Journal of Hospital Infection (2000) 44: 81–92

doi: 10.1053/jhin.1999.0688, available online at http://www.idealibrary.com on

REVIEW

Sampling of Aspergillus spores in air

G. Morris*, M. H. Kokki†, K. Anderson‡ and M. D. Richardson§
*Consultant in Environmental Health, Scottish Centre for Infection and Environmental Health, Glasgow, UK,
†Consultant in Infectious Diseases, National Public Health Institute, Helsinki, Finland, ‡Consultant in Respiratory
Medicine, Law Hospital, Carluke, UK, §Specialist in Clinical Mycology, University of Helsinki, Finland

Summary: Nosocomially acquired aspergillosis typically occurs in the setting of treatment for leukaemia
or other haematological malignancy. As Aspergillus species can be readily found in the environment, it has
been widely believed that aspergillosis occurs as a consequence of exogenous acquition of the fungus.
Stringent environmental controls in transplant units have included high-efficiency air filtration, positive-
pressure ventilation and frequent room air changes. Although there have been several well-documented
examples of aspergillosis outbreaks as a result of hospital demolition and reconstruction, it has not always
been possible to demonstrate elevated spore counts in clinical areas during building work. The sampling of
air for Aspergillus is very problematic. Careful attention must be given to the design of air sampler, sampling
protocols and an understanding of air sampling data. This review outlines many of the physical and environ-
mental parameters that influence meaningful air sampling and recommends a simple procedure that has been
tried and tested in many aspergillosis outbreaks. © 2000 The Hospital Infection Society

Keywords: Aspergillus spp.; Aspergillus spores, air samplers.

Introduction Many studies have attempted to evaluate the

importance of fungal aerobiology in relation to the
Fungal infections in immunocompromized patients
acquisition of invasive fungal infection by the com-
are not only difficult to treat, but are common and
promized patient.3–8 The objectives of air sampling
often unsuspected at the time of death.1 Recent
are stated in Table I. Most authors recommend
autopsy studies confirm this. For example, among
Aspergillus air counts of less than 5 colony forming
bone marrow transplant patients, invasive yeast
units (CFU)/m3 in protective isolation suites, and
infection or pulmonary aspergillosis were present in
counts of less than 0·1 CFU/m3 are desirable.
about one half of the early deaths. Many haematolo-
However, there are few standards for performing
gists who have treated such patients are acutely
and evaluating air counts in hospitals.9 The purpose
aware of the extremely high mortality of progressive
of this review and protocol is to provide background
fungal disease.
material to the various parameters that can influence
An analysis of an individual patient’s risk factors
air sampling and to recommend a procedure that has
for fungal infection and the type of fungus to which
been found useful in monitoring the level of air-
they are most susceptible, indicates the preventive
borne fungi in private dwellings and hospital envi-
strategies that are likely to be successful. These
ronments.
include the prevention of endogenous fungal infec-
tions, such as candidosis, by means of prophylaxis
Sampling of biological aerosols: some general con-
and prevention of exogenous infections (mostly air-
siderations
borne and caused by filamentous fungi) through
environmental protection and chemoprophylaxis.1,2 The considerations relevant to sampling Aspergillus
spp. spores in air are essentially the same considera-
Address for correspondence: Dr Malcolm Richardson, Mycology
tions which apply to sampling any aerosol. Aerosols
Unit, Department of Bacteriology & Immunology, Haartman
Institute, University of Helsinki, Haartmaninkatu 3, 00014, are made up of particles suspended in air with an
Helsinki, Finland. E-mail: malcolm.richardson@helsinki.fi upper size limit of approximately 25 µm. Because

0195–6701/00/020081 + 12 $35.00 © 2000 The Hospital Infection Society

82 G. Morris et al.

Table I Objectives of air sampling may be to study a single species or a group of

species, perhaps with certain shared characteristics.
To correlate outbreaks of invasive aspergillosis with hospital
construction/demolition Knowledge of the physical and biological character-
To identify potential sources of nosocomial aspergillosis, eg. potting istics of the target organism or organisms will influ-
soil, damp ceiling voids, damp fire proofing material, carpeting, etc. ence the choice of instrument. Biological
To predict environmental spore contamination from outside sources characteristics including capacity for survival during
To identify defects/breakdown in hospital ventilation/filtration systems*
aerial transport are important as are germination
To monitor cleaning procedures that may release bursts of airborne
Aspergillus conidia and growth requirements. In particular, biological
To determine the efficacy of HEPA filters in laminar flow facilities characteristics influence the choice of culture
To monitor efficacy of procedures to contain hospital building medium used in any instruments which operate by
work from hospital wards and other areas where high-risk patients trapping particles on an agar surface for subsequent
are managed
culturing. Air sampler design imparts certain aero-
To determine level of contamination prior to initial occupancy of
special controlled environments dynamic characteristics and thus sampling effi-
ciency is never uniform for any instrument across
*regular engineering maintenance of the air supply system (whether the range of particle sizes. Hence a knowledge of
HEPA-filtered or not) is more important than regular air sampling particle size, or more specifically, aerodynamic
diameter, is necessary to determine whether the
instrument will efficiently capture the target species.
they may be derived from many sources, aerosol
Whilst the physical size of a particle is obviously an
particles exhibit considerable compositional varia-
important factor in determining its aerodynamic
tion. They may, for example, be solid or liquid,
behaviour, other characteristics such as particle shape,
comprize organic and/or inorganic material or con-
density and surface irregularities are also important.
tain living organisms. Inevitably, some aerosol parti-
Prior to the development of the concept of aerody-
cles are potentially toxic, allergenic or infective to
namic diameter, linear size alone, as measured under
man. However, all aerosols, whatever their composi-
an optical microscope, was used as the measure of par-
tion, are governed by the same physical rules which
ticle size.12 Because particles come in many shapes,
determine their aerodynamic behaviour.10 Thus,
e.g., spheres, cubes, flakes or fibres, it has proved use-
many aspects of the behaviour of microbial particles
ful to create a unit for particle size as deposited in the
in air might be predicted from a knowledge of their
respiratory tract which takes cognisance of all the
physical attributes but biological features can also be
influences on deposition, and particularly on settle-
important as they influence the take off, aerial
ment and inertial impaction. This is known as the
transport and landing in addition to survival and
aerodynamic diameter (dae). This concept is derived
infectivity. Accordingly, successful isolation and
from the way the particle behaves when airborne and
subsequent identification of any microbial entity in
considers gravitational and inertial forces acting on
air requires a clear understanding of the physical
the particle proportional to its mass. It is defined as
and biological properties of the species under inves-
‘the diameter of a sphere of density (ρο) 1 g/cm3
tigation. The term aerobiology has been coined to
which settles through air with a velocity equal to that
describe the science of the aerial transport of micro-
of the actual particle under consideration’.12
organisms and other microscopic biological materi-
Appropriately then, dae reflects behaviour rather
als in air, their deposition and the ensuing
than linear diameter and is useful in understanding
consequences for life forms including the micro-
the behaviour of the non spherical irregular parti-
scopic entities themselves.10
cles which exist in the practical circumstance. The
As direct microscopic examination of spores sus-
aerodynamic diameter is less useful when particles
pended in air is wholly impractical, detection and
are very small (< 0·5 µm) because diffusion becomes
enumeration of spores in air requires that they be
an important deposition mechanism and this is
removed from the air to a surface where they can be
solely influenced by particle size as opposed to shape
examined under a microscope directly or after
or density.13 Aerodynamic diameter also loses its
growth in culture.11
utility in the larger size range (> 15 µm) because of
the very short residence time of larger particles in
Characteristics of the target species air. Nonetheless, because they exhibit significant
variation in shape, dae is an important measure of the
In some circumstances, the parameter of interest capacity of fungal spores to lodge in the respiratory
may be the total airspora whilst in others the aim tract or be trapped in an air sampler.11
Sampling of Aspergillus spores in air
One complicating factor derives from the fact that
particles absorb and lose water causing their size to
change with changing atmospheric humidity.14,15 It is
more appropriate then, to think of aerodynamic diam-
eter for a given species as falling within a range. In
practice, aerodynamic diameters have been estab-
lished by calculation from settling velocities. Lacey
and Dutkiewity (1976), using this method, calculated
the dae of Aspergillus fumigatus as 3·1 µm and of mixed
Penicillium species as 3·2 µm.16 Pasanen et al (1991)
reported aerodynamic diameters for viable fungal par-
ticles at different humidities, finding for example, a
range of aerodynamic diameters for Penicillium spp of
2·2–3·9 µm depending on humidity.14 Madelin and
Johnson (1992) conducted particle sizing of a number
of common indoor fungal isolates in a relative humid-
ity range of 40–98% using an aerodynamic particle
sizer.15 These, they recorded as 1·9–2·2 µm for
Aspergillus fumigatus, 2·6–3·0 µm for Penicillium
chrysogenum and 2·3–2·5 µm for Cladosporium cla-
dosporoides. Samson and van Reenen Hoekstra (1988)
reported corresponding physical diameters for these
species of 2·5–3 µm (A. fumigatus), 3–7 µm (P. chryso-
genum) and 2–4 (C. cladosporoides).17 A very important
observation in relation to respiratory deposition was
made by Lacey (1991) who noted that aggregated fun-
gal particles occurring in chains behaved similarly, in
aerodynamic terms to single fungal particles.18
Sampling period
In most circumstances air sampling is conducted to
characterize the airborne microbial environment or
some aspect of it. Often this is done in order to esti-
mate human exposure as part of an epidemiological
investigation. The requirement to present an inte-
grated assessment of exposure implies that the sam-
pling period should be long, perhaps hours or days.
Yet sampling over long periods masks short term
temporal or spatial variation in the airspora which
may be important in the situation being investigated.
In such cases a time discrimination of minutes or
even seconds may be required. Many instruments,
and particularly those which rely on culturing of
viable organisms to permit enumeration and identifi-
cation, are likely to become overloaded in contami-
nated environments and may only sample over short
periods to provide a snapshot of conditions.
Sample volume
Quantification of airborne organisms requires that
the volume of air from which the organisms were
83
collected is known. This permits the numbers pre-
sent to be expressed as a concentration per unit vol-
ume of air, perhaps as colony forming units per
cubic metre of air (CFUs/m3) where viability is of
interest as it is to, e.g., medical microbiologists or
plant breeders. Where both viable and non-viable
orgnisms are of interest e.g., to those investigating
allergic phenomena, the parameter of concern may
be the total number of spores per cubic metre mea-
surement. This requires a different type of instru-
ment and different laboratory techniques which do
not rely on spore viability. Some instruments incor-
porate a pump rated to deliver a particular flow rate
consistent with the aerodynamic characteristics of
the instrument. In others cases, a separate pump
must be used and this must be calibrated. The vol-
ume of air sampled is altered by reducing or extend-
ing the sample period. Choice of sampling volume
must be a compromize between the desire to obtain
a sample sufficiently large to be representative and
other practical considerations, most notably in the
case of instruments providing viable spore counts,
the requirement not to exceed the capacity of the
instrument. Skill in selecting the appropriate sam-
pling period will develop with increasing knowledge
of the environment under investigation, but initially
and in the absence of any insight into expected air-
borne concentrations, appropriate sampling peri-
ods/volume must be selected through trial and error.
Practical and economic considerations
With the exception of non-volumetric methods
which rely on settlement under gravity to collect
particles, most instruments require a power supply
to operate the pump. Any instrument which requires
a mains electricity supply is unsuitable in many out-
door situations. This has been addressed by some
manufacturers who provide a rechargable battery to
power the equipment. Whilst this confers infinitely
greater flexibility, true portability is also determined
by the weight and dimensions of the instrument.
Quite apart from their requirement for a separate
mains operated pump, large slit samplers, for exam-
ple, are construed by many as non portable due to
their size and weight. Such considerations may be
very important for extensive sampling programmes
conducted at various sites such as might be the case
in housing surveys.
Capital and running costs are also important con-
siderations as are robustness and reliability. Most
important however is the observation that, subject
to certain qualifications, operating a microbiological
84 G. Morris et al.

sampler is relatively straightforward. Enumeration,
and particularly identification, of airborne organ-
isms however, can be a skilled operation and, as
Gregory has pointed out, it is uneconomic to use the
time of a trained scientist with a good microscope on
inefficient sampling methods.11
Sampling instruments for enumerating and
identifying air spora
Types of sampler
It is convenient to consider microbiological sam-
plers for collecting organisms in air as falling into
several broad categories. The features of the differ-
ent types of sampler are summarized in Table II.
Many popular microbiological air samplers use the
principle of impaction to trap the organisms by
impacting them directly on to agar or another surface.
This group includes the Andersen Six-Stage Sampler
(Anderson Instruments Inc., Atlanta, Georgia), the
Casella Slit Sampler (Casella Ltd, Bedford, England)
and the Surface Air Systems (SAS) Sampler
(Cherwell Laboratories, Bicester, Oxon).
Some instruments operate by using centrifugal
acceleration to impact organisms on to agar as in the
case of the Reuter Centrifugal Sampler (RCS) sam-
pler (Biotest Folex, Birmingham, England) or on to
Table II Air samplers for quantitation of viable fungal spores
Sampler type Principle Flow rate
(litres/min)
liquid as for the Cyclone sampler (Aimer Ltd,
London). Despite the quite different design charac-
teristics these instruments are also impactors.
A further distinct group are the impingers which
operate by impinging organisms into liquid. This
group includes the single-stage midget and micro
impingers (SKC Ltd, Poole, Dorset), the multistage
May liquid impinger (AW Dixon, Beckenham,
Kent) and the Ace all-glass impinger-30 Sampler
(AG1–30) (Ace Glass, Vineland, New Jersey).
Certain instruments use electrostatic precipita-
tion to isolate particles from the air stream although
the principle of electrostatic precipitation is more
commonly seen in air cleaning applications. The
large volume Litton-type sampler (Sci-Med, Inc,
Eden Prairie, Madison) is an example of an air sam-
pling device which relies on this principle but it is
seldom used in indoor air sampling.
Filtration methods also find application in microbi-
ological air sampling. Such methods use either high or
low volume pumps such as those produced by Casella
Ltd of Bedford, U.K. to draw air through filters and in
so doing, trap airborne organisms on the filters. Filters
may be mounted in cassettes such as the Millipore cas-
sette (Millipore, Watford, Herts, UK). Other filter
options include cellulose-acetate and polycarbonate
filters (Nuclepore, Sterilin, Houslow, Middlesex) or
gelatin filters (Sartorius, Epsom, Surrey).
The literature also contains many accounts of
studies in which agar filled petri dishes were placed
horizontally to isolate airborne particles through
Cut-off diameter settlement prior to culturing.19
(d50)(um)

Sieve impactor Impaction 28·3 0·65–7·0

(Anderson) on to agar Impactors
plate
Samplers which operate by impacting particles from
Slit sampler Impaction 30–700 ~0·5 an airstream onto either an adhesive or agar surface
(e.g. Casella) on to rotating
agar plate
are the most widely used in indoor air surveys.
Because the air stream is drawn through by a pump,
Centrifugal Impaction 40 4·0 fan or aspirator, these samplers can be calibrated and
Impactor due to
(RCS) centrifugal
are hence volumetric. Impaction on to agar surfaces
acceleration is the more common method. Instruments are gener-
ally described as being single-stage or multi-stage. In
Impingers Impingement 12·5 0·3
(e.g. AGI) into liquid
single-stage instruments the air stream is directed
towards the surface of a single agar filled plate. The
P.B.I. SAS Impaction 90/180 2·0 air and the particles it carries accelerate on entering
Sampler on to agar
(Single stage plate
the instrument through a restricted inlet, a narrow
impaction) slit in the case of a slit sampler such as the Casella
Slit Sampler20 or a plate perforated with a number of
Settle plates Gravity Non-volumetric N/A
uniform diameter holes as in the case of eg. the SAS
Contact plates Surface Non-volumetric N/A sampler.21 The rapid change in the air direction as it
Sampling approaches the collecting surface at right angles
Sampling of Aspergillus spores in air
causes particles to be thrown from the stream to
impact on the agar surface with an efficiency which
depends on the velocity of the air and the size of the
particles. In multi-stage or “cascade” impactors such
as the Andersen Six-Stage Sampler the air is
directed through a stack of perforated or sieve plates
each with 400 uniform holes.22 As with a single stage
impactor, particles are again deposited from the air
stream onto an agar surface positioned below the
perforated plate as the air stream rapidly changes
direction. However, in a multi-stage impactor, the air
stream (minus the particles deposited on the upper
plate) proceeds to the next stage where it is drawn
though a second plate, this time with smaller diame-
ter holes, imparting a greater velocity to the air and
causing smaller particles to be deposited from the
airstream. The sequence is repeated for each plate in
the stack. The total size range of the particles which
can be collected over all the stages of an Anderson
Six-Stage impactor is about 0·3 µm to 15 µm.10 This
represents rather well the range of particle sizes
which might present a hazard to the human respira-
tory tract. A capacity to separate particles according
to size is important in the context of human health.
In the case of the Andersen Sampler, sometimes
termed the N6, there are six stages each capable of
impacting particles in a different size range. Non-
respirable particles are deposited on the top two
plates and, the smaller respirable particles which
would reach the alveoli are deposited on the lower
plates.19 Most published data on the presence of fun-
gal particles in non industrial environments has been
obtained using Andersen samplers.23 The advantage
of direct impaction on to an agar surface lies in the
fact that the agar plates can be incubated without
further treatment. This means that colonies grow
directly from collected viable airborne particles. The
statistical probability that a colony may be derived
from more than a single colony forming unit passing
through a hole is catered for by the application of
probability tables supplied by the manufacturer. If,
however, the environment is very heavily contami-
nated enumeration is impossible as the plate becomes
overloaded. This problem can be overcome by draw-
ing a smaller volume of air. Growth of colonies on an
agar surface permits their identification using gross
colony morphology and microscopic techniques.
An enduring problem of impactors when sam-
pling bacteria and the smaller fungal spores has been
that the small size and resultant low inertia of the
particles has demanded that that the airstream
achieves a high velocity to permit impaction on to
the capture surface. Pumps capable of doing this
85
have tended to be bulky, noisy and require consider-
able power.24 Slit samplers and the Andersen
Samplers have traditionally used separate mains
operated pumps. Impactors introduced more
recently have addressed this problem by using inte-
gral pumps operated from a rechargeable battery. An
example of this latter category is the Surface Air
Sytems (SAS) Sampler.
Centrifugal acceleration
The principle of centrifugal acceleration is used in
certain types of instrument to remove particles from
the sampled air. In the most commonly used instru-
ment, the RCS sampler, airborne particles are drawn
in by an impeller and thereafter impacted onto an
agar coated plastic strip lining the internal periphery
of the impeller housing.24
Agar strips are subsequently removed for incuba-
tion. Whilst operating according to rather different
aerodynamic principles, RCS samplers are impactors
and share some of the advantages and disadvantages
of other impactors described above. The results
obtained using RCS instruments must however be
regarded with caution. Macher and First (1983)
found an instrument drawing 210 L/min to be com-
paratively inefficient for particle sizes below 15 µm, a
range of considerable importance in human respira-
tory deposition.25 Because the airstream enters and
leaves the instrument through the same point, flow
rate is difficult to evaluate making estimations of
efficacy controversial.
A second type of instrument relying on centrifugal
acceleration collects the airborne particles in liquid.
The sampler, known as the cyclone sampler, mixes
the incoming air with a liquid supplied via a needle
fed gravitationally or through a peristaltic pump.26
The mixture is drawn tangentially into an inverted
cone. The airstream spirals down to the base of the
sampler before being drawn up the centre of the
instrument to the outlet.19 Particles are deposited on
the internal wall of the sampler and washed to a col-
lection point at the base of the instrument. The liquid
containing micro-organisms can then be used as an
inoculum. Several cyclones of differing design can be
used to obtain particle size differentiation.
Impingement into liquids
These samplers work by drawing air through liquid
causing the airborne particles to become suspended.
As with impactors, impingers can be classified as
single-stage and multi-stage. Single stage impingers
86
differ from straightforward bubblers in that a small
flask carries a wide inlet tube the inner end of which
is fused to a piece of capillary tube which dips at
least 5 mm into the flask (in the original form) and
terminates at least 4 mm from the bottom of the
flask. The capillary tube is a limiting orifice and
thus controls the flow rate under suction from an
attached pump.11
The multistage impinger (SKC Ltd, Poole,
Dorset) confers advantages over the single stage
device by having a gentler flow which is less damaging
to particles and in its capacity to separate the retained
particles into three particle size ranges corresponding
to retention within (a) the upper respiratory tract, (b)
the brochi and bronchioles and (c) the alveolar region
of the human respiratory system. Sampled air passes
through three liquid filled chambers at three different
speeds (10 L/min, 20 L/min and 55 L/min). Particles
collected in the first two chambers are impacted on to
sintered glass discs washed by liquid. In the third
stage particles are impinged tangentially into the liq-
uid.19 Thus multi-stage impingers have the advantage
of minimizing damage to microbes and improving
collection efficiency.11
Electrostatic precipitation
Movement of charged particles in electrical fields
has been used for dust extraction from airstreams.
Berry (1941) is credited with recognising the value of
applying this principle to microbiological sampling.27
Different designs for microbiological samplers have
been described.11,19 The Litton-type sampler draws
400–1000 litres of air/min. The charged particles are
attracted to a rotating aluminium disc which carries
the opposite charge. The disc is coated with a thin
liquid film which moves centrifugally over the disk
to a collection channel placed around the perimeter
before being recirculated. The solution obtained can
be used as an inoculum.
Filtration
Passing air through a filter causes particles to be
trapped on the filter medium. Micro-organisms col-
lected can be resuspended in an aqueous solution
and used as an inoculum. Polycarbonate membrane
filters are particularly suitable due to the ease with
which collected particles can be removed. The use of
black polycarbonate filters and subsequent staining
with acridine orange causes viable microbial cells to
fluoresce orange, allowing them to be counted
directly under fluorescence microscopy.28 When
G. Morris et al.
collected on polycarbonate or cellulose acetate mem-
brane filters, micro-organisms can be viewed
directly under a microscope. Mounting and coating
of polycarbonate filters allows examination under
electron microscopy.19 Porous gelatin filters can be
used to trap micro-organisms. The filters can then
be dissolved and the trapped micro-organisms in
solution used as an inoculum.29
Large volume filtration techniques can be used to
trap airborne particles for biochemical or immuno-
logical assay.30
Gravity sedimentation methods
Gravity sedimentation methods such as the open
petri dish (OPD) method combine both gravita-
tional and inertial processes. Burge and Soloman
(1996) commented that because air is never still, set-
tlement under gravity might play a relatively small
part in particle collection.31 Indeed they went as far
as to characterise a gravity slide or plate as “a contin-
ually changing inertial collector with a gravity com-
ponent that varies inversely with wind speed and
turbulance”. Gravity settlement methods, presum-
ably for reasons of low cost, have been widely used
but are defective in that they preferentially select
larger particles. Results can also be misleading due
to shadowing or turbulent deposition. Most impor-
tantly the method is not volumetric and provides
qualitative rather than quantitative results. Verhoeff
et al. (1989) considered it to have some merit
because its capacity to monitor over longer periods
than most volumetric methods allow it to provide
what they described as “an integrated assessment of
exposure”.32
Contact plates
Valuable information about the types of fungi in a
particular environment can be obtained by sampling
the accumulated dust on various surfaces such as
tables, floors, horizonatal blinds, fan blades and
guards. Using the same contact plates of Czapek-
Dox agar as described below for the SAS air sam-
pler, a unit area of surface can be sampled,
incubated and then the CFUs enumerated.
Choosing a sampler for Aspergillus species
Many factors require to be considered in choosing a
sampling instrument for use in a particular situation.
Some have been highlighted above and can now be
applied in the context of Aspergillus spp spores.
Sampling of Aspergillus spores in air
Inevitably other issues remain rather specific to the
circumstances of the user and are less amenable to
quantification. Amongst these are the resources
available for purchase, the nature and scale of labora-
tory support and the environment under investiga-
tion. A further consideration is that in practice, any
sampler may require to be used in several applica-
tions. Finally, personal preference and experience of
an instrument in use may greatly influence choice.
It quickly emerges, even from the most cursory
review of studies which characterise the indoor fun-
gal environment that a wide range of samplers and
sampling techniques are able to isolate Aspergillus
species from air. This observation is of itself com-
paratively unhelpful as it gives no real clue as to
comparative efficiency. Several investigators have
sought to evaluate the efficiency of air sampling
instruments either in isolation or comparatively
applying performance criteria such as total yield
(expressed as a concentration per unit volume of
air), number of species isolated and the coefficient
of variation between parallel or consecutive samples
(as a measure of instrument precision).21,32,33
There is sufficient agreement within the litera-
ture to suggest that certain instruments consistently
trap more organisms from the environment and are,
in absolute terms, more efficient. Some instruments,
such as the six stage Andersen N6 cascade sampler or
the Casella slit samplers have been used over a long
period of time and are regarded as efficient and
robust. Indeed much of our knowledge of the aero-
biology of occupational and other environments has
accrued through the use of these instruments.
Studies comparing the efficiency of different types
of instrument in terms of their capacity to trap
spores of a particular fungal species are less com-
mon and knowledge here often derives more from
theoretical calculation based on information on the
aerodynamic characteristics of the instrument and
the target species or from experimental work using
aerosols of known particle size.
Accordingly, in selecting a sampler for isolating
and enumerating Aspergillus spp. spores in air it is
important to consider relevant biological and physi-
cal characteristics.
Biological considerations
Aspergillus spores are intended for dissemination in
air and share with most fungal spores a robustness and
ability to resist impact damage and desiccation which
means that sampling and enumeration techniques
which might be unsuitable for more vulnerable
87
particles are ostensibly suitable for Aspergillus spores.
Nonetheless, whilst biological considerations may be
less relevant to the method employed to capture
Aspergillus spores it is relevant to the selection of the
medium used in impactors which rely on culturing to
enumerate and identify viable spores from the catch.
The minimum water activity for growth is influenced
by pH of the substrate and particularly by tempera-
ture. It varies too, according to species, but in the case
of Aspergillus spp, the minimum Aw is accepted by
most commentators to be around 0·78 with an
optimum of around 0·97.34–36 Sui (1951) found that
the range of relative humidity for spore germination
on nutrient gelatin of seven different species of
Aspergillus fell between 64% and 84%.37
Physical considerations
Allowing for the fact that atmospheric humidity will
influence aerodynamic diameter, it is reasonable to
express the aerodynamic diameter of Aspergillus spp
spores as falling in the range 1·9–3·2 µm.14–17 In addi-
tion to permitting the spores to penetrate the pul-
monary defences and reach the alveolar spaces where
they may germinate and form hyphae, their size also
places them at the lower end of the aerosol particle
size range and thus limits the choice of sampler.38
The possibility of grouping of several spores in a
single clump also merits consideration as this would
clearly influence aerodynamic behaviour in sampling
and site of deposition within the respiratory tract.19,31
Gregory (1973) mentions that spore clumping is a
recognised phenomenon with certain spores particu-
larly with Cladosporium spp. and a few other types
notably Ustilago.11 This, he took to denote failure of
the spores to separate at the time of liberation rather
than secondary aggregation during aerial transport,
it being considered that many spores will be kept
apart by like charges at the time of liberation. Lacey
additionally observed that with Aspergillus fumigatus,
the chains were so tightly bound together that they
might break into clumps of chains with aerodynamic
diameter determined by the size of the clump which
would presumably vary.18 Kozakiewicz (1989) com-
mented on the significant variation in surface orna-
mentation within the same genera, highlighting
Aspergillus spp as an example.39 Surface ornamenta-
tion is a primary determinant of aggregation. Thus,
clumping inevitably occurs to some extent with
Aspergillus spp. This has implications for particle
capture within instruments.
It can be appreciated that, irrespective of the phys-
ical principles on which they operate, instruments can
88
be classified according to whether they are intended
to measure only viable airborne particles or both
viable and non-viable particles. Filtration methods,
some gravitational settlement techniques, liquid
impingers and electostatic precipitators all gather
both viable and non-viable particles although subse-
quent laboratory procedures can often permit enu-
meration of viable particles alone. A clear advantage
is that, where the air is very contaminated, the large
number of trapped spores will not exceed the capaity
of the instrument and enumeration of the catch is
possible using serial dilutions. A very pertinent con-
sideration with regard to Aspergillus is that the focus
of interest is normally the viable spores because they
have the capacity to infect. Viable spore sampling
techniques, i.e., those which capture spores on an agar
surface permit the catch of organisms to be cultured
without further laboratory preparation. The develop-
ment of colonies also facilitates enumeration and
identification.
Despite its widespread use, the open Petri dish
method of air sampling, relying, as it does on sedi-
mentation is non-volumetric and inefficient for
small particles. Whilst aggregations of Aspergillus
spp spores will inevitably settle on horizontal sur-
faces or spores may impact when carried in rapidly
moving air currents, open Petri dish methods cannot
be recommended for Aspergillus spores and will not
be discussed further here.
Cascade samplers, and in particular the Andersen
N6 samplers have been widely used and their effi-
ciency in the sampling of smaller particles is recog-
nised as an advantage, particularly when allied to
their capacity for particle size differentiation which
can be important in many applications. Slit samplers
too, have been evaluated and found to be efficient in
their capacity to capture small particles. The larger
slit samplers in particular the large model Casella
draw some 700 litres of air per minute which may be
an attractive feature in some hospital environments
where there are low levels of airborne contamination.
Unfortunately both these instruments have the
disadvantage of a separate mains operated pump
and the Casella, in particular, is bulky and heavy.
Both instruments also require significant quantities
of media. It is possible to operate the Andersen
sampler using only those stages suitable for capture
of smaller airborne particles thus reducing the
quantity of media required but this does not over-
come the inconvenience of an external pump which
requires to be calibrated to align with the aerody-
namic properties of the instrument. The supremacy
of slit to agar samplers and the cascade samplers
G. Morris et al.
would suggest that, on the grounds of particle cap-
ture efficiency alone, these would be the natural
choice for microbiological sampling. These instru-
ments are however costly and the lack parcticality
for monitoring external or otherwise inaccessible
areas. For sampling Aspergillus spp. spores, it is nec-
essary at least to consider those instruments which
capture viable particles and are powered by rechar-
gable batteries as these would appear to address
some of the problems encountered with other
impactors. This group is exemplified by the Reuter
Centrifugal Sampler and the Surface Air Systems
Microbiological sampler. Both instruments have
been demonstrated to perform consistently below
the level of the Casella slit sampler and the
Andersen samplers in terms of the total number of
organisms isolated.21,32 This does not of itself rule
these out as the instrument of choice but the find-
ings of Clark and colleagues (1981) that collection
efficiency of the RCS falls off rapidly for particles
below 15 µm and to approximately 50% for particles
below 5 µm suggested it is unsuitable for the capture
of Aspergillus spp spores in air.24
The SAS sampler is a simple single-stage
impactor in which air is directed towards an agar
filled contact plate of 50-mm diameter positioned
behind the cover plate. The air is drawn into the
instrument through a perforated cover plate which,
in standard form, is perforated with 219 holes of
1-mm diameter. Particles leaving the air stream
impact on the agar surface and thereafter the plate
may be removed for subsequent incubation prior to
enumeration and identification of colonies. The vol-
ume of air drawn into the instrument is regulated by
pre-setting the period of operation using an integral
timer. Capture efficiency in both quoted flow rates
(180 l min–1 and 90 l min–1) falls to around 50% for
particles of 2 µm (approximating to the size of the
smaller Aspergillus spp spores). The instrument is
light, portable, robust and sufficiently versatile to
sample in inaccessible areas or be pointed to detect a
microbiological leak. The authors have found the
180 l min–1 SAS sampler to be practical and reliable
when used in large epidemiological studies in the
home environment and during outbreak investiga-
tions in hospital environments. The performance of
the sampler and variations of it has been evaluated
in comparison to other instruments.21,32 An impor-
tant aerodynamic difference which affects capture
efficiency is in the speed imparted to the incoming
air with a markedly lower velocity in the case of the
SAS than, say, the Six-Stage Andersen instrument.
This is explained by the greater area of the combined
Sampling of Aspergillus spores in air
orifices in the perforated cover plate of the SAS
compared to the slit and Andersen samplers. A
smaller inlet area offers greater resistance which can
only be overcome using powerful pumps. Hence,
low airspeed and reduced efficiency particularly in
the impaction of smaller particles, may be an
inevitable consequence of portability and conve-
nience of the SAS sampler. The key question in this
context is whether the lesser efficiency rules out the
SAS as the instrument of choice for sampling
Aspergillus spp spores in the way it would seem to do
for the RCS sampler.
The performance of the SAS in standard 220 hole
format and a version with 260 holes with a Bourdillon
slit sampler have been compared.21 The version with
260 holes used a 90 mm agar filled contact plate as
opposed to the standard 50 mm. contact plate. The
most important characteristics of an air sampler have
been defined as being the effective volume-rate of
sampling and the range of particle sizes over which
this is maintained.21 The tests showed that the effec-
tive sampling volume of both instruments remained
nearly constant over the particle sizes likely to be
encountered during environmental sampling. But
that collection efficiency fell off for particle sizes
below 4 µm and at 2 µm reduced to 50%. Hence, effi-
ciency of the SAS with a rated volume of 180 L/min
is 50% for the smaller Aspergillus spp spores. It has
also been seen that the high air flows of the samplers
made them particularly suited to the measurement of
low levels of micro-organisms.21
In subsequent work conducted on behalf of the
manufacturers a later version of the SAS sampler
operating on the same principles but drawing 90
litres per minute was evaluated by the Centre for
Aerobiological Research at Porton Down.40 Instead
of using the 220, 1-mm diameter hole cover plate,
the test used a cover plate with 0·75-mm holes. The
study found that the reduction in the hole size
resulted in an instrument which was 100% effective
for collecting particles in the size range of
Aspergillus spp spores. Unfortunately this is a spe-
cial adaptation and is not available as standard
equipment.
Whilst on first appraisal it might appear that the
simple substitution of a new coverplate would create
the ideal instrument, certainly for Aspergillus spp
spores, it must be remembered that the greater air
velocities created by the smaller diameter holes
would reduce the volume sampling rate whilst the
increased air velocity a would render the instrument
less suitable for general microbiological air sampling
where less robust organisms are of interest.
89
The writers’ experience of the standard 180 l
min-1 SAS instrument has shown that it regularly
captures Aspergillus spp. spores. We nonetheless
recognise that it may appear illogical to recommend
an instrument which may be only 50% efficient for
particles of the size of the target organism.
However, the greatest advantage conferred by use of
the SAS, particularly in 180 l min–1 form is its high
flow rate which is particularly suited to environ-
ments where there may be low concentrations of the
target organism.21 A further justification must be
that the instrument is commonly used permitting
comparison of monitoring conducted in different
locations. The purchase price too is comparatively
low, yet in the authors’ experience it has proved
robust and reliable. The accepted efficiency deficit
in the critical 2 µm range requires, in our view, to be
set against the significant advantages offered in
terms of practicality.
The efficiency gained by using a modified SAS
has been highlighted above yet this is not a practical
option as the instrument is not available as standard
in this form. Accordingly, the method set out below
is recommended for sampling Aspergillus spp. spores
in air.
A recommended method for the sampling of
Aspergillus species spores in air
Sampling to isolate and enumerate Aspergillus spp
spores in air should be conducted as follows:-
Materials and equipment
Sampling Instrument: Surface Air Systems micro-
biological air sampler.
Manufacturer: Pool Bioanalyse Italian (PBI),
Milan Italy
U.K. Supplier: Cherwell Laboratories,
Churchill Rd., Bicester,
Oxon, U.K.
Collection Rate: 180 1 min–1
Medium: Czapek Dox Agar (Oxoid)
Method
Sampling Procedure (general)
The air sample is aspirated through the instrument
at a nominal rate of 180 litres per minute for a pres-
elected period of between 20 seconds and 6 minutes
giving a volume range between 60 litres and 1080
litres. The airflow is directed towards the agar
surface of a 50 mm diameter contact plate which
90
contains 12·5 mL of agar so that particles whose
aerodynamic diameter causes them to leave the
airstream are deposited on the agar surface. The
plate is then removed for incubation.
Sampling location
Sampling location is dependent on circumstances
but should accord broadly with the breathing zone
of potentially affected personnel. The choice of
sampling height is 1·2 metres for room hygiene, with
other samples taken for exploratory purposes near
suspected or potential sources of contamination.
Multiple samples are preferable to single samples as
they will highlight temporal and spatial variation in
spore levels within any environment.
Selection of sampling time
Selection of an appropriate sampling period is vital
to the success of the sampling operation. If the sam-
pling time selected is long in a heavily contaminated
environment then the colonies, once cultivated, can
only be expressed as exceeding a particular number.
Where confluent growth occurs the colonies may
even be uncountable. A chart is supplied with the
instrument to assist in selection of the sampling
time which, on the basis of an assumption regarding
the level of contamination in the environment per-
mits the number of sampling units (periods of 20) to
be estimated. In practice, whilst some knowledge of
the levels of contamination may build up over time,
certainly initially the selection of the sampling
period must largely be done by trial and error.
Sampling steps
1. Unscrew the top cover plate avoiding contact with the
inner or outer surfaces of the drilled area. The cover
plate should be cleaned after each use.
2. Insert a contact plate with Czapek Dox agar (Oxoid
Basingstoke UK) with the lid still in place, remove the
contact plate lid and replace the instrument cover plate.
3. Set the digital selector on the instrument to zero units.
4. Switch on the battery pack.
5. Turn the timer to the desired setting.
6. Press the instrument start button.
7. Following completion of the sampling the instrument
will switch off. The cover plate can then be removed
and the exposed contact plate agar surface immediate-
ly covered by replacing the contact plate lid.
8. The contact plate should then be removed for in
cubation.
Laboratory procedure
1. On receipt of the contact plates, these are placed in a
pre-heated incubator to 28°C for 48 hrs to permit ger-
mination and colony formation.
G. Morris et al.
2. The plates are then microscopically examined at × 100
magnification to enumerate colonies growing on the
plate.
3. Identification of fungal colonies is based on colony
characteristics and micromorphological characteristics
ascertained through microscopic examination at × 400
magnification.
4. Specimens for examination should be prepared using a
wet needle mount using lactophenol with cotton blue
stain (0·75%).
5. A colour key is available for the specific identification
of different Aspergillus spp. grown on Czapek Dox
agar or broth.
N.B. Czapek Dox is recognised as a suitable medium
for isolation and culturing of Aspergillus spp., whilst
permitting growth of the majority of airborne fun-
gal species. This permits general levels of fungal
contamination to be simultaneously appraised as an
indicator of building hygiene.
Enumerating the colony forming units
There is a possibility that any colony which grows
on the contact plate derives from more than one
colony forming unit passing through a single hole in
the cover plate. This possibility increases with the
number of colonies on the plate and for higher
counts a correction factor is applied according to the
following formula:—
Pr = N(1/N+ 1/(N–1) + 1/(N–2)+ . . . . . . . . . 1/(N–r+ 1))
Where
Pr is the probable statistical total
N is the number of holes in the sampling head
r is the number of colonies counted
Tables have been prepared by the manufacturer
which can be read off to give a value for P once r has
been established.
The number of colony forming units calculated
from the above formula is normally expressed as
CFUs/m3. This is calculated using the formula:—
Adjusted colony count on plate × 1000
X =
Volume of air drawn into sampler (litres)
The interpretation of air sampling data and recom-
mendations for intervention are given in Table III.
Conclusions
In published outbreaks of invasive aspergillosis,
local fungal contamination of ducts, grids and filters
may release spores intermittently as may nearby
demolition and building works. Although in some
Sampling of Aspergillus spores in air 91

Table III Interpretation of air sampling data and recommendations

Levels of fungal spores vary by several orders of magnitude during the course of a day due to:
Activity levels in any one particular area
Fluctuations in temperature
Fluctuations in humidity
Fluctuations in air flow
Changes in light level

A single air sample will often underestimate the fungal contamination in the air:
multiple air sampling has to be performed
No strict numerical guidelines are available which are appropriate for assessing
whether the contamination in a particular location is acceptable or not but the
following threshold levels have been recorded:
Outdoor air: total fungal count: 103 to 105 CFU/m3
Aspergillus: 0·2–3·5 conidia/m3
Note: seasonal variation recognised
HEPA filtered air (> 95% efficiency and > 10 air changes per hour): < 0·1 CFU/m3
No air filtration: 5·0 conidia/m3
Construction/defective ventilation: 2·3–5·9 conidia/m3
If total fungal count exceeds 1·0 CFU/m3 on several occasions the air systems
or procedural practice in patient areas requires intensive evaluation.

Further investigation of sources of contamination is warranted in the following circumstances:

Total indoor counts are greater than outdoor counts
Comparison of indoor and outdoor levels of fungal organisms show one of the following:
Organisms are present in the indoor sample and not in the outdoor sample
The predominant organisms found in the indoor sample is different from the
predominant organism in the outdoor sample
A monoculture of an organism is found in the indoor sample. It may be
absent from samples taken in other areas of the building
Persistently high counts

If persistently high counts are recorded, or nosocomial invasive aspergillosis suspected or

confirmed: identify source of contamination by sampling:
dust
fabrics
ventilation ducts/screens/fans
ceiling voids
kitchen areas
excreta of roosting birds in close proximity of windows
Restrict food stuffs known to carry Aspergillus conidia, eg. black pepper
Consider painting contaminated surfaces with copper-8-quinolinolate
Start appropriate antifungal prophylaxis or pre-emptive therapy if not already used
Perform an intensive retrospective review of microbiological, histopathological and
post-mortem records for other cases.
Alert clinicians caring for high risk patients to the possibility of infection
Establish a system for prospective surveillance of patients and their environment for additional cases
If further cases arise in the absence of a nosocomial source consider monitoring home
environments of patients pre admission.

outbreaks of aspergillosis the patient’s isolates may
have been different from those in the environment,
some degree of control is more important than mea-
suring air counts, because these are taken only at one
point in time within the ward or clinic. If an out-
break of infection has occurred, it reflects an episode
of air contamination that happened some days or
weeks before. ‘Normal’ air counts are therefore not a
reason for complacency and a search for a specific
source of air contamination is required, together
with continued vigilance against the introduction of
large numbers of spores into the patient’s room.
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