ISO 16000-22:2025
(Main)Indoor air — Part 22: Detection and quantification of fungal biomass by fungal β-N-acetylhexosaminidase enzyme activity
Indoor air — Part 22: Detection and quantification of fungal biomass by fungal β-N-acetylhexosaminidase enzyme activity
This document specifies requirements for the sampling and analysis of air, surface or bulk material samples analysed by fluorometric detection of an enzyme activity present in filamentous fungi[1] (US Patent No. 6,372,446) to quantitatively determine the total fungal biomass density. It describes the analytical procedure that can be performed on-site or in a laboratory. This method does not enumerate or differentiate genera or species of fungi.
Air intérieur — Partie 22: Détection et quantification de la biomasse fongique par caractérisation de l’activité de l'enzyme fongique β-N-acétyl-hexosaminidase
General Information
Standards Content (Sample)
International
Standard
ISO 16000-22
First edition
Indoor air —
2025-02
Part 22:
Detection and quantification of
fungal biomass by fungal β-N-
acetylhexosaminidase enzyme
activity
Air intérieur —
Partie 22: Détection et quantification de la biomasse fongique
par caractérisation de l’activité de l'enzyme fongique β-N-acétyl-
hexosaminidase
Reference number
© ISO 2025
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle of enzyme-targeted fluorescent detection . 2
5 Apparatus and materials . 3
6 Reagents . 4
7 Sampling . 4
7.1 Surface sampling.4
7.2 Porous materials .4
7.2.1 High-density materials (e.g. cementitious materials, such as concrete, plaster,
mortar) .4
7.2.2 Low-density materials (e.g. insulation materials such as glass wool or mineral
wool).4
7.3 Air sampling .5
8 Preparation of the fluorometer . 5
9 Analysis . . 5
9.1 General .5
9.2 Surface samples .5
9.3 Analysis of porous materials (both high- and low-density materials) .5
9.4 Analysis of air samples .6
Annex A (informative) Reaction time and temperature correction and example data
interpretation categories . 7
Annex B (informative) Activated sampling — Protocol for air sampling .10
Bibliography .12
iii
Foreword
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iv
Introduction
Fungi from different taxonomic groups form filamentous cells (mycelium) and asexual spores (conidia). 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
fungi 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.
Fungi spores are widely distributed in the outdoor environment and, therefore, also occur in varying
concentrations indoors. The growth of fungi in indoor environments, however, is considered to be a hygiene
problem because epidemiological studies have revealed that dampness and/or mould growth in homes is
closely related to health problems affecting the occupants.
Harmonized methods for sampling, detection and enumeration of moulds, including standards for sampling
strategies, are important for the comparative assessment of indoor mould problems. Before doing any
[14]
measurements, a plan for the measurement strategy (on the basis of ISO 16000-19 ) should be made.
This document describes the measurement of fungal material by enzymatic biochemical analysis.
It describes a rapid quantitative method to determine the total fungal material in air, on surfaces or
in material samples by measuring a naturally occurring enzyme found in the chitinolytic system of all
[1][2][3][4][5][6][7][8][9][10]11]
filamentous fungi (β-N-acetylhexosaminidase or NAHA (EC 3.2.1.52)).
It describes the analytical procedure that can be performed on-site or in a laboratory and refers to applicable
sampling procedures for air, surfaces and material samples.
This method does not enumerate or differentiate genera or species of fungi such as those found in
[12] [13] [15]
ISO 16000-17 , ISO 16000-18 and ISO 16000-20 .
It is a quantitative method used to rapidly assess conditions found in indoor spaces or post-remediation.
v
International Standard ISO 16000-22:2025(en)
Indoor air —
Part 22:
Detection and quantification of fungal biomass by fungal β-N-
acetylhexosaminidase enzyme activity
1 Scope
This document specifies requirements for the sampling and analysis of air, surface or bulk material samples
[1]
analysed by fluorometric detection of an enzyme activity present in filamentous fungi (US Patent
No. 6,372,446) to quantitatively determine the total fungal biomass density. It describes the analytical
procedure that can be performed on-site or in a laboratory. This method does not enumerate or differentiate
genera or species of fungi.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
enzyme
substance, produced by living cells that acts as a catalyst to bring about a specific biochemical reaction
3.2
filamentous fungus
fungus growing in the form of filaments of cells known as hyphae
Note 1 to entry: The term “filamentous fungi” differentiates fungi with hyphal growth from yeasts.
[15]
[SOURCE: ISO 16000-20:2014, 2.3 ]
3.3
mould
filamentous fungi (3.2) from several taxonomic groups; namely Ascomycota, Basidiomycota,
Mucoromycota, and their asexual states
Note 1 to entry: Mould form different types of spores depending on the taxonomic group they belong to, namely
conidiospores (conidia), sporangiospores, basidiospores or ascospores.
[15]
[SOURCE: ISO 16000-20:2014, 2.6 , modified — “Basidiomycota, Mucoromycota, and their asexual states”
replaced “Zygomycota, and their anamorphic states former known as Deuteromycota or fungi imperfecti”.
“basidiospores” added to Note 1 to entry.]
3.4
mycelium
branched hyphae network
[16]
[SOURCE: ISO/TS 10832:2009, 3.5 ]
4 Principle of enzyme-targeted fluorescent detection
The principle of enzyme-targeted fluorescent detection (ETFD) is to identify and target a specific naturally
occurring enzyme (β-n-acetylhexosaminidase) present in the cell walls of the fungi, and therefore present
in all fungal particles including hyphae, hyphal fragments, mycelium and spores. This enzyme is used by the
fungal cells to act on certain substances or substrates in the environment and break them down.
Enzyme activity follows Michaelis Menten kinetics, and if the amount of substrate is in surplus during the
reaction, the rate of reaction is constant (V ). If the enzyme reaction is running for a certain amount of
max
time (e.g. 30 min), the amount of substrate formed by the reaction will depend on:
— the Michaelis Menten constant (K );
m
— the temperature;
— the enzyme concentration.
K is a constant, and if the temperature is kept constant during the reaction time, the amount of product
m
formed will be linear with time and proportional to the concentration of enzyme. The concentration of
enzyme depends on the concentration of fungi, and the concentration of product is therefore, proportional
to the fungal concentration.
The technology described in this method uses this naturally occurring reaction by using an artificial
substrate that mimics what the enzyme would act on in the environment. Inside this artificial substrate, an
inert fluorescent molecule (fluorophore) is embedded. The signature of this particular fluorophore (4-MU)
is that it absorbs light at 365 nm wavelength and emits equal
...
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