to the search for the targeted component of the
bioaerosol (see 5.1).
5.4 Measurement options
The following approaches can be used to measure microorganisms and microbial compounds:
— direct counting of microbial cells by microscopy including culturable, non-culturable but viable and
non-viable ones;
— enumeration of microbial cells and cell aggregates by culturing on agar media (the culturable
number);
— quantification of cellular components of microorganisms, from viable, non-viable or disintegrated
microorganisms, for example, constituents of cell structure such as endotoxin, glucans and
ergosterol;
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— quantification of primary metabolites (such as ATP or chitinase from fungi) which can serve as
markers for microorganisms or of their vital activity;
— quantification of secondary metabolites (for example, mycotoxins and volatile organic compounds
(VOCs)) which derive from microorganisms and carried by other particles in the bioaerosol.
5.5 Uncertainty of measurement
The uncertainty of measurement originates from the sampling procedure, samples preservation and the
analytical method. Sampling procedures and analytical methods shall be validated. Reproducibility shall
be determined.
NOTE Validation of methods for measurements of microorganisms can be limited by lack of reference
materials and/or reference methods. However, the study of method characteristics can be assessed in laboratory
assays with microbial cultures, liquid solutions and experimental bioaerosols.
5.6 Variability of exposure level
The variability in exposure levels
...
SLOVENSKI STANDARD
oSIST prEN 13098:2018
01-september-2018
[Not translated]
Workplace exposure - Measurement of airborne micro-organisms and microbial
compounds - General requirements (Revision of EN 13098:2000)
Exposition am Arbeitsplatz - Messung von luftgetragenen Mikroorganismen und
mikrobiellen Bestandteilen - Allgemeine Anforderungen (Überarbeitung von EN
13098:2000)
Exposition sur les lieux de travail - Mesurage des micro-organismes et des composés
microbiens en suspension dans l’air - Exigences générales (révision de l’EN
13098:2000)
Ta slovenski standard je istoveten z: prEN 13098
ICS:
07.100.99 Drugi standardi v zvezi z Other standards related to
mikrobiologijo microbiology
13.040.30 Kakovost zraka na delovnem Workplace atmospheres
mestu
oSIST prEN 13098:2018 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 13098:2018
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oSIST prEN 13098:2018
DRAFT
EUROPEAN STANDARD
prEN 13098
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2018
ICS 07.100.99; 13.040.30 Will supersede EN 13098:2000
English Version
Workplace exposure - Measurement of airborne micro-
organisms and microbial compounds - General
requirements (Revision of EN 13098:2000)
Exposition am Arbeitsplatz - Messung von
luftgetragenen Mikroorganismen und mikrobiellen
Bestandteilen - Allgemeine Anforderungen
(Überarbeitung von EN 13098:2000)
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 137.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 13098:2018 E
worldwide for CEN national Members.
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oSIST prEN 13098:2018
prEN 13098:2018 (E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Symbols and abbreviations . 9
5 Measurement of microorganisms and microbial compounds . 10
5.1 Biological agents and biological properties . 10
5.2 Aim of measurement . 10
5.3 Measurement strategy . 10
5.3.1 General . 10
5.3.2 Specification of the objectives for measurement . 10
5.3.3 Specification of the measurement task . 10
5.3.4 Collection of background information . 11
5.3.5 Sampling strategy. 11
5.4 Measurement options . 11
5.5 Uncertainty of measurement . 12
5.6 Variability of exposure level . 12
6 Sampling . 12
6.1 Principles and general requirements . 12
6.2 Sampler . 13
6.2.1 Categories . 13
6.2.2 Requirements . 13
6.3 Pumps . 13
6.4 Operator skills . 13
6.5 Transport and storage of samples . 13
6.5.1 Transport . 13
6.5.2 Storage at the laboratory. 14
6.6 Sampling documentation . 14
7 Analytical method . 15
7.1 Requirements . 15
7.2 Validation . 15
7.3 Documentation . 15
7.3.1 General information . 15
7.3.2 Specific information . 16
7.4 Determination of culturable fraction. 17
7.5 Determination of direct cell count by microscopy . 17
7.5.1 General . 17
7.5.2 Epifluorescence microscopy and light microscopy . 17
7.5.3 Scanning electron microscopy . 17
7.6 Determination of microbial compounds . 17
8 Expression of results . 17
8.1 General . 17
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8.2 Cultivation methods. 17
8.3 Microscopic methods. 18
8.4 Microbial compounds . 18
9 Test report . 18
Annex A (informative) Recommendations for selection of measuring procedures . 19
Annex B (informative) Sampling form example . 32
Annex C (normative) Determination of airborne microorganisms by cultivation . 34
C.1 General . 34
C.2 Requirements . 34
C.2.1 Suspension media and dilution media . 34
C.2.2 Cultivation media. 34
C.2.3 Cultivation temperature and incubation period . 34
C.2.4 Colony counts . 35
C.2.5 Identification . 35
Annex D (informative) List of generic media . 36
Annex E (informative) Formula and calculation examples for colony counting . 37
E.1 Calculation. 37
E.2 Examples . 38
Bibliography . 40
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oSIST prEN 13098:2018
prEN 13098:2018 (E)
European foreword
This document (prEN 13098:2018) has been prepared by Technical Committee CEN/TC 137
“Assessment of workplace exposure to chemical and biological agents”, the secretariat of which is held
by DIN.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 13098:2000.
The major technical changes between this European Standard and the previous edition are as follows:
a) document title changed;
b) list of measurable bioaerosol compounds extended;
c) new measuring techniques added;
d) new definitions for “allergen”, “cell count of microorganisms”, “glucan”, “microbial compound”,
“mycotoxin”, and “RFc-recombinant” added;
e) existing definitions technically revised, where necessary;
f) terms and definitions already referred to in EN 1540 deleted;
g) 5.3 on “Measurement strategy” improved by providing more details;
h) Annex A updated with regard to new techniques and methods;
i) Annex B updated with new compounds that can be measured;
j) Annex C updated with regard to new counting strategies and identification methods;
k) new Annex E on “Formula and calculation examples for colony counting” added;
l) Bibliography updated;
m) whole document editorially and technically revised.
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Introduction
Representative assessment of occupational exposure to airborne microbial organisms or compounds is
challenging. However, because of potential health consequences following exposure, it is important to
be able to evaluate and control exposure. The sampling equipment used can introduce its own critical
limitations, such as the assessment of the health-related aerosol fractions. Some sampling equipment is
capable only of measuring culturable microorganisms, while others allow the characterization of both,
the total number of microbial cells and the culturable fraction. Both, preservation of samples and
analytical procedures can induce difficulties and uncertainties due to changes of microbial population
and/or unwanted interferences. However, by adhering to the principles outlined in this European
Standard for choice of sampling and analytical procedures, these uncertainties can be reduced and
controlled, allowing comparable and representative measurements to be made.
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1 Scope
This document specifies general requirements for the measurement of microorganisms and microbial
compounds.
This document provides also guidelines for the assessment of workplace exposure to airborne
microorganisms including the determination of total number and culturable number of microorganisms
and microbial compounds in the workplace atmosphere.
This document does not apply to the measurement of viruses.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 482, Workplace exposure — General requirements for the performance of procedures for the
measurement of chemical agents
EN 1540, Workplace exposure - Terminology
EN ISO 13137, Workplace atmospheres - Pumps for personal sampling of chemical and biological agents -
Requirements and test methods (ISO 13137)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 1540 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
actinomycetes
filamentous Gram-positive, aerobic or anaerobic bacteria belonging to the phylum Actinobacteria
Note 1 to entry: Filamentous actinomycetes form a branching network of thin filaments called a mycelium. Most
actinomycetes replicate by conidia-like spores which can easily be made airborne.
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3.2
allergen
substance that can cause an allergic reaction in sensitized person
Note 1 to entry: Allergens from microbiological origin are usually proteins or glycoproteins derived from fungi
or bacteria.
3.3
bacteria
large group of prokaryotic microorganisms with one chromosome in a nuclear region and which
replicate only asexually by cell division
Note 1 to entry: Different cell-wall chemistry is used for the classification of Gram-positive and Gram-negative
bacteria. Morphological criteria divide into spheres (cocci) and rods. Some species produce endospores as
survival units.
3.4
biological preservation efficiency
capability of a sampler to maintain the viability of airborne microorganisms during collection and also
to keep the microbial products intact
3.5
cell count of microorganisms
number of microorganisms determined as single organisms (or a corresponding measure)
Note 1 to entry Both, the viable and the non-viable micro-organisms are included
3.6
colony forming unit
CFU
unit by which the culturable number (3.7) is given
Note 1 to entry: One colony forming unit can originate from one single microorganism, an aggregate of many
microorganisms or from one or many microorganisms attached to one particle.
Note 2 to entry: The number of outgrown colonies can depend on cultivation conditions.
3.7
culturable number
number of microorganisms, single cells or aggregates able to form colonies on a solid nutrient medium
3.8
elevated level
level above normal background level of microorganisms in a specified environment
3.9
endotoxin
constituent of the external membrane of Gram-negative bacteria (lipopolysaccharide), consisting of a
complex lipid, lipid A, which is covalently bound to a polysaccharide
Note 1 to entry “Free endotoxin” is liberated after cell death and by budding from living cells. Lipid A is the
active (toxic) part and is a potent pro-inflammatory substance and can induce febrile, bronchial and other
symptoms in exposed workers. The composition and the toxicity of endotoxin differ between species
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3.10
endotoxin unit
unit standardized against the defined reference material, reference standard endotoxin
3.11
filtration
collection of particles suspended in gas or liquid by flow through a porous medium
3.12
fungi
diverse group of eukaryotic microorganisms with membrane-bound nucleus comprising several
chromosomes
Note 1 to entry Multiplication is mainly asexual but several groups replicate also by sexual spores. Filamentous
fungi (moulds) grow in lengthy hyphae and form compact tufts called mycelia. Asexual spores (conidia) are easily
made airborne. Yeasts are usually unicellular, of spherical shape and their cells multiply sexually or asexually by
budding
3.13
glucan
polysaccharide molecule present in the cell walls of eukaryotes and prokaryotes including most molds,
upper fungi, yeasts, algae and certain bacteria
Note 1 to entry Glucan is also referred as (1,3)-β-D-glucan
3.14
impaction
collection of airborne particles accelerated through a nozzle or orifice on a surface by inertia effect
3.15
impingement
combination of impaction onto a surface and subsequent dispersion into a liquid medium
3.16
Limulus Amoebocyte Lysate
enzymes extracted from the blood cells of the horse shoe crab (Limulus polyphemus) that are activated
by endotoxin
3.17
microbial compound
cell or cell wall component or metabolite of microbial origin
Note 1 to entry Endotoxins, glucans, mycotoxins and enzymes are examples of microbial compounds. Microbial
DNA is also included in this definition
3.18
microorganism
microbiological entity of any type, cellular or non-cellular, capable of replication or of transferring
genetic material, or entities that have lost these properties
Note 1 to entry The term microorganism covers the term “biological agent” defined in EN 1540
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3.19
mycotoxin
toxic secondary metabolite produced by fungi
Note 1 to entry One fungal species can produce many different mycotoxins, and several species can produce
the same mycotoxin
3.20
physical sampling efficiency
capability of the sampler to collect particles with specific sizes suspended in workplace air
3.21
RFc-recombinant
synthetic version of Factor C, an element found in the blood cells of horseshoe crabs
Note 1 to entry Factor C is the essential biological component of bacterial endotoxin testing
3.22
sieve sampler
multi-orifice impactor
3.23
total sampling efficiency
product of physical sampling efficiency (3.20) and biological preservation efficiency (3.4)
3.24
viable number of microorganisms
number of microorganisms having a potential for metabolic activity
Note 1 to entry A viable microorganism is not necessarily culturable (also called “viable but not culturable”),
which means that the number of culturable microorganisms might only be part of the viable number
4 Symbols and abbreviations
ATP adenosine triphosphate
CFU colony forming unit
CV coefficient of variation
DNA desoxyribonucleic acid
ELISA enzyme-linked immunosorbent assay
EU endotoxin unit
GSD geometric standard deviation
LAL Limulus Amoebocyte Lysate
LPS lipopolysaccharide
PCR polymerase chain reaction
SEM scanning electron microscope
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5 Measurement of microorganisms and microbial compounds
5.1 Biological agents and biological properties
Bioaerosols can contain different microorganisms and/or microbial compounds originating from these.
Microorganisms can be classified in different taxonomic groups including Gram-positive and Gram-
negative bacteria, actinomycetes, fungi, protozoa, algae and viruses. These can be further classified to
family, genus or species level. Immunologic reactions, e.g. allergic and/or toxic reactions, can result
from exposure to microorganisms and microbial compounds.
5.2 Aim of measurement
The measurement of microorganisms and microbial compounds in the workplace air has two
objectives:
n) To assess workers´ exposure, and/or
o) to assess the biological characteristics of air at different locations and/or at different times and
over different time periods.
It is essential to state the purpose of the measurement and how the results will be interpreted.
NOTE Measurement tasks can be to locate sources emitting microorganisms and/or microbial compounds, to
measure a worker’s daily or work shift exposure, to identify peaks in exposure, to test the efficiency of control
measures, or to control actions taken to diminish the exposure. It is essential to verify that the sampling and
analysis corresponds to the assessment objectives and to the search for the targeted component of the bioaerosol
(see 5.1). It can be useful to be able to measure both viable and non-viable bioaerosols.
5.3 Measurement strategy
5.3.1 General
The measurement strategy describes the action plan that allows for interpretation of measurement
data. The action plan shall consist of the following:
a) specification of the objectives for measurement (see 5.3.2);
b) specification of the measurement task (see 5.3.3);
c) collection of background information (see 5.3.4);
d) sampling strategy (see 5.3.5).
5.3.2 Specification of the objectives for measurement
The objectives for measurement shall be given and a strategy settled that is adapted to the objectives. It
is also to be considered that no OELs are available for the interpretation of measurement data.
5.3.3 Specification of the measurement task
Examples for measurement tasks are to
— locate the sources emitting microorganisms and/or microbial compounds,
— measure a worker’s daily or work shift exposure,
— identify peak exposure,
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— control actions taken to diminish the exposure,
— test the efficiency of control measures.
5.3.4 Collection of background information
Background information should comprise
— the identification of potential onsite sources,
— expected biological agents,
— expected concentrations or exposure levels of biological agents,
— the variability of the biological agents in space, time and particle size.
5.3.5 Sampling strategy
The sampling strategy shall specify the design of a sampling plan which describes what should be
measured and where, when and how measurement should be done.
The sampling plan usually includes:
— the definition of the target biological agent(s) and the corresponding analytical method(s),
— the determination of the sampling method(s) (sampling device, collection substrate, flow rate etc.),
— the type of sampling (static sampling/personal sampling),
— the setting of sampling parameters (location, number, frequency and duration),
— requirements on sample transportation.
NOTE 1 It can be useful to know the environmental conditions (e.g. relative humidity, temperature, wind
speed), especially when extreme environmental conditions are to be expected.
NOTE 2 Passive methods (e.g. surface swabs, electrostatic dust collectors) can be applied to complement air
sampling information.
For assessment of measuring results reference samples, such as outdoor air, non-exposed workplace air
(e.g. from another office in the same workplace area) or workplace air before starting of work activity,
shall be taken.
It is essential to verify that the sampling and analysis corresponds to the assessment objectives and to
the search for the targeted component of the bioaerosol (see 5.1).
5.4 Measurement options
The following approaches can be used to measure microorganisms and microbial compounds:
— direct counting of microbial cells by microscopy including culturable, non-culturable but viable and
non-viable ones;
— enumeration of microbial cells and cell aggregates by culturing on agar media (the culturable
number);
— quantification of cellular components of microorganisms, from viable, non-viable or disintegrated
microorganisms, for example, constituents of cell structure such as endotoxin and glucans;
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— quantification of primary metabolites (such as ATP or chitinase from fungi) which can serve as
markers for microorganisms or of their vital activity;
— quantification of secondary metabolites (for example, mycotoxins) which derive from
microorganisms and carried by other particles in the bioaerosol.
5.5 Uncertainty of measurement
The uncertainty of measurement originates from the sampling procedure, samples preservation and the
analytical method. Sampling procedures and analytical methods shall be validated. Reproducibility shall
be determined.
NOTE Validation of methods for measurements of microorganisms can be limited by lack of reference
materials and/or reference methods. However, the study of method characteristics can be assessed in laboratory
assays with microbial cultures, liquid solutions and experimental bioaerosols.
5.6 Variability of exposure level
The variability in exposure levels of microorganisms over time and between worker or working areas
can be very high and much higher than the precision of measuring procedures.
NOTE Geometric standard deviations (GSD) of 4 to 6, for a measurement with a duration of 8 h are not
unusual. As a consequence, the uncertainty in the estimation of long-term exposure from a single measurement is
high.
5 −3
For example, if the geometric mean is 4 × 10 m microorganisms and GSD = 5, then the 95 %
43− 73−
×10 m 10 m
confidence interval of one measurement is 1,6 to . Shorter sampling periods will
further increase the uncertainty.
Identification of the causes of the variability can help to reduce the measurement effort by targeted
sampling of special tasks or exposure situations.
6 Sampling
6.1 Principles and general requirements
Sampling of bioaerosols should be made in accordance with the principles of sampling to asses
...
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