SIST ISO 16000-17:2013
Indoor air - Part 17: Detection and enumeration of moulds - Culture-based method
Indoor air - Part 17: Detection and enumeration of moulds - Culture-based method
This part of ISO 16000 specifies a method for the detection and enumeration of moulds by cultivation after sampling by impaction according to ISO 16000-18 or by filtration according to ISO 16000-16. It is also suitable for cultivation of moulds from material suspensions or from direct plating.
Air intérieur - Partie 17: Détection et dénombrement des moisissures - Méthode par culture
L'ISO 16000-17:2008 sp�cifie une m�thode de d�tection et de d�nombrement des moisissures par culture apr�s �chantillonnage par impaction, selon l'ISO 16000-18:—, ou par filtration, selon l'ISO 16000-16. Elle convient �galement � la culture des moisissures � partir de mati�res en suspension ou de culture directe sur bo�te de Petri.
Notranji zrak - 17. del: Ugotavljanje prisotnosti in števila gliv - Metoda, temelječa na kulturi
Ta del standarda ISO 16000 določa metodo za ugotavljanje prisotnosti in števila gliv s kultivacijo po vzorčenju z impakcijo, kot je opisano v standardu IS0 16000-18, ali s filtracijo, kot je opisano v standardu ISO 16000-16. Prav tako je primerna za kultivacijo gliv iz suspenzij materiala ali neposrednih nanosov.
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INTERNATIONAL ISO
STANDARD 16000-17
First edition
2008-12-15
Indoor air —
Part 17:
Detection and enumeration of moulds —
Culture-based method
Air intérieur —
Partie 17: Détection et dénombrement des moisissures — Méthode par
culture
Reference number
ISO 16000-17:2008(E)
©
ISO 2008
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ISO 16000-17:2008(E)
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ii © ISO 2008 – All rights reserved
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ISO 16000-17:2008(E)
Contents Page
Foreword. iv
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Definitions . 1
4 Principle. 3
5 Apparatus . 3
6 Culture media and diluents. 3
6.1 Dicloran 18 % glycerol agar (DG18 agar) . 4
6.2 Malt-extract agar . 4
6.3 Potato dextrose agar . 5
6.4 Saline solution . 5
6.5 Saline solution with polysorbate 80. 5
7 Procedure . 6
7.1 General. 6
7.2 Processing of filters . 6
7.3 Incubation. 7
7.4 Examination and counting. 7
7.5 Identification of mould species . 8
7.6 Calculation and expression of results. 8
8 Test report . 11
Annex A (informative) Specific characteristics of mould spores. 12
Annex B (informative) Exchange of samples for validation of the cultivation method . 15
Bibliography . 20
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ISO 16000-17:2008(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 16000-17 was prepared by Technical Committee ISO/TC 146, Air quality, Subcommittee SC 6, Indoor air.
ISO 16000 consists of the following parts, under the general title Indoor air:
⎯ Part 1: General aspects of sampling strategy
⎯ Part 2: Sampling strategy for formaldehyde
⎯ Part 3: Determination of formaldehyde and other carbonyl compounds — Active sampling method
⎯ Part 4: Determination of formaldehyde — Diffusive sampling method
⎯ Part 5: Sampling strategy for volatile organic compounds (VOCs)
⎯ Part 6: Determination of volatile organic compounds in indoor and test chamber air by active sampling on
®
Tenax TA sorbent, thermal desorption and gas chromatography using MS/FID
⎯ Part 7: Sampling strategy for determination of airborne asbestos fibre concentrations
⎯ Part 8: Determination of local mean ages of air in buildings for characterizing ventilation conditions
⎯ Part 9: Determination of the emission of volatile organic compounds from building products and
furnishing — Emission test chamber method
⎯ Part 10: Determination of the emission of volatile organic compounds from building products and
furnishing — Emission test cell method
⎯ Part 11: Determination of the emission of volatile organic compounds from building products and
furnishing — Sampling, storage of samples and preparation of test specimens
⎯ Part 12: Sampling strategy for polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins
(PCDDs), polychlorinated dibenzofurans (PCDFs) and polycyclic aromatic hydrocarbons (PAHs)
⎯ Part 13: Determination of total (gas and particle-phase) polychlorinated dioxin-like biphenyls (PCBs) and
polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/PCDFs) — Collection on sorbent-backed filters
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ISO 16000-17:2008(E)
⎯ Part 14: Determination of total (gas and particle-phase) polychlorinated dioxin-like biphenyls (PCBs) and
polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/PCDFs) — Extraction, clean-up and analysis by
high-resolution gas chromatography and mass spectrometry
⎯ Part 15: Sampling strategy for nitrogen dioxide (NO )
2
⎯ Part 16: Detection and enumeration of moulds — Sampling by filtration
⎯ Part 17: Detection and enumeration of moulds — Culture-based method
⎯ Part 23: Performance test for evaluating the reduction of formaldehyde concentrations by sorptive
building materials
⎯ Part 24: Performance test for evaluating the reduction of volatile organic compounds and carbonyl
compounds without formaldehyde concentrations by sorptive building materials
The following parts are under preparation:
⎯ Part 18: Detection and enumeration of moulds — Sampling by impaction
⎯ Part 19: Sampling strategy for moulds
⎯ Part 25: Determination of the emission of semi-volatile organic compounds by building products — Micro-
chamber method
⎯ Part 28: Sensory evaluation of emissions from building materials and products
The following parts are planned:
⎯ Part 20: Detection and enumeration of moulds — Sampling from house dust
⎯ Part 21: Detection and enumeration of moulds — Sampling from materials
⎯ Part 22: Detection and enumeration of moulds — Molecular methods
⎯ Part 27: Standard method for the quantitative analysis of asbestos fibres in settled dust
Furthermore,
⎯ ISO 12219-1 (under preparation), Indoor air — Road vehicles — Part 1: Whole vehicle test chamber —
Specification and method for the determination of volatile organic compounds in car interiors,
⎯ ISO 16017-1, Indoor, ambient and workplace air — Sampling and analysis of volatile organic compounds
by sorbent tube/thermal desorption/capillary gas chromatography — Part 1: Pumped sampling, and
⎯ ISO 16017-2, Indoor, ambient and workplace air — Sampling and analysis of volatile organic compounds
by sorbent tube/thermal desorption/capillary gas chromatography — Part 2: Diffusive sampling
focus on volatile organic compound (VOC) measurements.
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ISO 16000-17:2008(E)
Introduction
Mould is a common name for filamentous fungi from different taxonomic groups [Zygomycetes, Ascomycetes
(Ascomycota), 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 2 µm to10 µm, some up to 30 µm and a very 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 (Stachybotrys, Fusarium) and less mobile.
Mould spores are widely distributed in the outdoor environment and, therefore, also occur in varying
concentrations indoors. Growth of moulds in indoor environments, however, should be considered a public
health problem because epidemiological studies have revealed that dampness and/or mould growth in homes
and health impairment of occupants are closely related.
Standardised methods for sampling, detection and enumeration of moulds including standards for sampling
strategies are important for comparative assessment of mould problems indoors. Before taking any
measurements, a measurement strategy is required.
[5] [6]
The procedure specified in this part of ISO 16000 is based on VDI 4253-2 and VDI 4300-10 .
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INTERNATIONAL STANDARD ISO 16000-17:2008(E)
Indoor air —
Part 17:
Detection and enumeration of moulds — Culture-based method
WARNING — The use of this part of ISO 16000 may involve hazardous materials, operations and
equipment. This part of ISO 16000 does not purport to address any safety problems associated with
its use. It is the responsibility of the user of this part of ISO 16000 to establish appropriate safety and
health practices and determine the applicability of regulatory limitations prior to use.
1 Scope
This part of ISO 16000 specifies a method for the detection and enumeration of moulds by cultivation after
sampling by impaction according to ISO 16000-18 or by filtration according to ISO 16000-16. It is also suitable
for cultivation of moulds from material suspensions or from direct plating.
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.
ISO 8199, Water quality — General guidance on the enumeration of micro-organisms by culture
ISO 16000-16, Indoor air — Part 16: Detection and enumeration of moulds — Sampling by filtration
1)
ISO 16000-18, Indoor air — Part 18: Detection and enumeration of moulds — Sampling by impaction
3 Definitions
For the purpose of this part of ISO 16000, the following terms and definitions apply:
3.1
filamentous fungus
fungus growing in the form of filaments of cells known as hyphae
NOTE 1 Hyphae aggregated in bundles are called mycelia.
NOTE 2 The term “filamentous fungi” differentiates fungi with hyphal growth from yeasts.
[ISO 16000-16:2008]
1) To be published.
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ISO 16000-17:2008(E)
3.2
filtration
collection of particles suspended in gas or liquid by flow through a porous medium
[4]
[EN 13098:2000 ]
NOTE In this part of ISO 16000, filtration is understood as the separation of microorganisms or moulds from a
defined volume of air by means of filters.
3.3
indirect method
〈air quality〉 resuspension of deposited microorganisms with subsequent plating of aliquots on a suitable
culture medium, incubation and counting of colonies growing under the conditions selected
3.4
colony forming unit
cfu
unit by which the culturable number of microorganisms is expressed
[4]
[EN 13098:2000 ]
NOTE 1 One colony forming unit can originate from one single microorganism, from aggregates of many
microorganisms as well as from one or many microorganisms attached to one particle.
NOTE 2 The number of colonies can depend on cultivation conditions.
3.5
cultivation
〈air quality〉 growing of microorganisms on culture media
[ISO 16000-16:2008]
3.6
microorganism
any microbiological entity, cellular or non-cellular, capable of replication or of transferring genetic material, or
entities that have lost these properties
[4]
[EN 13098:2000 ]
3.7
moisture indicator
〈air quality〉 mould in indoor environments preferring relatively high humidity for growth and therefore indicating
moisture problems when present
3.8
secondary colony
colony which does not originate from the “primary” sampling of airborne spores but from a spore released from
a colony growing on the agar plates
3.9
mould
〈air quality〉 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]
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ISO 16000-17:2008(E)
4 Principle
Agar plates (DG18 agar and malt-extract agar or potato dextrose agar) obtained from sampling by impaction
are incubated directly at (25 ± 3) °C.
Filters obtained from sampling by filtration are re-suspended in saline solution (0,9 % mass fraction NaCl) with
2)
0,01 % polysorbate 80 . Decimal dilutions of the suspension are prepared and aliquots spread on DG18 agar
as well as on malt-extract agar or potato dextrose agar (indirect method). 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 and counted. The extent of identification depends on the
objective of the investigation.
5 Apparatus
Usual microbiological laboratory equipment, and in particular the following.
5.1 Incubator, vibration free, thermostatically controlled at (25 ± 3) °C.
5.2 Incubator, vibration free, thermostatically controlled at (36 ± 2) °C.
5.3 Incubator, vibration free, thermostatically controlled at (45 ± 2) °C.
5.4 Refrigerator, thermostatically controlled at (5 ± 3) °C.
5.5 pH meter, with an accuracy of ± 0,1 pH unit.
5.6 Microbiological safety cabinet, Class II, for user and product protection.
5.7 Water bath, capable of being maintained at 35 °C to 40 °C with shaker.
3)
5.8 Test tube shaker, e.g. Vortex shaker .
5.9 Petri dishes, vented, sterile, of diameter ~90 mm.
5.10 Autoclave, capable of operating at (115 ± 3) °C and at (121 ± 3) °C.
6 Culture media and diluents
Use reagents of recognized analytical grade, unless other grades can be shown to lead to similar results, and
only distilled or deionised water or water of equivalent purity.
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.
2) Polysorbate 80 is equivalent to polyoxyethylenesorbitan monooleate or polyethylene glycol sorbitan monooleate.
Tween is an example of a suitable product available commercially. This information is given for the convenience of users
of this International Standard and does not constitute an endorsement by ISO of this product.
3) Example of a suitable product available commercially. This information is given for the convenience of users of this
International Standard and does not constitute an endorsement by ISO of this product. Equivalent products may be used if
they can be shown to produce similar results.
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ISO 16000-17:2008(E)
6.1 Dicloran 18 % glycerol agar (DG18 agar)
The components are listed in Table 1.
Table 1 — Composition of dicloran 18 % glycerol agar (DG18 agar)
Component Quantity
4)
Peptone 5,0 g
Glucose 10,0 g
Potassium dihydrogen phosphate (KH PO) 1,0 g
2 4
Magnesium sulfate heptahydrate (MgSO ·7HO) 0,5 g
4 2
a
Dicloran (2,6-dichloro-4-nitroaniline) 0,2 % volume fraction in ethanol 1,0 ml
Chloramphenicol 0,1 g
b
Glycerol 220 g
Agar 15,0 g
Water 1 000 ml
a
Final concentration in medium: 0,002 g/l.
b
18 % mass fraction of ~1 220 g final mass = ~220 g.
Add minor ingredients and agar to ~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.
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 fungi from growing on this agar.
NOTE DG18 agar is suitable for the detection of a wide spectrum of xerophilic (i.e. preferring dryness) fungi. Glycerol
reduces the water activity, a , to 0,95. Chloramphenicol inhibits bacteria, especially gram-negative bacteria. Dicloran
H O
2
inhibits the spread of fast-growing mould colonies and thus prevents overgrowing of slow-growing colonies.
6.2 Malt-extract agar
The components are listed in Table 2.
Table 2 — Composition of malt-extract agar
Component Quantity
Malt extract 30,0 g
Peptone from soy 3,0 g
Agar 15,0 g
Water 1 000 ml
4) 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 for comparison of recovery and of morphological appearance of the colonies are, therefore, important.
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ISO 16000-17:2008(E)
NOTE The addition of Chloramphenicol (0,05 g/l) may be necessary if samples contain high concentrations of
bacteria. This is usually not the case for samples of indoor air but bacteria may be present in high numbers in material and
dust samples.
Add ingredients and agar in the water and dissolve by boiling. Sterilise in an autoclave at (115 ± 3) °C for
(10 ± 1) min. After sterilisation, the pH shall correspond to 5,5 ± 0,2 at 25 °C. Dispense aliquots of about 20 ml
into Petri dishes.
Plates of malt-extract agar in bags will keep for up to one month at (5 ± 3) °C in the dark.
Many commercial malt-extract agars with different compositions are available. Ensure that the ingredients
correspond to the composition given above.
6.3 Potato dextrose agar
The components are listed in Table 3.
Table 3 — Composition of potato dextrose agar
Component Quantity
Potato extract 4,0 g
Glucose 20,0 g
Agar 15,0 g
Water 1 000 ml
NOTE The addition of chloramphenicol (0,05 g/l) may be necessary if samples contain high concentrations of
bacteria.
Add ingredients and agar to the water and dissolve by boiling. Sterilise in an autoclave at (115 ± 3) °C for
(10 ± 1) min. After sterilisation, the pH shall correspond to 5,6 ± 0,2 at 25 °C. Dispense aliquots of about 20 ml
into Petri dishes.
Plates of potato dextrose agar in bags will keep for up to one month at (5 ± 3) °C in the dark.
6.4 Saline solution
The components are listed in Table 4.
Table 4 — Composition of saline solution
Component Quantity
Sodium chloride (NaCl) 8,5 g
Water 1 000 ml
Dissolve the NaCl in the water and sterilise in an autoclave at (121 ± 3) °C for (15 ± 1) min.
6.5 Saline solution with polysorbate 80
The components are listed in Table 5.
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ISO 16000-17:2008(E)
Table 5 — Composition of saline solution with polysorbate 80
Component Quantity
Sodium chloride (NaCl) 8,5 g
Polysorbate 80 0,1 g
Water 1 000 ml
Dissolve the NaCl in the water and sterilise in an autoclave at (121 ± 3) °C for (15 ± 1) min. Allow to cool and
add the polysorbate 80.
7 Procedure
7.1 General
Samples to be analysed are either agar plates from sampling by impaction according to ISO 16000-18 or
filters from sampling by filtration according to ISO 16000-16. Samples from direct plating or material
suspensions can be treated accordingly.
7.1.1 Samples from impaction
Agar plates are directly incubated (see 7.3).
7.1.2 Samples from filtration
Filters are resuspended and aliquots spread on agar plates (see 7.2) followed by incubation (see 7.3).
Process the samples in the laboratory preferably without delay, but not later than 48 h after the end of
sampling. Store samples in the laboratory in the dark at a temperature not exceeding the incubation
temperature (< 25 °C), protected against adverse influences (humidity, desiccation, contamination). Document
the storage conditions.
Carry out all procedures under conditions that protect the samples from any contamination. Check aseptic
conditions regularly by controls and results shall be documented.
7.2 Processing of filters
7.2.1 General
Airborne moulds deposited on filters are processed using the indirect plating method.
NOTE Aggregates of either spores or aggregates of particles may be dissolved by suspension as well as by dilution,
which may result in higher numbers of colonies after incubation. An aggregate of 30 spores may result in 30 colonies
under these circumstances. By contrast, a reduction of the number of colonies may occur if the detachment of the mould
spores from the filters is incomplete or by damage of the fungal cells during processing in the laboratory.
7.2.2 Resuspension
In the aseptic atmosphere of a safety cabinet (5.6), transfer the filters (gelatin and polycarbonate filter) into a
sterile container containing 5 ml of saline solution with polysorbate 80 (see 6.5) using a sterile pair of tweezers.
Shake (5.8) the filters steeped in this solution in their container intensively in a horizontal position in a water
bath maintained at 35 °C to 40 °C (5.7) for 15 min. Make sure that the spore-loaded surface of the filter lies
flat and faces upwards and can move freely within the suspension during shaking.
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ISO 16000-17:2008(E)
Process the sample according to 7.2.3 within 1 h after suspension.
7.2.3 Dilution
Based on the original suspension, set up a serial dilution series.
Immediately prior to dilution, shake the suspension for 1 min on a test tube shaker (5.8). Add 1 ml of the
suspension to 9 ml saline solution (6.4) using a sterile disposable pipette or a cotton-stuffed glass pipette. In
the same way, carry out two further dilution steps resulting in dilutions 1:10, 1:100, and 1:1 000.
The number of dilution steps and the dilution intervals should be adapted to the expected mould concentration
and the specific measurement task. It may be necessary to set up additional dilution steps.
7.2.4 Plating
Plate 0,1 ml of the original suspension and the dilutions (7.2.3) in parallel on DG18 agar (6.1) as well as on
malt-extract agar (6.2) or potato dextrose agar (6.3). Use at least two parallel plates for each dilution and
incubation temperature (7.3).
If, according to the sampling protocol, low concentrations of moulds are expected, 1 ml of the original solution
can be plated on four plates for each agar medium (using 250 µl per plate) to enhance the sensitivity of the
method.
Determine laboratory blank samples for all dilution steps.
7.3 Incubation
Incubate agar plates upside up in incubators at (25 ± 3) °C for 7 days. DG18 agar plates may require
prolonged incubation times — 10 days — especially if differentiation of fungi is anticipated.
For special purposes (e.g. thermotolerant Aspergillus spp.), malt-extract agar plates or potato dextrose agar
plates may additionally be incubated at (36 ± 2) °C. For selective cultivation of Aspergillus fumigatus,
incubation temperatures of (45 ± 2) °C are used.
CAUTION — At incubation temperatures above 25 °C, take special care to avoid desiccation of the
agar plates.
Incubate agar plates in such way that sufficient oxygen will allow optimal mould growth, e.g. avoid incubation
in tight polyethylene bags. Incubate agar plates in a vibration-free incubator, to minimize the risk of secondary
colony inoculation due to spreading of spores. Also avoid extensive airflows in view of desiccation of the agar
plates.
7.4 Examination and counting
Examine agar plates for the first time after 2 days to 3 days and subsequently in regular intervals for up to at
least 7 days.
Count thermotolerant moulds (36 °C or 45 °C) after 1 day to 3 days, as they grow very rapidly.
Handling of the agar plates may lead to distribution of spores on the plate resulting in secondary colonies in
the course of incubation. Take care not to count secondary colonies.
The optimal range for genus/species identification and quantification using a standard culture plate with a
diameter of ~90 mm is between 20 and 40 colonies. For quantitative results, at least 10 colonies of the
respective genus/species should be on the agar plate and a maximum number of 100 colonies in total. Some
moulds may spread very fast inhibiting the growth of other colonies (e.g. Rhizopus, Chrysonilia, Mucor,
Botrytis) — even on plates containing dicloran — and the capacity of the plate can run down even at lower
colony counts.
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ISO 16000-17:2008(E)
Record the maximum number of colonies counted within the 7 days of incubation for each agar medium and
sampling volume (impaction) or dilution (filtration), respectively.
7.5 Identification of mould species
Identification of the moulds growing on the agar plates is essential for most questions concerning mould
problems in indoor environments. Identification is based on macroscopic and microscopic morphological
characteristics. These characteristics are usually better identified on malt-extract agar or potato dextrose agar
compared to DG18 agar.
NOTE Malt-extract agar and potato dextrose agar have been shown to give comparable results for identification of
mould genera and species.
The extent of identification depends on the purpose of the investigation.
To detect sources of mould growth indoors, it is important
...
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Air intérieur - Partie 17: Détection et dénombrement des moisissures - Méthode par cultureIndoor air - Part 17: Detection and enumeration of moulds - Culture-based method13.040.20Kakovost okoljskega zrakaAmbient atmospheresICS:Ta slovenski standard je istoveten z:ISO 16000-17:2008SIST ISO 16000-17:2013en,fr01-december-2013SIST ISO 16000-17:2013SLOVENSKI
STANDARD
SIST ISO 16000-17:2013
Reference numberISO 16000-17:2008(E)© ISO 2008
INTERNATIONAL STANDARD ISO16000-17First edition2008-12-15Indoor air — Part 17: Detection and enumeration of moulds — Culture-based method Air intérieur — Partie 17: Détection et dénombrement des moisissures — Méthode par culture
SIST ISO 16000-17:2013
ISO 16000-17:2008(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.
COPYRIGHT PROTECTED DOCUMENT
©
ISO 2008 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's member body in the country of the requester. ISO copyright office Case postale 56 • CH-1211 Geneva 20 Tel.
+ 41 22 749 01 11 Fax
+ 41 22 749 09 47 E-mail
copyright@iso.org Web
www.iso.org Published in Switzerland
ii © ISO 2008 – All rights reserved
SIST ISO 16000-17:2013
ISO 16000-17:2008(E) © ISO 2008 – All rights reserved iiiContents Page Foreword.iv Introduction.vi 1 Scope.1 2 Normative references.1 3 Definitions.1 4 Principle.3 5 Apparatus.3 6 Culture media and diluents.3 6.1 Dicloran 18 % glycerol agar (DG18 agar).4 6.2 Malt-extract agar.4 6.3 Potato dextrose agar.5 6.4 Saline solution.5 6.5 Saline solution with polysorbate 80.5 7 Procedure.6 7.1 General.6 7.2 Processing of filters.6 7.3 Incubation.7 7.4 Examination and counting.7 7.5 Identification of mould species.8 7.6 Calculation and expression of results.8 8 Test report.11 Annex A (informative)
Specific characteristics of mould spores.12 Annex B (informative)
Exchange of samples for validation of the cultivation method.15 Bibliography.20
SIST ISO 16000-17:2013
ISO 16000-17:2008(E) iv © ISO 2008 – All rights reserved Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 16000-17 was prepared by Technical Committee ISO/TC 146, Air quality, Subcommittee SC 6, Indoor air. ISO 16000 consists of the following parts, under the general title Indoor air: ⎯ Part 1: General aspects of sampling strategy ⎯ Part 2: Sampling strategy for formaldehyde ⎯ Part 3: Determination of formaldehyde and other carbonyl compounds — Active sampling method ⎯ Part 4: Determination of formaldehyde — Diffusive sampling method ⎯ Part 5: Sampling strategy for volatile organic compounds (VOCs) ⎯ Part 6: Determination of volatile organic compounds in indoor and test chamber air by active sampling on Tenax TA® sorbent, thermal desorption and gas chromatography using MS/FID ⎯ Part 7: Sampling strategy for determination of airborne asbestos fibre concentrations ⎯ Part 8: Determination of local mean ages of air in buildings for characterizing ventilation conditions ⎯ Part 9: Determination of the emission of volatile organic compounds from building products and furnishing — Emission test chamber method ⎯ Part 10: Determination of the emission of volatile organic compounds from building products and furnishing — Emission test cell method ⎯ Part 11: Determination of the emission of volatile organic compounds from building products and furnishing — Sampling, storage of samples and preparation of test specimens ⎯ Part 12: Sampling strategy for polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polycyclic aromatic hydrocarbons (PAHs) ⎯ Part 13: Determination of total (gas and particle-phase) polychlorinated dioxin-like biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/PCDFs) — Collection on sorbent-backed filters SIST ISO 16000-17:2013
ISO 16000-17:2008(E) © ISO 2008 – All rights reserved v⎯ Part 14: Determination of total (gas and particle-phase) polychlorinated dioxin-like biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDDs/PCDFs) — Extraction, clean-up and analysis by high-resolution gas chromatography and mass spectrometry ⎯ Part 15: Sampling strategy for nitrogen dioxide (NO2) ⎯ Part 16: Detection and enumeration of moulds — Sampling by filtration ⎯ Part 17: Detection and enumeration of moulds — Culture-based method ⎯ Part 23: Performance test for evaluating the reduction of formaldehyde concentrations by sorptive building materials ⎯ Part 24: Performance test for evaluating the reduction of volatile organic compounds and carbonyl compounds without formaldehyde concentrations by sorptive building materials The following parts are under preparation: ⎯ Part 18: Detection and enumeration of moulds — Sampling by impaction ⎯ Part 19: Sampling strategy for moulds ⎯ Part 25: Determination of the emission of semi-volatile organic compounds by building products — Micro-chamber method ⎯ Part 28: Sensory evaluation of emissions from building materials and products The following parts are planned: ⎯ Part 20: Detection and enumeration of moulds — Sampling from house dust ⎯ Part 21: Detection and enumeration of moulds — Sampling from materials ⎯ Part 22: Detection and enumeration of moulds — Molecular methods ⎯ Part 27: Standard method for the quantitative analysis of asbestos fibres in settled dust Furthermore, ⎯ ISO 12219-1 (under preparation), Indoor air — Road vehicles — Part 1: Whole vehicle test chamber — Specification and method for the determination of volatile organic compounds in car interiors, ⎯ ISO 16017-1, Indoor, ambient and workplace air — Sampling and analysis of volatile organic compounds by sorbent tube/thermal desorption/capillary gas chromatography — Part 1: Pumped sampling, and ⎯ ISO 16017-2, Indoor, ambient and workplace air — Sampling and analysis of volatile organic compounds by sorbent tube/thermal desorption/capillary gas chromatography — Part 2: Diffusive sampling focus on volatile organic compound (VOC) measurements.
SIST ISO 16000-17:2013
ISO 16000-17:2008(E) vi © ISO 2008 – All rights reserved Introduction Mould is a common name for filamentous fungi from different taxonomic groups [Zygomycetes, Ascomycetes (Ascomycota), 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 2 µm to10 µm, some up to 30 µm and a very 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 (Stachybotrys, Fusarium) and less mobile. Mould spores are widely distributed in the outdoor environment and, therefore, also occur in varying concentrations indoors. Growth of moulds in indoor environments, however, should be considered a public health problem because epidemiological studies have revealed that dampness and/or mould growth in homes and health impairment of occupants are closely related. Standardised methods for sampling, detection and enumeration of moulds including standards for sampling strategies are important for comparative assessment of mould problems indoors. Before taking any measurements, a measurement strategy is required. The procedure specified in this part of ISO 16000 is based on VDI 4253-2 [5] and VDI 4300-10 [6].
SIST ISO 16000-17:2013
INTERNATIONAL STANDARD ISO 16000-17:2008(E) © ISO 2008 – All rights reserved 1Indoor air — Part 17: Detection and enumeration of moulds — Culture-based method WARNING — The use of this part of ISO 16000 may involve hazardous materials, operations and equipment. This part of ISO 16000 does not purport to address any safety problems associated with its use. It is the responsibility of the user of this part of ISO 16000 to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 1 Scope This part of ISO 16000 specifies a method for the detection and enumeration of moulds by cultivation after sampling by impaction according to ISO 16000-18 or by filtration according to ISO 16000-16. It is also suitable for cultivation of moulds from material suspensions or from direct plating. 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. ISO 8199, Water quality — General guidance on the enumeration of micro-organisms by culture ISO 16000-16, Indoor air — Part 16: Detection and enumeration of moulds — Sampling by filtration ISO 16000-18, Indoor air — Part 18: Detection and enumeration of moulds — Sampling by impaction 1) 3 Definitions For the purpose of this part of ISO 16000, the following terms and definitions apply: 3.1 filamentous fungus fungus growing in the form of filaments of cells known as hyphae NOTE 1 Hyphae aggregated in bundles are called mycelia. NOTE 2 The term “filamentous fungi” differentiates fungi with hyphal growth from yeasts. [ISO 16000-16:2008]
1) To be published. SIST ISO 16000-17:2013
ISO 16000-17:2008(E) 2 © ISO 2008 – All rights reserved 3.2 filtration collection of particles suspended in gas or liquid by flow through a porous medium [EN 13098:2000 [4]] NOTE In this part of ISO 16000, filtration is understood as the separation of microorganisms or moulds from a defined volume of air by means of filters. 3.3 indirect method 〈air quality〉 resuspension of deposited microorganisms with subsequent plating of aliquots on a suitable culture medium, incubation and counting of colonies growing under the conditions selected 3.4 colony forming unit cfu unit by which the culturable number of microorganisms is expressed [EN 13098:2000 [4]] NOTE 1 One colony forming unit can originate from one single microorganism, from aggregates of many microorganisms as well as from one or many microorganisms attached to one particle. NOTE 2 The number of colonies can depend on cultivation conditions. 3.5 cultivation 〈air quality〉 growing of microorganisms on culture media [ISO 16000-16:2008] 3.6 microorganism any microbiological entity, cellular or non-cellular, capable of replication or of transferring genetic material, or entities that have lost these properties [EN 13098:2000 [4]] 3.7 moisture indicator 〈air quality〉 mould in indoor environments preferring relatively high humidity for growth and therefore indicating moisture problems when present 3.8 secondary colony colony which does not originate from the “primary” sampling of airborne spores but from a spore released from a colony growing on the agar plates 3.9 mould 〈air quality〉 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] SIST ISO 16000-17:2013
ISO 16000-17:2008(E) © ISO 2008 – All rights reserved 34 Principle Agar plates (DG18 agar and malt-extract agar or potato dextrose agar) obtained from sampling by impaction are incubated directly at (25 ± 3) °C. Filters obtained from sampling by filtration are re-suspended in saline solution (0,9 % mass fraction NaCl) with 0,01 % polysorbate 80 2). Decimal dilutions of the suspension are prepared and aliquots spread on DG18 agar as well as on malt-extract agar or potato dextrose agar (indirect method). 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 and counted. The extent of identification depends on the objective of the investigation. 5 Apparatus Usual microbiological laboratory equipment, and in particular the following. 5.1 Incubator, vibration free, thermostatically controlled at (25 ± 3) °C. 5.2 Incubator, vibration free, thermostatically controlled at (36 ± 2) °C. 5.3 Incubator, vibration free, thermostatically controlled at (45 ± 2) °C. 5.4 Refrigerator, thermostatically controlled at (5 ± 3) °C. 5.5 pH meter, with an accuracy of ± 0,1 pH unit. 5.6 Microbiological safety cabinet, Class II, for user and product protection. 5.7 Water bath, capable of being maintained at 35 °C to 40 °C with shaker. 5.8 Test tube shaker, e.g. Vortex shaker 3). 5.9 Petri dishes, vented, sterile, of diameter ~90 mm. 5.10 Autoclave, capable of operating at (115 ± 3) °C and at (121 ± 3) °C. 6 Culture media and diluents Use reagents of recognized analytical grade, unless other grades can be shown to lead to similar results, and only distilled or deionised water or water of equivalent purity. 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.
2) Polysorbate 80 is equivalent to polyoxyethylenesorbitan monooleate or polyethylene glycol sorbitan monooleate. Tween is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product. 3) Example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product. Equivalent products may be used if they can be shown to produce similar results. SIST ISO 16000-17:2013
ISO 16000-17:2008(E) 4 © ISO 2008 – All rights reserved 6.1 Dicloran 18 % glycerol agar (DG18 agar) The components are listed in Table 1. Table 1 — Composition of dicloran 18 % glycerol agar (DG18 agar) Component Quantity Peptone 4) 5,0 g Glucose 10,0 g Potassium dihydrogen phosphate (KH2PO4) 1,0 g Magnesium sulfate heptahydrate (MgSO4·7H2O) 0,5 g Dicloran (2,6-dichloro-4-nitroaniline) 0,2 % volume fraction in ethanol 1,0 ml a Chloramphenicol 0,1 g Glycerol 220 g b Agar 15,0 g Water 1 000 ml a Final concentration in medium: 0,002 g/l. b 18 % mass fraction of ~1 220 g final mass = ~220 g.
Add minor ingredients and agar to ~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. 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 fungi from growing on this agar. NOTE DG18 agar is suitable for the detection of a wide spectrum of xerophilic (i.e. preferring dryness) fungi. Glycerol reduces the water activity, aH2O, to 0,95. Chloramphenicol inhibits bacteria, especially gram-negative bacteria. Dicloran inhibits the spread of fast-growing mould colonies and thus prevents overgrowing of slow-growing colonies. 6.2 Malt-extract agar The components are listed in Table 2. Table 2 — Composition of malt-extract agar Component Quantity Malt extract 30,0 g Peptone from soy 3,0 g Agar 15,0 g Water 1 000 ml
4) 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 for comparison of recovery and of morphological appearance of the colonies are, therefore, important. SIST ISO 16000-17:2013
ISO 16000-17:2008(E) © ISO 2008 – All rights reserved 5NOTE The addition of Chloramphenicol (0,05 g/l) may be necessary if samples contain high concentrations of bacteria. This is usually not the case for samples of indoor air but bacteria may be present in high numbers in material and dust samples. Add ingredients and agar in the water and dissolve by boiling. Sterilise in an autoclave at (115 ± 3) °C for (10 ± 1) min. After sterilisation, the pH shall correspond to 5,5 ± 0,2 at 25 °C. Dispense aliquots of about 20 ml into Petri dishes. Plates of malt-extract agar in bags will keep for up to one month at (5 ± 3) °C in the dark. Many commercial malt-extract agars with different compositions are available. Ensure that the ingredients correspond to the composition given above. 6.3 Potato dextrose agar The components are listed in Table 3. Table 3 — Composition of potato dextrose agar Component Quantity Potato extract 4,0 g Glucose 20,0 g Agar 15,0 g Water 1 000 ml
NOTE The addition of chloramphenicol (0,05 g/l) may be necessary if samples contain high concentrations of bacteria. Add ingredients and agar to the water and dissolve by boiling. Sterilise in an autoclave at (115 ± 3) °C for (10 ± 1) min. After sterilisation, the pH shall correspond to 5,6 ± 0,2 at 25 °C. Dispense aliquots of about 20 ml into Petri dishes. Plates of potato dextrose agar in bags will keep for up to one month at (5 ± 3) °C in the dark. 6.4 Saline solution The components are listed in Table 4. Table 4 — Composition of saline solution Component Quantity Sodium chloride (NaCl) 8,5 g Water 1 000 ml
Dissolve the NaCl in the water and sterilise in an autoclave at (121 ± 3) °C for (15 ± 1) min. 6.5 Saline solution with polysorbate 80 The components are listed in Table 5. SIST ISO 16000-17:2013
ISO 16000-17:2008(E) 6 © ISO 2008 – All rights reserved Table 5 — Composition of saline solution with polysorbate 80 Component Quantity Sodium chloride (NaCl) 8,5 g Polysorbate 80 0,1 g Water 1 000 ml
Dissolve the NaCl in the water and sterilise in an autoclave at (121 ± 3) °C for (15 ± 1) min. Allow to cool and add the polysorbate 80. 7 Procedure 7.1 General Samples to be analysed are either agar plates from sampling by impaction according to ISO 16000-18 or filters from sampling by filtration according to ISO 16000-16. Samples from direct plating or material suspensions can be treated accordingly. 7.1.1 Samples from impaction Agar plates are directly incubated (see 7.3). 7.1.2 Samples from filtration Filters are resuspended and aliquots spread on agar plates (see 7.2) followed by incubation (see 7.3). Process the samples in the laboratory preferably without delay, but not later than 48 h after the end of sampling. Store samples in the laboratory in the dark at a temperature not exceeding the incubation temperature (< 25 °C), protected against adverse influences (humidity, desiccation, contamination). Document the storage conditions. Carry out all procedures under conditions that protect the samples from any contamination. Check aseptic conditions regularly by controls and results shall be documented. 7.2 Processing of filters 7.2.1 General Airborne moulds deposited on filters are processed using the indirect plating method. NOTE Aggregates of either spores or aggregates of particles may be dissolved by suspension as well as by dilution, which may result in higher numbers of colonies after incubation. An aggregate of 30 spores may result in 30 colonies under these circumstances. By contrast, a reduction of the number of colonies may occur if the detachment of the mould spores from the filters is incomplete or by damage of the fungal cells during processing in the laboratory. 7.2.2 Resuspension In the aseptic atmosphere of a safety cabinet (5.6), transfer the filters (gelatin and polycarbonate filter) into a sterile container containing 5 ml of saline solution with polysorbate 80 (see 6.5) using a sterile pair of tweezers. Shake (5.8) the filters steeped in this solution in their container intensively in a horizontal position in a water bath maintained at 35 °C to 40 °C (5.7) for 15 min. Make sure that the spore-loaded surface of the filter lies flat and faces upwards and can move freely within the suspension during shaking. SIST ISO 16000-17:2013
ISO 16000-17:2008(E) © ISO 2008 – All rights reserved 7Process the sample according to 7.2.3 within 1 h after suspension. 7.2.3 Dilution Based on the original suspension, set up a serial dilution series. Immediately prior to dilution, shake the suspension for 1 min on a test tube shaker (5.8). Add 1 ml of the suspension to 9 ml saline solution (6.4) using a sterile disposable pipette or a cotton-stuffed glass pipette. In the same way, carry out two further dilution steps resulting in dilutions 1:10, 1:100, and 1:1 000. The number of dilution steps and the dilution intervals should be adapted to the expected mould concentration and the specific measurement task. It may be necessary to set up additional dilution steps. 7.2.4 Plating Plate 0,1 ml of the original suspension and the dilutions (7.2.3) in parallel on DG18 agar (6.1) as well as on malt-extract agar (6.2) or potato dextrose agar (6.3). Use at least two parallel plates for each dilution and incubation temperature (7.3). If, according to the sampling protocol, low concentrations of moulds are expected, 1 ml of the original solution can be plated on four plates for each agar medium (using 250 µl per plate) to enhance the sensitivity of the method. Determine laboratory blank samples for all dilution steps. 7.3 Incubation Incubate agar plates upside up in incubators at (25 ± 3) °C for 7 days. DG18 agar plates may require prolonged incubation times — 10 days — especially if differentiation of fungi is anticipated. For special purposes (e.g. thermotolerant Aspergillus spp.), malt-extract agar plates or potato dextrose agar plates may additionally be incubated at (36 ± 2) °C. For selective cultivation of Aspergillus fumigatus, incubation temperatures of (45 ± 2) °C are used. CAUTION — At incubation temperatures above 25 °C, take special care to avoid desiccation of the agar plates. Incubate agar plates in such way that sufficient oxygen will allow optimal mould growth, e.g. avoid incubation in tight polyethylene bags. Incubate agar plates in a vibration-free incubator, to minimize the risk of secondary colony inoculation due to spreading of spores. Also avoid extensive airflows in view of desiccation of the agar plates. 7.4 Examination and counting Examine agar plates for the first time after 2 days to 3 days and subsequently in regular intervals for up to at least 7 days. Count thermotolerant moulds (36 °C or 45 °C) after 1 day to 3 days, as they grow very rapidly. Handling of the agar plates may lead to distribution of spores on the plate resulting in secondary colonies in the course of incubation. Take care not to count secondary colonies. The optimal range for genus/species identification and quantification using a standard culture plate with a diameter of ~90 mm is between 20 and 40 colonies. For quantitative results, at least 10 colonies of the respective genus/species should be on the agar plate and a maximum number of 100 colonies in total. Some moulds may spread very fast inhibiting the growth of other colonies (e.g. Rhizopus, Chrysonilia, Mucor, Botrytis) — even on plates containing dicloran — and the capacity of the plate can run down even at lower colony counts. SIST ISO 16000-17:2013
ISO 16000-17:2008(E) 8 © ISO 2008 – All rights reserved Record the maximum number of colonies counted within the 7 days of incubation for each agar medium and sampling volume (impaction) or dilution (filtration), respectively. 7.5 Identification of mould species Identification of the moulds growing on the agar plates is essential for most questions concerning mould problems in indoor environments. Identification is based on macroscopic and microscopic morphological characteristics. These characteristics are usually better identified on malt-extract agar or potato dextrose agar compared to DG18 agar. NOTE Malt-extract agar and potato dextrose agar have been shown to give comparable results for identification of mould genera and species. The extent of identification depends on the purpose of the investigation. To detect sources of mould growth indoors, it is important to recognise differences in species/genera composition between indoors and outdoors as well as moisture indicators e.g. Aspergillus versicolor or Chaetomium spp. Identification to species level within certain genera (e.g. Phialophora, Trichoderma, Acremonium, Chaetomium, Penicillium and Fusarium) is not recommended for this purpose as it is very difficult and scarcely achievable in routine analysis. Detailed species identification may, however, be important for investigations related to health problems. In this case, species identification should be anticipated even fo
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NORME ISO
INTERNATIONALE 16000-17
Première édition
2008-12-15
Air intérieur —
Partie 17:
Détection et dénombrement des
moisissures — Méthode par culture
Indoor air —
Part 17: Detection and enumeration of moulds — Culture-based method
Numéro de référence
ISO 16000-17:2008(F)
©
ISO 2008
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ISO 16000-17:2008(F)
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ISO 16000-17:2008(F)
Sommaire Page
Avant-propos. iv
Introduction . vii
1 Domaine d'application. 1
2 Références normatives . 1
3 Termes et définitions. 1
4 Principe. 3
5 Appareillage . 3
6 Milieux de culture et diluants . 3
6.1 Gélose dichloran-glycérol 18 % (Gélose DG18) . 4
6.2 Gélose à l'extrait de malt. 4
6.3 Gélose dextrosée à la pomme de terre. 5
6.4 Solution saline . 5
6.5 Solution saline au polysorbate 80. 5
7 Mode opératoire . 6
7.1 Généralités . 6
7.2 Traitement des filtres. 6
7.3 Incubation. 7
7.4 Examen et dénombrement. 7
7.5 Identification des espèces de moisissures. 8
7.6 Calcul et expression des résultats. 9
8 Rapport d'essai . 12
Annexe A (informative) Caractéristiques spécifiques des spores de moisissures. 13
Annexe B (informative) Échange d'échantillons pour validation de la méthode de culture . 16
Bibliographie . 21
© ISO 2008 – Tous droits réservés iii
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ISO 16000-17:2008(F)
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes nationaux de
normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est en général confiée
aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude a le droit de faire partie du
comité technique créé à cet effet. Les organisations internationales, gouvernementales et non
gouvernementales, en liaison avec l'ISO participent également aux travaux. L'ISO collabore étroitement avec
la Commission électrotechnique internationale (CEI) en ce qui concerne la normalisation électrotechnique.
Les Normes internationales sont rédigées conformément aux règles données dans les Directives ISO/CEI,
Partie 2.
La tâche principale des comités techniques est d'élaborer les Normes internationales. Les projets de Normes
internationales adoptés par les comités techniques sont soumis aux comités membres pour vote. Leur
publication comme Normes internationales requiert l'approbation de 75 % au moins des comités membres
votants.
L'attention est appelée sur le fait que certains des éléments du présent document peuvent faire l'objet de
droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable de ne
pas avoir identifié de tels droits de propriété et averti de leur existence.
L'ISO 16000-17 a été élaborée par le comité technique ISO/TC 146, Qualité de l'air, sous-comité SC 6, Air
intérieur.
L'ISO 16000 comprend les parties suivantes, présentées sous le titre général Air intérieur:
⎯ Partie 1: Aspects généraux de la stratégie d'échantillonnage
⎯ Partie 2: Stratégie d'échantillonnage du formaldéhyde
⎯ Partie 3: Dosage du formaldéhyde et d'autres composés carbonylés — Méthode par échantillonnage actif
⎯ Partie 4: Dosage du formaldéhyde — Méthode par échantillonnage diffusif
⎯ Partie 5: Stratégie d'échantillonnage pour les composés organiques volatils (COV)
⎯ Partie 6: Dosage des composés organiques volatils dans l'air intérieur des locaux et enceintes d'essai par
®
échantillonnage actif sur le sorbant Tenax TA , désorption thermique et chromatographie en phase
gazeuse utilisant MS/FID
⎯ Partie 7: Stratégie d'échantillonnage pour la détermination des concentrations en fibres d'amiante en
suspension dans l'air
⎯ Partie 8: Détermination des âges moyens locaux de l'air dans des bâtiments pour caractériser les
conditions de ventilation
⎯ Partie 9: Dosage de l'émission de composés organiques volatils de produits de construction et d'objets
d'équipement — Méthode de la chambre d'essai d'émission
⎯ Partie 10: Dosage de l'émission de composés organiques volatils de produits de construction et d'objets
d'équipement — Méthode de la cellule d'essai d'émission
iv © ISO 2008 – Tous droits réservés
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ISO 16000-17:2008(F)
⎯ Partie 11: Dosage de l'émission de composés organiques volatils de produits de construction et d'objets
d'équipement — Échantillonnage, conservation des échantillons et préparation d'échantillons pour essai
⎯ Partie 12: Stratégie d'échantillonnage des polychlorobiphényles (PCB), des polychlorodibenzo-p-dioxines
(PCDD), des polychlorodibenzofuranes (PCDF) et des hydrocarbures aromatiques polycycliques (HAP)
⎯ Partie 13: Dosage des polychlorobiphényles (PCB) de type dioxine et des polychlorodibenzo-p-dioxines
(PCDD)/polychlorodibenzofuranes (PCDF) totaux (en phase gazeuse et en phase particulaire) —
Collecte sur des filtres adsorbants
⎯ Partie 14: Dosage des polychlorobiphényles (PCB) de type dioxine et des polychlorodibenzo-p-dioxines
(PCDD)/polychlorodibenzofuranes (PCDF) totaux (en phase gazeuse et en phase particulaire) —
Extraction, purification et analyse par chromatographie en phase gazeuse haute résolution et
spectrométrie de masse
⎯ Partie 15: Stratégie d'échantillonnage du dioxyde d'azote (NO )
2
⎯ Partie 16: Détection et dénombrement de moisissures — Échantillonnage par filtration
⎯ Partie 17: Détection et dénombrement de moisissures — Méthode par culture
⎯ Partie 23: Essai de performance pour l'évaluation de la réduction des concentrations en formaldéhyde
par des matériaux de construction sorptifs
⎯ Partie 24: Essai de performance pour l'évaluation de la réduction des concentrations en composés
organiques volatils et en composés carbonyles sans formaldéhyde, par des matériaux de construction
sorptifs
Les parties suivantes sont en cours de préparation:
⎯ Partie 18: Détection et dénombrement de moisissures — Échantillonnage par impaction
⎯ Partie 19: Stratégie d'échantillonnage des moisissures
⎯ Partie 25: Détermination de l'émission de composés organiques semi-volatiles des produits de
construction — Méthode de la micro chambre
⎯ Partie 28: Évaluation sensorielle des émissions des matériaux et des produits de construction
Les parties suivantes sont prévues:
⎯ Partie 20: Détection et dénombrement des moisissures — Échantillonnage à partir de poussières
domestiques
⎯ Partie 21: Détection et dénombrement des moisissures — Échantillonnage à partir de matériaux
⎯ Partie 22: Détection et dénombrement des moisissures — Méthodes moléculaires
⎯ Partie 27: Méthode normalisée pour l'analyse quantitative des fibres d'amiante dans la poussière
En outre,
⎯ l'ISO 12219-1 (en préparation), Air intérieur — Véhicules routiers — Partie 1: Enceinte d'essai d'un
véhicule complet — Spécification et méthode de détermination des composés organiques volatils dans
les habitacles de voitures
© ISO 2008 – Tous droits réservés v
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ISO 16000-17:2008(F)
⎯ l'ISO 16017-1, Air intérieur, air ambiant et air des lieux de travail — Échantillonnage et analyse des
composés organiques volatils par tube à adsorption/désorption thermique/chromatographie en phase
gazeuse sur capillaire — Partie 1: Échantillonnage par pompage
⎯ l'ISO 16017-2, Air intérieur, air ambiant et air des lieux de travail — Échantillonnage et analyse des
composés organiques volatils par tube à adsorption/désorption thermique/chromatographie en phase
gazeuse sur capillaire — Partie 2: Échantillonnage par diffusion
traitent plus particulièrement des mesurages liés aux composés organiques volatils (COV).
vi © ISO 2008 – Tous droits réservés
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ISO 16000-17:2008(F)
Introduction
Le terme «moisissure» est le nom commun des champignons filamenteux appartenant à différents groupes
taxonomiques [Zygomycètes, Ascomycètes (Ascomycota) et Deutéromycètes]. Ils forment un mycélium
(hyphes) et des spores, à savoir des conidiospores (conidies), des sporangiospores ou des ascospores, qui
les rendent visibles à l'œil nu. La plupart des spores mesurent de 2 µm à 10 µm, certaines atteignent 30 µm et,
dans de rares cas, certaines peuvent mesurer jusqu'à 100 µm. Les spores de certains genres de moisissure
sont de petite taille et se mettent facilement en suspension dans l'air (par exemple, Aspergillus, Penicillium)
tandis que d'autres sont plus grandes et/ou intégrées à une matrice visqueuse (Stachybotrys, Fusarium), ce
qui les rend moins mobiles.
Les spores de moisissures sont disséminées un peu partout dans l'environnement extérieur et se retrouvent
ainsi en concentration variable à l'intérieur des bâtiments. Il convient toutefois de considérer la croissance des
moisissures dans les environnements intérieurs comme un problème d'hygiène. En effet, des études
épidémiologiques ont montré que l'humidité et/ou la croissance des moisissures dans les logements sont
étroitement liées aux problèmes de santé affectant les habitants.
L'existence de méthodes normalisées pour l'échantillonnage, la détection et le dénombrement des
moisissures, y compris des normes relatives à des stratégies d'échantillonnage, est importante pour
l'évaluation comparative des problèmes liés aux moisissures à l'intérieur des bâtiments. Avant de procéder à
tout mesurage, une stratégie d'échantillonnage est nécessaire.
[5]
Le mode opératoire spécifié dans la présente partie de l'ISO 16000 repose sur les guides VDI 4253-2 et
[6]
VDI 4300-10 .
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NORME INTERNATIONALE ISO 16000-17:2008(F)
Air intérieur —
Partie 17:
Détection et dénombrement des moisissures — Méthode par
culture
AVERTISSEMENT — La présente partie de l'ISO 16000 peut impliquer l'utilisation de produits et la
mise en œuvre de modes opératoires et d'appareillages à caractère dangereux. La présente partie de
l'ISO 16000 n'a pas pour but d'aborder tous les problèmes de sécurité liés à son utilisation. Il incombe
à l'utilisateur de la présente partie de l'ISO 16000 d'établir des pratiques d'hygiène et de sécurité
appropriées et de déterminer l'applicabilité des restrictions réglementaires, avant utilisation.
1 Domaine d'application
La présente partie de l'ISO 16000 spécifie une méthode de détection et de dénombrement des moisissures
par culture après échantillonnage par impaction, selon l'ISO 16000-18, ou par filtration, selon l'ISO 16000-16.
Elle convient également à la culture des moisissures à partir de matières en suspension ou de culture directe
sur boîte de Petri.
2 Références normatives
Les documents de référence suivants sont indispensables pour l'application du présent document. Pour les
références datées, seule l'édition citée s'applique. Pour les références non datées, la dernière édition du
document de référence s'applique (y compris les éventuels amendements).
ISO 8199, Qualité de l'eau — Lignes directrices générales pour le dénombrement des micro-organismes sur
milieu de culture
ISO 16000-16, Air intérieur — Partie 16: Détection et dénombrement des moisissures — Échantillonnage par
filtration
ISO 16000-18, Air intérieur — Partie 18: Détection et dénombrement des moisissures — Échantillonnage par
1)
impaction
3 Termes et définitions
Pour les besoins du présent document, les termes et définitions suivants s'appliquent.
3.1
champignon filamenteux
champignon poussant sous la forme de filaments de cellules connus sous le nom de hyphes
NOTE 1 Les hyphes agrégées en faisceaux sont appelées mycélia.
NOTE 2 Le terme «champignon filamenteux» distingue les champignons à hyphes des levures.
[ISO 16000-16:2008]
1) À publier.
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ISO 16000-17:2008(F)
3.2
filtration
prélèvement de particules en suspension dans un gaz ou dans un liquide par passage à travers un milieu
poreux
[4]
[EN 13098:2000 ]
NOTE Dans la présente partie de l'ISO 16000, la filtration désigne la séparation des micro-organismes ou des
moisissures d'un volume défini d'air au moyen de filtres.
3.3
méthode indirecte
〈qualité de l'air〉 remise en suspension des micro-organismes sédimentés suivie de la mise en culture des
aliquotes sur un milieu de culture adapté, de l'incubation et du dénombrement des colonies se développant
dans les conditions choisies
3.4
unité formant colonie
ufc
unité exprimant le nombre de micro-organismes cultivables
[4]
[EN 13098:2000 ]
NOTE 1 Une unité formant colonie peut provenir d'un seul micro-organisme, d'agrégats de nombreux
micro-organismes ou d'un ou plusieurs micro-organismes liés à une particule.
NOTE 2 Le nombre de colonies formées dépend des conditions de culture.
3.5
culture
〈qualité de l'air〉 croissance de micro-organismes sur milieux de culture
[ISO 16000-16:2008]
3.6
micro-organisme
toute entité microbiologique, cellulaire ou non, capable de se répliquer ou de transférer son matériel génétique
ou entité ayant perdu ces propriétés
[4]
[EN 13098:2000 ]
3.7
indicateur d'humidité
〈qualité de l'air〉 moisissure en environnements extérieurs dont la croissance est favorisée par une humidité
relativement élevée et indiquant donc l'existence de problèmes d'humidité
3.8
colonie secondaire
colonie qui ne provient pas du prélèvement initial de spores en suspension dans l'air mais de spores libérées
par une colonie se développant sur les boîtes de gélose
3.9
moisissure
〈qualité de l'air〉 champignon filamenteux appartenant à différents groupes taxonomiques, à savoir
Zygomycètes, Ascomycètes (Ascomycota) et Deutéromycètes (champignons imparfaits)
NOTE Les moisissures forment différents types de spores selon le groupe taxonomique auquel elles appartiennent, à
savoir des conidiospores (conidies), des sporangiospores ou des ascospores.
[ISO 16000-16:2008]
2 © ISO 2008 – Tous droits réservés
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ISO 16000-17:2008(F)
4 Principe
Des boîtes de gélose (gélose DG18 et gélose à l'extrait de malt ou gélose dextrosée à la pomme de terre)
obtenues par échantillonnage par impaction sont directement incubées à (25 ± 3) °C.
Les filtres issus du prélèvement par filtration sont remis en suspension en solution saline (à 0,9 % en fraction
2)
massique de NaCl) avec 0,01 % de polysorbate 80 . Des dilutions décimales de la suspension sont
préparées et les aliquotes étalées sur gélose DG18 et sur gélose à l'extrait de malt ou sur gélose dextrosée à
la pomme de terre (méthode indirecte). Les boîtes de gélose sont incubées à (25 ± 3) °C. Pour des
applications spécifiques, il est possible d'incuber les boîtes à (36 ± 2) °C (par exemple, pour Aspergillus spp.,
thermotolérant) ou à (45 ± 2) °C (Aspergillus fumigatus).
Après incubation, les colonies de moisissures sont identifiées et dénombrées. Le niveau d'identification
dépend de l'objectif de l'étude.
5 Appareillage
Matériel courant de laboratoire de microbiologie et, en particulier, ce qui suit.
5.1 Incubateur, sans vibrations, thermostaté à (25 ± 3) °C.
5.2 Incubateur, sans vibrations, thermostaté à (36 ± 2) °C.
5.3 Incubateur, sans vibrations, thermostaté à (45 ± 2) °C.
5.4 Réfrigérateur, thermostaté à (5 ± 3) °C.
5.5 pH-mètre, d'une précision de ± 0,1 unité de pH.
5.6 Enceinte de sécurité microbiologique, de classe II, destinée à la protection de l'utilisateur et des
produits.
5.7 Bain-marie, pouvant être maintenu entre 35 °C et 40 °C avec agitateur.
3)
5.8 Agitateur secoueur pour tubes à essai, par exemple agitateur Vortex .
5.9 Boîtes de Petri, aérées, stériles, d'environ 90 mm de diamètre.
5.10 Autoclave, capable de fonctionner à (115 ± 3) °C et à (121 ± 3) °C.
6 Milieux de culture et diluants
Utiliser des réactifs de qualité analytique reconnue, sauf si des réactifs de qualité différente ont démontré
qu'ils donnent les mêmes résultats, et utiliser uniquement de l'eau distillée ou déionisée ou de l'eau de pureté
équivalente.
Il est conseillé d'utiliser des substrats déshydratés disponibles dans le commerce, à condition qu'ils soient
conformes à la description donnée. Ils doivent être préparés conformément aux instructions du fabricant.
2) Le polysorbate 80 est l’équivalent du monooléate de polyoxyéthylène sorbitane ou monooléate de polyéthylène glycol
sorbitane. Le Tween est un exemple de produit adapté disponible dans le commerce. Cette information est donnée à titre
indicatif aux utilisateurs de la présente partie de l’ISO 16000 et ne constitue en aucun cas une recommandation de ce
produit par l’ISO.
3) Exemple de produit approprié disponible sur le marché. Cette information est donnée à l'intention des utilisateurs de
la présente partie de l'ISO 16000 et ne signifie nullement que l'ISO approuve ou recommande l'emploi exclusif du produit
ainsi désigné. Des produits équivalents peuvent être utilisés s'il est démontré qu'ils conduisent aux mêmes résultats.
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ISO 16000-17:2008(F)
6.1 Gélose dichloran-glycérol 18 % (Gélose DG18)
Les composants sont listés dans le Tableau 1.
Tableau 1 — Composition de la gélose dichloran-glycérol 18 % (Gélose DG 18)
Composant Quantité
)
4
Peptone 5,0 g
Glucose 10,0 g
Dihydrogénophosphate de potassium (KH PO) 1,0 g
2 4
Sulfate de magnésium heptahydraté (MgSO ,7HO) 0,5 g
4 2
a
Dichloran (2,6-dichloro-4-nitroaniline) à 0,2 % en fraction volumique dans l'éthanol 1,0 ml
Chloramphénicol 0,1 g
b
Glycérol 220 g
Gélose 15,0 g
Eau 1 000 ml
a
Concentration finale du milieu: 0,002 g/l.
b
18 % en fraction massique de la masse finale d'environ 1 220 g, soit environ 220 g.
Ajouter les ingrédients en quantité minoritaire et la gélose dans environ 800 ml d'eau et dissoudre par
ébullition. Compléter à 1 000 ml et ajouter 220 g de glycérol. Stériliser dans un autoclave à (121 ± 3) °C
pendant (15 ± 1) min. Après stérilisation, le pH doit être de 5,6 ± 0,2 à 25 °C. Répartir des aliquotes d'environ
20 ml dans des boîtes de Petri.
Placées dans des sachets, les boîtes de gélose DG18 se conservent une semaine à (5 ± 3) °C et à l'obscurité.
La gélose DG18 présente une activité de l'eau réduite définie. Veiller à ne pas réduire davantage l'activité de
l'eau par dessiccation au risque d'empêcher la croissance des champignons sur cette gélose.
NOTE La gélose DG18 convient à la détection d'un large spectre de champignons xérophiles (c'est-à-dire qui
préfèrent la sécheresse). Le glycérol réduit l'activité de l'eau a à 0,95. Le chloramphénicol inhibe les bactéries, en
H O
2
particulier les bactéries Gram négatives. Le dichloran inhibe la prolifération des colonies des moisissures à croissance
rapide et empêche ainsi l'envahissement par les colonies à croissance lente.
6.2 Gélose à l'extrait de malt
Les composants sont listés dans le Tableau 2.
Tableau 2 — Composition de la gélose à l'extrait de malt
Composant Quantité
Extrait de malt 30,0 g
Peptone de soja 3,0 g
Gélose 15,0 g
Eau 1 000 ml
NOTE L'ajout de chloramphénicol (à 0,05 g/l) peut être nécessaire si les échantillons contiennent de fortes
concentrations de bactéries. Ce n'est généralement pas le cas des échantillons d'air intérieur mais les bactéries peuvent
être présentes en grand nombre dans les échantillons de matériaux et de poussières.
4) Différentes peptones sont utilisées par différents fabricants (par exemple, la peptone de caséine, la peptone
mycologique). Cela n’a habituellement aucune influence sur les résultats quantitatifs des mesurages, mais peut avoir une
influence sur l’apparence des colonies. Des contrôles positifs de comparaison de rétablissement et d’apparence
morphologique des colonies sont, par conséquent, importants.
4 © ISO 2008 – Tous droits réservés
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ISO 16000-17:2008(F)
Ajouter les ingrédients et la gélose dans l'eau et dissoudre par ébullition. Stériliser dans un autoclave à
(115 ± 3) °C pendant (10 ± 1) min. Après stérilisation, le pH doit être de 5,5 ± 0,2 à 25 °C. Répartir des
aliquotes d'environ 20 ml dans des boîtes de Petri.
Placées dans des sachets, les boîtes de gélose à l'extrait de malt se conservent un mois à (5 ± 3) °C et à
l'obscurité.
De nombreuses géloses à l'extrait de malt de différentes compositions sont disponibles dans le commerce.
Vérifier que les ingrédients correspondent à la composition indiquée ci-dessus.
6.3 Gélose dextrosée à la pomme de terre
Les composants sont listés dans le Tableau 3.
Tableau 3 — Composition de la gélose dextrosée à la pomme de terre
Composant Quantité
Extrait de pomme de terre 4,0 g
Glucose 20,0 g
Gélose 15,0 g
Eau 1 000 ml
NOTE L'ajout de chloramphénicol (à 0,05 g/l) peut être nécessaire si les échantillons contiennent de fortes
concentrations de bactéries.
Ajouter les ingrédients et la gélose dans l'eau et dissoudre par ébullition. Stériliser dans un autoclave à
(115 ± 3) °C pendant (10 ± 1) min. Après stérilisation, le pH doit être de 5,6 ± 0,2 à 25 °C. Répartir des
aliquotes d'environ 20 ml dans des boîtes de Petri.
Placées dans des sachets, les boîtes de gélose dextrosée à la pomme de terre se conservent un mois à
(5 ± 3) °C et à l'obscurité.
6.4 Solution saline
Les composants sont listés dans le Tableau 4.
Tableau 4 — Composition de la solution saline
Composant Quantité
Chlorure de sodium (NaCl) 8,5 g
Eau 1 000 ml
Dissoudre le NaCl dans l'eau et stériliser dans un autoclave à (121 ± 3) °C pendant (15 ± 1) min.
6.5 Solution saline au polysorbate 80
Les composants sont listés dans le Tableau 5.
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ISO 16000-17:2008(F)
Tableau 5 — Composition de la solution saline au polysorbate 80
Composant Quantité
Chlorure de sodium (NaCl) 8,5 g
Polysorbate 80 0,1 g
Eau 1 000 ml
Dissoudre le NaCl dans l'eau et stériliser dans un autoclave à (121 ± 3) °C pendant (15 ± 1) min. Laisser
refroidir et ajouter le polysorbate 80.
7 Mode opératoire
7.1 Généralités
Les échantillons à analyser sont soit des boîtes de gélose issues de l'échantillonnage par impaction, selon
l'ISO 16000-18, soit des filtres obtenus lors de l'échantillonnage par filtration, selon l'ISO 16000-16. Les
échantillons issus de mise en culture directe ou de la mise en suspension de matériaux peuvent être traités
en conséquence.
7.1.1 Échantillons obtenus par impaction
Les boîtes de gélose sont incubées directement (voir 7.3).
7.1.2 Échantillons obtenus par filtration
Les filtres sont remis en suspension et des aliquotes sont réparties dans des boîtes de gélose (voir 7.2) qui
sont ensuite incubées (voir 7.3).
Traiter les échantillons au laboratoire, de préférence, sans délai et en tout cas dans les 48 h suivant la fin de
l'échantillonnage. Conserver les échantillons au laboratoire, à l'obscurité, à une température ne dépassant
pas la température d'incubation (< 25 °C), à l'abri des conditions défavorables (humidité, dessiccation,
contamination). Consigner les conditions de conservation.
Suivre tous les modes opératoires dans des conditions empêchant la contamination des échantillons. Vérifier
régulièrement les conditions aseptiques par des contrôles dont les résultats doivent être consignés.
7.2 Traitement des filtres
7.2.1 Généralités
Les moisissures en suspension dans l'air déposées sur les filtres sont traitées par la méthode de mise en
culture indirecte.
NOTE Les agrégats de spores ou de particules peuvent être dissous par mise en suspension ou par dilution, ce qui
peut entraîner un plus grand nombre de colonies après incubation. Dans ces circonstances, un agrégat de 30 spores peut
donner lieu à 30 colonies. À l'inverse, une réduction du nombre de colonies peut se produire si le détachement des spores
de moisissures des filtres est incomplet ou si les cellules de champignons sont endommagées au cours du traitement en
laboratoire.
7.2.2 Remise en suspension
Sous atmosphère aseptique d'une enceinte de sécurité (5.6), transférer les filtres (filtre en gélatine et en
polycarbonate) dans un récipient stérile contenant 5 ml de solution saline au polysorbate 80 (voir 6.5) à l'aide
d'une paire de pinces stériles.
6 © ISO 2008 – Tous droits réservés
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ISO 16000-17:2008(F)
Agiter (5.8) vigoureusement les filtres dans cette solution, en position horizontale, dans un bain-marie (5.7)
maintenu à une température entre 35 °C et 40 °C, pendant 15 min. S'assurer que la surface du filtre chargée
en spores est à plat et orientée vers le haut et que le filtre peut se déplacer librement dans la suspension lors
de l'agitation.
Traiter l'échantillon conformément à 7.2.3 dans l'heure suivant la suspension.
7.2.3 Dilution
À partir de la suspension initiale, préparer une série de dilutions.
Immédiatement avant la dilution, agiter la suspension pendant 1 min sur un agitateur secoueur pour tubes à
essai (5.8). Ajouter 1 ml de suspension à 9 ml de solution saline (6.4) à l'aide d'une p
...
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