CEN/TS 16115-1:2011

SLOVENSKI STANDARD
01-december-2011
.DNRYRVW]XQDQMHJD]UDND0HULWYHELRDHURVRORYGHO'RORþHYDQMHJOLY]
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Ambient air quality - Measurement of bioaerosols - Part 1: Determination of moulds using
filter sampling systems and cultivation based analyses
Luftbeschaffenheit - Messen von Bioaerosolen - Teil 1: Bestimmung von Schimmelpilzen
mittels Probenahme auf Filtern und kulturellem Nachweis
Qualité de l'air ambiant - Mesurage de bioaérosols - Dosage des moisissures à l'aide de
systèmes de prélèvement sur filtres et d'analyses de cultures
Ta slovenski standard je istoveten z: CEN/TS 16115-1:2011
ICS:
13.040.20 Kakovost okoljskega zraka Ambient atmospheres
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

TECHNICAL SPECIFICATION
CEN/TS 16115-1
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
April 2011
ICS 13.040.20
English Version
Ambient air quality - Measurement of bioaerosols - Part 1:
Determination of moulds using filter sampling systems and
culture-based analyses
Qualité de l'air ambiant - Mesurage de bioaérosols - Partie Luftbeschaffenheit - Messen von Bioaerosolen - Teil 1:
1: Dosage des moisissures à l'aide de systèmes de Bestimmung von Schimmelpilzen mittels Probenahme auf
prélèvement sur filtres et d'analyses de cultures Filtern und kulturellem Nachweis
This Technical Specification (CEN/TS) was approved by CEN on 4 October 2010 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2011 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16115-1:2011: E
worldwide for CEN national Members.

Contents Page
Foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 Symbols and abbreviations .8
5 Basic principle of the method .8
6 Sampling .9
7 Culture-based analyses . 15
8 Performance characteristics and minimum requirements . 22
9 Quality assurance . 23
10 Trouble shooting during sampling and analyses . 23
Annex A (informative) Example for a validated sampling device . 26
Annex B (informative) Recovery of spores on gelatine filters in combination with polycarbonate
filters . 32
Annex C (informative) Examples for sampling and analyses reports . 35
Annex D (informative) Membrane filtration technique . 41
Annex E (informative) Calculation by weighted mean . 42
Bibliography . 44

Foreword
This document (CEN/TS 16115-1:2011) has been prepared by Technical Committee CEN/TC 264 “Air
quality”, the secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: 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, Romania, Slovakia, Slovenia,
Spain, Sweden, Switzerland and the United Kingdom.
Introduction
Airborne particles of biological origin are called bioaerosols. Depending on the emission source bioaerosols
vary in composition; one component of ambient bioaerosols with possible ecological and health relevance can
be moulds. Natural and anthropogenic sources for mould spores are widely distributed in the environment.
Anthropogenic sources can for example be agriculture and construction activities or waste treatment.
Mould is a common name for filamentous fungi from different taxonomic groups (Zygomycetes, Ascomycetes,
Deuteromycetes). They form a mycelium (hyphae) and spores – namely conidiospores (conidia),
sporangiospores or ascospores – by which they become visible macroscopically. Most spores are in the size
range of 2 µm to 10 µm, some up to 30 µm and only few up to 100 µm. Spores of some mould genera are
small and become airborne very easily (e.g., Aspergillus, Penicillium) while others are bigger and/or
embedded in a slime matrix (e.g., Stachybotrys, Fusarium) and less mobile.

The procedure described in this document is based on VDI 4252 Part 2 [1], VDI 4253 Part 2 [2] and is related
to the ISO standards on indoor air ISO 16000-16 [3] and ISO 16000-17 [4].
1 Scope
This Technical Specification describes the measurement of moulds in ambient air in order to identify, quantify
and characterize bioaerosol pollution in ambient air resulting from emissions from different sources.
The method described specifies the sampling of moulds as part of the suspended particulate matter (SPM,
here particles with aerodynamic diameter up to ca. 30 µm) using a filter sampling system with gelatine/poly-
carbonate filter combination followed by the culture-based analyses on DG18 agar. The sampling duration can
be varied between 10 min to 24 h. The health effect of bioaerosols is not limited to any particle fraction,
therefore, this document describes the sampling of moulds as part of the suspended particulate matter as a
convention method.
NOTE The sampling method described in this document in principle is likely to be appropriate for the sampling of
actinomycetes and other spore-forming bacteria (resistant to desiccation). For these species a special analytical
procedure using different culture media should be applied, but this is not within the scope of this document.
The standard method set out in this Technical Specification is accepted by convention as reference method.
The measured quantity, here the number of colony forming units per cubic meter (CFU/m ), is determined by
the inlet design of the sampling head, the associated operational parameters and the analytical procedure.
Standardized methods for sampling, detection and enumeration of moulds including standards for sampling
strategies are important for comparative assessment of moulds in ambient air. Before doing any
measurements a plan for the measurement strategy is necessary (see CEN/TS 16115-2 [5]).
WARNING — The use of this Technical Specification may involve hazardous materials, operations and
equipment. This Technical Specification does not purport to address all the safety problems
associated with its use. It is the responsibility of the user of this standard to establish appropriate
safety and health practices and determine the applicability of regulatory limitations prior to use.
2 Normative references
The following referenced documents are indispensable for the application 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 ISO 8199:2007, Water quality ― General guidance on the enumeration of micro-organisms by culture
(ISO 8199:2005)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
aerodynamic diameter
diameter of a sphere of density 1 g/cm³ with the same terminal velocity due to gravitational force in calm air as
the particle, under the prevailing conditions of temperature, pressure and relative humidity
[ISO 7708:1995, 2.2 [6]]
3.2
ambient air
outdoor air in the lower troposphere excluding workplace air
[EN 14907:2005, 3.1.1 [7]]
3.3
analytical blank value
value determined by a blank sample covering the analytical procedure to ensure that no significant
contamination occurs during the complete analytical procedure including autoclaving, agar preparation,
suspension and extraction of the filters, dilution, incubation, counting, etc.
3.4
bioaerosol
airborne particles of biological origin
[EN 13098:2000, 3.3 [8]].
NOTE Bioaerosols in the sense of this document are all aggregations of particles in the atmosphere to which fungi
(spores, conidia, fragments of hyphae), bacteria, viruses and/or pollen as well as their cell membrane components and
metabolites (e.g. endotoxins, mycotoxins) are attached or that consist of the above mentioned components.
3.5
biological sampling efficiency
biological preservation efficiency
capacity of the sampler to maintain the viability of the airborne microorganisms during collection and also to
keep the microbial products intact
[EN 13098:2000, 3.4 [8]]
NOTE The biological sampling efficiency considers the sampling stress occurring during sampling and analysis in
addition to the physical sampling efficiency. It refers to the proportion (in percent) of collected organisms which have not
lost the ability to be cultured subsequently. It is strain- and species specific.
3.6
colony count
number of all visible colonies of microorganisms on a culture medium after incubation under the selected
conditions
3.7
Colony Forming Unit
CFU
unit by which the culturable number of microorganisms is expressed
[EN 13098:2000, 3.5 [8]].
NOTE 1 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 The number of outgrowing colonies depends on cultivation conditions.
3.8
culture-based analyses
cultivation
growing of microorganisms on culture media
[ISO 16000-16:2008, 3.6 [3]]
NOTE The prerequisites for the detection are the abilities to grow and propagate.
3.9
face velocity
air flow rate divided by the face area
NOTE 1 The face velocity is expressed in metres per second.
[Adapted from EN 779:2002, 3.11 [9]]
NOTE 2 In this document, the face velocity is defined as the volume flow rate divided by the effective filter area.
3.10
field blank value
value determined by a blank sample covering the complete measurement procedure including preparation,
sampling, transport and analyses to ensure that no significant contamination has occurred during all steps of
measurement and to check that the operator can achieve a quantification level adapted to the task
NOTE A field blank sample is a sample taken in an identical manner as the real sample, but without sucking air
through the sampling device. The resulting blank represents the number of CFU entering the sample simply by handling
the filter during sampling. The results of the field blanks are not used for correction of measurement results but to detect
sampling errors.
3.11
filtration
sampling of particles suspended in gas or liquid by flow through a porous medium
[EN 13098:2000, 3.11 [8]]
NOTE In this document, filtration is understood as the separation of moulds from a defined volume of air by means of
filters.
3.12
indirect method
suspension of deposited microorganisms with subsequent plating of aliquots on a suitable culture medium,
incubation and counting of colonies growing under the conditions selected
[Adapted from ISO 16000-17:2008, 3.3 [4]]
3.13
microbial air pollution
concentrations of airborne microorganisms that exceed natural concentrations or differ in type from the
naturally occurring mircoorganisms
3.14
microorganism
microbial entity, either cellular or non cellular, that is capable of multiplication or transfer of genetic material, or
entities that have lost these properties
[EN 13098:2000, 3.16 [8]]
3.15
mould
filamentous fungi from several taxonomic groups namely Zygomycetes, Ascomycetes (Ascomycota) and
Deuteromycetes (fungi imperfecti)
NOTE Moulds form different types of spores depending on the taxonomic group they belong to, namely
conidiospores (conidia), sporangiospores or ascospores.
[ISO 16000-16:2008, 3.9 [3]]
3.16
physical sampling efficiency
capacity of the sampling device to collect particles with specific sizes suspended in ambient air
[Adapted from EN 13098:2000, 3.17 [8]]
3.17
PM2,5
fraction of suspended particulate matter which passes through a size-selective inlet with a 50 % cut-off efficiency
at 2,5 µm aerodynamic diameter
[EN 14907:2005, 3.1.5 [7]]
NOTE By convention, the size-selective standard inlet design prescribed in EN 14907:2005, 5.1.2, used at the flow
rate given in EN 14907:2005, 5.1.5, possesses the required characteristics in order to sample the PM fraction in ambient
2,5
air.
3.18
PM10
fraction of suspended particulate matter which passes through a size-selective inlet with a 50 % cut-off efficiency
at 10 µm aerodynamic diameter
NOTE Definition in analogy to PM2,5; adapted from EN 14907:2005, 3.1.5 [7]]
3.19
Suspended Particulate Matter
SPM
notion of all particles surrounded by air in a given, undisturbed volume of air
[EN 14907:2005, 3.1.6 [7]]
NOTE The bioaerosol sampling head shows a mean cut-off value of 30 µm aerodynamic diameter without a rigid
upper separation limit due to its construction design and the specified flow rate, both determining the face velocity at the
filter.
3.20
total sampling efficiency
product of the physical sampling efficiency and the biological preservation efficiency
[EN 13098:2000, 3.19 [8]]
4 Symbols and abbreviations
Not applicable.
5 Basic principle of the method
5.1 Sampling
In this document the measurement object are the airborne moulds as part of the suspended particulate matter
(SPM, here particles with aerodynamic diameter up to approximately 30 µm).
During filtration, a defined air quantity is sucked through a filter – on or in which separation of the suspended
particles occurs (see Annex A). Airborne moulds are collected on gelatine filters resulting in a high total
sampling efficiency. Polycarbonate filters are used below the gelatine filters as supporting and protective filters
to enhance stability (see Annex B).
Ambient air sampling devices validated for different fractions of particles are commercially available and are
widely used for ambient air particle sampling according to national guidelines and European Standards:
 VDI 2463 Part 7 [11] and VDI 2463 Part 8 [12] for suspended particulate matter;
 EN 12341:1998 for PM10 [10];
 EN 14907:2005 for PM2,5 [7].
For sampling of bioaerosols an adapted bioaerosol sampling head, e.g. with larger filter diameters, has been
developed resulting in changes of the face velocity at the filter compared with the standards given above.
NOTE 1 The sampling head was modified in respect of the reference method described in the national guideline VDI
2463 Part 8 [12]. The applicability of this adapted sampling head for bioaerosols has been confirmed by comparison
measurements in 2001 (see Annex A), whereas the reference sampling head according to VDI 2463 Part 8 was validated
in 1987/88 during an international WRAC validation campaign (WRAC = Wide range aerosol classifier) [13; 14].
The reason for the modification of the standardised sampling head for suspended particulate matter was the
reduction of the face velocity in order to decrease the sampling stress. Additionally, the modifications enable
the use of disposable or sterilizable filter holders ensuring aseptic conditions during the handling of filter and
sampling head and avoiding carry-over effects and contaminations.
In a comparison measurement campaign it was shown that the adapted bioaerosol sampling head described
in A.1 using a filter with a diameter of 8 cm (effective filter diameter of 7 cm) resulting in a face velocity of
3 3
approximately 20 cm/s (19,5 cm/s to 23,8 cm/s depending on the flow rate of 2,7 m /h to 3,3 m /h) gave
comparable results to the non-modified standardised sampling head for sampling SPM (here up to 30 µm)
with regard to the physical sampling efficiency. Additionally a validation trial using this bioaerosol sampling
head for the detection of moulds in ambient air under real conditions including sampling and analyses was
performed (see A.2) showing comparable biological sampling efficiency. The performance characteristics and
minimum requirements of the bioaerosol sampling head are given in Clause 8.
In general, any sampling head can be used, that assures a comparable physical sampling efficiency of the
SPM fraction SPM (here up to 30 µm) and a comparable biological sampling efficiency with regard to the
bioaerosol sampling head described in this document. Additionally, aseptic handling of the filter shall be
assured und contaminations shall be avoided.
NOTE 2 If only the inhalable fraction is of interest, PM10 sampling can be performed. In this case fractions of
bioaerosols which are not inhalable but can cause irritation by contact e.g. to mucous membranes are missed. When
using the filter combination gelatine/polycarbonate filters with PM resp. PM sampling heads, the physical requirements
2,5 10
for particle sampling (cut-off efficiency, flow rate, etc., see EN 14907 [7] resp. EN 12341 [10]) apply.
After sampling the mould spores are cultured and counted according Clause 7.
5.2 Analyses
With the methods described here, mesophilic and thermotolerant moulds are quantified by culture-based
analyses of the viable and culturable propagules on selective agar. The quantitative determination of the
mould concentration is performed by counting the visually recognisable colonies. The density of the colonies
grown on the culture medium shall always allow proper enumeration of the colonies. The density of the
colonies results from the number of dilution steps after suspension of the cells from the filter. Therefore, in
principal several dilution steps need to be plated out.
6 Sampling
6.1 Sampling equipment
The following components are needed:
6.1.1 Stand, to position the bioaerosol sampling head at the sampling height needed.
)
6.1.2 Bioaerosol sampling head , to position the filter holder with the inserted filters in a hanging position,
if necessary.
1)
Sampling heads for bioaerosols as well as complete sampling devices are commercially available from several
manufacturers, e.g. Leckel, Berlin/Germany; Derenda, Stahnsdorf/Germany; Digitel, Hegnau/Switzerland. This information
is given for the convenience of users of this Technical Specification and does not constitute an endorsement by CEN of
the product named.
A bent pipe or hose connection can be used to connect the bioaerosol sampling head to the sampling device.
If using the bioaerosol sampling head described here, the inner diameter of the pipe or hose shall be 8 mm to
10 mm. The length of the connecting hose should not exceed 1,5 m. If using other sampling heads, these
dimensions shall be adapted accordingly in order to ensure the physical requirements (e.g, face velocity, flow
rate, see Table 3).
6.1.3 Filter holder, sterile (disposable or sterilizable), to insert the filters.
2) 3)
6.1.4 Filter, gelatine filter , sterile, pore size 3 µm, and polycarbonate filter , sterile, pore size 0,8 µm
(see Annex B).
The physical sampling efficiency of both gelatine and polycarbonate filters shall be > 95 % for moulds resp.
particles with an aerodynamic diameter range of > 1 µm, using a flow velocity at the filter of v = 21,7 cm/s ±
10 %. The combination of the two filters ensures a sampling efficiency of 99 % (Annex B).
6.1.5 Vacuum pump, ensuring a constant flow rate during continuous operation.
The flow rate has to be adapted in order to achieve a face velocity at the filter of 22 cm/s ± 10 %.
NOTE 1 If a filter with a diameter of 8 cm (effective diameter of 7 cm) is used, this face velocity is achieved by a flow
rate of about 3 m /h ± 10 %. During the sampling duration the filter resistance may increase therefore is recommended to
use a pump with a capacity of approximately 6 m /h.
NOTE 2 This method has been validated for a face velocity at the filter of 21,7 cm/s (see Annex A).
6.1.6 Gas volume meter, to determine the gas volume sucked at the bioaerosol sampling head, in
operating cubic meters.
Display accuracy of the flow rate: 0,01 m /h.
NOTE The use of volumetric measuring systems should take into account the manufacturer's specifications with
regard to the prevailing conditions during sampling, e.g. difference pressure between ambient and operating conditions,
temperature, humidity.
6.1.7 Timer, for presetting time and duration of sampling.
6.1.8 Protective housing, to protect the sampling device from harmful environmental conditions (optional).
The distance between the upper edge of the protective housing and the lower edge of the bioaerosol sampling
head should be at least 40 cm.
6.1.9 Commonly used devices for measuring ambient air conditions and operating conditions, e.g.
temperature, humidity, pressure.
For long term measurements a data logger may be necessary.
6.2 Materials
6.2.1 Container, sterile, for filter containment during transport, e.g. Petri dishes.
6.2.2 Container, insulated, for sample transport.
6.2.3 Disposable protective gloves, to avoid contamination and ensure occupational safety.
6.2.4 Disinfectant, e.g. iso-propanol or ethanol (70 %, volume content).

2)
Gelatine filters are commercially manufactured by Sartorius Stedim Biotech GmbH, Goettingen, Germany, however,
they are available from many suppliers. This information is given for the convenience of users of this Technical
Specification and does not constitute an endorsement by CEN of the product named.
3)
Polycarbonate filters are available from many manufacturers.
6.2.5 Tweezers, sterile, to handle the filters.
6.2.6 Thermometer, pressure gauge, hygrometer, to measure ambient air conditions during sampling
and transportation.
6.3 Sampling procedure
6.3.1 Preparation for sampling
The required number of measurement devices with accessories or other equipment shall be prepared in
accordance with the measurement task and the measurement strategy resulting thereof. It is recommended to
check the equipment for completeness and functionality using a check list.
The calibration validity of the sampling device shall be verified; otherwise new calibration shall be conducted
prior to the beginning of the measurements (see 6.5). The correct function of the sampling equipment shall be
documented in the sampling report.
Exclusively sterile filters and sterile filter holders shall be used for the measurements. If factory sterile
disposable filters with filter holders are not available, then the sterilized filters shall be inserted in the filter
holder at the laboratories safety cabinet and packed in sterility. For this purpose, first the polycarbonate filter
and afterwards the gelatine filter shall be placed in the filter holder. Thereby, special attention shall be paid
that the filters are tightly inserted into the filter holder. Filter sterility shall be guaranteed up to the moment of
sampling. During transport, the filters shall be protected from dust, heat and strong vibrations.
Assemble the sampling device according to Figure 1. A detailed example of a suitable sampling device is
given in Annex A.
Key
1 Bioaerosol sampling head
2 Gas volume meter (e.g. orifice plate, thermal mass flow rate meter)
3 Electronic circuit for conversion into operating cubic metres
4 Display for sampling volume V in operating cubic metres
B
5 Vacuum pump
6 Timer
7 Filter for abraded material
8 Protective housing
Figure 1 — Schematic set-up of the sampling device
The gas volume meter shall be connected between the pump and the bioaerosol sampling head in order to
determine the sampling volume without falsification caused by the leakage flow rate of the pump. The volume
of the sampled air is displayed in operating cubic metres and a display accuracy of 0,01 m³. For this purpose
the state variables temperature T and pressure p within the gas volume meter and in the air are being
continuously registered, and the sampling volume as measured by the gas volume meters is being converted
electronically using Equation (1):
T p
A G
V =V (1)
B G
T p
G A
where
V is the sampling volume in operating cubic metres referenced to ambient air conditions, in m ;
B
V is the sampling volume measured by a gas volume meter, in m ;
G
T is the ambient air temperature, in K;
A
T is the temperature within the gas volume meter, in K;
G
p is the ambient air pressure, in Pa;
A
p is the air pressure within the gas volume meter, in Pa.
G
6.3.2 Sampling
The bioaerosol sampling head is normally installed at a height of 1,5 m to 2,5 m with the filter in a hanging
position according to Figure 1.
During the complete sampling procedure aseptic techniques shall be ensured as far as possible. The sterile
filter holders and sterile filters shall be mounted on the sampling device without any contamination (preferably
by using disposable gloves). Prior to placing the filter holders the filters shall be visually inspected for integrity
and exact, air-tight fitting of the seat. This check shall be repeated after removal of the filter holder from the
sampling device.
The sampling device shall be started in accordance with the manufacturer's operating instructions.
The measurement task and all sampling details shall be recorded in the sampling report (see 6.3.4). In
addition all specific characteristics, anomalies and interferences shall be recorded that may be relevant to the
measurement result (e.g. odour perception, type and location of possible additional emission sources,
performance variations of the emission source(s) subject to the investigation, dust turbulence caused by
passing vehicles). A sketch shall depict the surroundings of the measurement location. A sampling report can
be found in Annex C as an example.
During sampling, the flow rate of the sampled air shall not be reduced by more than 10 % as a result of the
increasing filter loading.
NOTE There are self regulating sampling systems which maintain a constant flow rate and thereby avoid these
deviations.
Influences that could modify the measurement result shall be avoided. For example, no person is allowed to
stand within the range of the flow towards the sampling device.
After sampling, the filters and filter holders shall be removed from the sampling device by using disposable
gloves, and the filters shall be checked again for integrity. They shall be sealed in order to avoid any
secondary contamination (see 6.3.5).
A minimum of one field blank shall be taken per measurement day. The field blank should be taken preferably
in the middle of all measurements performed during one day. A field blank is a sample taken in an identical
manner as the real sample, but without sucking air through the sampling device. For this purpose a sterile
filter holder with filter is placed in the bioaerosol sampling head with the pump switched-off, then removed,
packed and analytically processed; a prolonged exposure of the filter to the ambient air shall be avoided. The
resulting field blank represents the number of CFU entering the sample simply by handling the filter during
sampling.
6.3.3 Sampling duration
The sampling duration is determined by the measurement task, and is generally between 10 min and 24 h.
This sampling duration was tested during the national validation campaign (see A.2). In this validation
campaign the lower limit of the sampling duration was given by the physical characteristics of the sampling
device (volume flow stability). The filter combination has proved successfully under different ambient air
conditions, also during rain, and it was shown that the humidity sensitive gelatine filter retained its applicability
due to the utilization of the polycarbonate filter as protective filter for 24 h. Longer sampling durations shall be
validated.
During sampling the flow rate of the sampled air shall not be reduced by more than 10 % as a result of the
increasing filter loading.
6.3.4 Sampling report
The samples shall be labelled for unique identification.
A sampling report shall be filled in for each sample before (or just after) sampling.
The report shall at least indicate:
 date and time of sampling;
 name and address of the operator;
 site or facility type, activities and interferences during the sampling;
 measurement task and sampling location (geographic coordinates);
 type of sampling device used;
 sampling volume, flow rate and duration of sampling;
 meteorological parameters as air temperature, air pressure, relative humidity. wind direction, wind
velocity, and climatic conditions;
 name of the person taking the sample.
The purpose of the analysis and, if applicable, a list of parameters to analyze are also needed as they may
subsequently help the laboratory with the choice of methods. Other details can be necessary (e.g. any
observations on phenomena which could affect the concentration of airborne moulds, see 6.3.2).
An example of a sampling report is given in Annex C.
6.3.5 Transport and storage
The samples shall be processed in the laboratory preferably without delay, but not later than 48 h after the
end of sampling. The loaded filters shall be protected from disturbing influences (sunshine, humidity or
desiccation, heat and dust, etc.) and shall be transported with the sampling side facing upwards in sealed
containers (see 6.2). The temperature during transport shall not exceed the incubation temperature (< 25 °C).
The conditions during transport (temperature, humidity, duration) shall be recorded.
The samples shall be stored in the laboratory in the dark at a temperature not exceeding the incubation
temperature (< 25 °C). The samples shall be protected against adverse influences (humidity, desiccation,
contamination). The storage conditions shall be documented.
If necessary, the samples shall be cooled during transport and storage, but care shall be taken not to freeze
them.
6.4 Sampling efficiency and method limitations
The limitations of the method are determined by the physical and biological sampling efficiency.
Physical sampling efficiency of both gelatine and polycarbonate filters is in excess of 95 % for moulds with an
aerodynamic diameter range of > 1 µm using a face velocity at the filter of v = 21,7 cm/s (Annex A and Annex
B). The combination of the two filters ensures a sampling efficiency of 99 %
Biological sampling efficiency is mainly influenced by desiccation. The effect of desiccation is not constant, but
depends on sampling duration, temperature, relative humidity and other parameters like season as well as on
the type of mould. Fortunately, most mould spores are relatively insensitive towards desiccation, so that the
filter method can usually be successfully applied in this field [15].
6.5 Laboratory calibration, function check in the field and maintenance
6.5.1 General
The sampling equipment should be checked and serviced regularly according to the manufacturer's
instructions. Particular emphasis should be placed on the calibration and the check of the flow rate. The
calibration of the sampling devices is done in the laboratory (see 6.5.2), function check is performed in the
field before starting the measurement (see 6.5.3).
6.5.2 Calibration of flow rate in the laboratory and maintenance
On a regular basis (e.g. twice a year) the complete sampling device shall be checked with regard to quality
assurance criteria. Hereby the flow rate of the sampling device is controlled against a reference volume
measuring device (e.g. mass flow meter, gas volume meter etc., certified by national calibration institute)
which is only used for this purpose in the laboratory. Also a general maintenance of the sampling device is to
be done during these checks according to the manufacturer's recommendations, this can concern clogged
filters of the pump, spare parts and consumables like gaskets, etc.
The volume flow rate of the sampling device against the reference volume measuring device shall be inside a
range of ± 5 %. If allowed according to the manufacturer's manual a flow adjustment may be performed. If this
is not allowed or not achievable the defective sampling device shall not be used any longer until it has been
repaired.
6.5.3 Function check in the field
The usual verification of the flow rate in the field shall be performed before starting the measurement.
A function check prior to the sampling process shall be conducted to assure that the sampling device after
transport to the sampling site is working properly. The main tasks are checks of the flow rate and for leaks. In
order to check the flow rate a test sampling head with test filters is connected to the sampling device. At the
inlet of the sampling head the testing volume measuring device (e.g. mass flow meter, gas volume meter etc.)
is connected using an adapter. Then the pump is switched on and the flow rate is observed. The flow rate
(calculated or directly readable) at the volume measuring device of the sampling device and the flow rate
(calculated or directly readable) at the testing volume measuring device shall be comparable. The tolerance
shall be in maximum 5 % and the flow rate shall be within the range of 2,7 m³/h to 3,3 m³/h as given in Table
3.
The data shall be recorded. If no valid data are achieved, the sampling device shall not be used for the
measurement and shall be labelled, e.g. "defective".
7 Culture-based analyses
7.1 Quantification equipment
7.1.1 Microbiological laboratory equipment
Usual microbiological laboratory equipment, and in particular:
a) autoclave, capable of operating at (115 ± 3) °C and (121 ± 3) °C;
b) incubator thermostatically controlled at (25 ± 3) °C;
c) incubator thermostatically controlled at (36 ± 2) °C;
d) incubator thermostatically controlled at (45 ± 2) °C;
e) refrigerator thermostatically controlled at (5 ± 3) °C;
f) pH meter with an accuracy of ± 0,1;
g) microbiological safety cabinet Class 2 (laminar flow cabinet);
h) water bath (35 °C to 40 °C) with shaker; ®
4)
i) test tube shaker (e.g. Vortex shaker );
j) test tubes, preferably disposable;
k) orbital shaker (speed: 150 r/min);
l) petri dishes, vented, sterile, diameter approximately 9 cm;
m) sterilized container (e.g. Erlenmeyer flask with minimum bottom diameter of 8 cm).
7.1.2 Culture media and diluents
7.1.2.1 General
All reagents and chemicals shall be of analytical grade. Other grades of chemicals may be used providing
they can be shown to lead to the same results. Water used shall be distilled or of equivalent quality.
Use of commercially available, dehydrated substrates is encouraged, provided they comply with the
descriptions given. They shall be prepared according to the instructions from the manufacturer. Alternatively
DG18 agar plates may also be bought ready for use at specialised manufacturers or may be prepared by the
laboratory according to Table 1. It shall be assured, that the composition of commercially available DG18 agar
plates is identical to the requirements of Table 1.
®
4)
Vortex shaker is an example of a suitable product available commercially. This is a product that is identified by a
trade market name. It has many manufacturers. This information is given for the convenience of users of this Technical

Specification and does not constitute an endorsement by CEN of the product named.
7.1.2.2 Dichloran 18 % glycerol agar (DG18 agar)
Table 1 — Formulation of the DG18 agar (pH = 5,6 ± 0,2) [16]
5)
5,0 g
Peptone
Glucose 10,0 g
Potassium dihydrogen phosphate (KH PO ) 1,0 g
2 4
Magnesium sulphate (MgSO  7 HO) 0,5 g
4 2
Dichlorane (2,6-dichlor-4-nitroanilin) 1,0 ml
0,2 % in ethanol (content by volume)
Chloramphenicol 0,1 g
a)
Glycerol 220 g
Agar 15,0 g
Distilled water 1 000 ml
or water of comparable quality

a)
18 mass percent of approximately 1 220 g final mass = approximately 220 g.

NOTE DG18 agar is suitable for the detection of a wide spectrum of xerophilic (i.e. preferring dryness) moulds.
Glycerol reduces the water activity a to 0,95. Chloramphenicol inhibits bacteria, especially gram-negative bacteria.
w
Dichlorane inhibits the spreading of fast growing mould colonies and thus prevents overgrowing of slow growing colonies.
Add ingredients, except glycerol, and agar as specified in Table 1 in ca. 800 ml water and dissolve by boiling.
Make up to 1 000 ml and add 220 g glycerol. Sterilise in an autoclave at (121 ± 3) °C for (15 ± 1) min. After
sterilisation, the pH shall correspond to 5,6 ± 0,2 at 25 °C. Dispense aliquots of about 20 ml in Petri dishes.
The pH of the agar is 5,6 ± 0,2 (at 25 °C), which is ensured by the given buffer composition in Table 1.
Plates of DG18 agar in bags will keep for up to one week at (5 ± 3) °C in the dark. DG18 agar has a defined
reduced water activity. Take care to avoid further reduction in water activity by desiccation because this may
prevent moulds from growing on this agar. ®
7.1.2.3 Saline Solution with Polysorbate 80
For suspension of the filters and for the dilution series a saline solution (0,85 % w/v NaCl) with 0,01 % v/v ®
6)
Polysorbate 80 is used. Dissolve the NaCl in the water and sterilise in an autoclave at (121 ± 3) °C for
® ®
6) 6)
(15 ± 1) min. Allow to cool and add the Polysorbate 80 . Due to the viscosity of the Polysorbate 80 it is
recommended to use a positive-displacement pipette or to weigh the required amount using a calibrated and
appropriate balance.
5)
Different peptones are used by different manufacturers (e.g. casein peptone, mycological peptone). This does usually
not influence the quantitative results of the measurements but may have an influence on the appearance of the colonies.
Positive controls using microbiological reference strains or natural samples for comparison of recovery and of

morphological appearance of the colonies are, therefore, important. ®
6)
Polysorbate 80 is equivalent to Polyoxyethylenesorbitan monooleate or Polyethylene glycol sorbitan monooleate. ®
Polysorbate 80 is an example of a suitable product available commercially. This chemical is a product that is identified by
a trade market name. It is unique and has a sole manufacturer, however, it is available from many suppliers. This
information is given for the convenience of users of this Technical Specification and does not constitute an endorsement

by CEN of the product named.
7.2 Processing of filters
7.2.1 General
Process the samples in the laboratory preferably without delay, but not later than 48 h after the end of
sampling.
Airborne moulds deposited on filters are processed using the indirect plating method. The filters obtained from ®
6)
sampling by filtration are re-suspended in saline solution (0,85 % NaCl) with 0,01 % Polysorbate 80 (see
7.1.2.3). Decimal dilutions of the suspension are prepared and aliquots are spread on DG18 agar. Agar plates
are incubated at (25 ± 3) °C. For special purposes plates can be incubated at (36 ± 2) °C (e.g. thermotolerant
Aspergillus spp.) or (45 ± 2) °C (Aspergillus fumigatus).
After incubation, mould colonies are identified by colony morphology and counted. The extent of identification
depends on the objective of the investigation. The identification requires appropriately trained personnel (see
also 7.4).
NOTE Aggregates should be separated and distributed equally by suspension and dilution as far as possible. This
cannot be ensured completely, therefore the measured concentrations may differ from the expected d
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