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AIR SAMPLING METHODS

Methods of Air Sampling

The various methods of air sampling include:
1. Impingement which is trapping of airborne particles in a liquid matrix.
2. Impaction which is forced deposition of airborne particles on a solid surface.
3. Centrifugation which is mechanically forced deposition of airborne particles using inertial forces of
gravity.
4. Filtration which is trapping of airborne particles by size exclusion.
5. Deposition which is collection of airborne particles using only naturally occurring deposition process.

The two most commonly used devices for microbial air sampling are all glass AGI-30 impinger and
Anderson 6-stage impaction sampler. Apart from these, the Reuter air sampler (based on centrifugation)
is also commonly used for small scale air sampling.

Impingement
Impingement into Liquid – Impingement in liquids:

In this method, the air is drawn through a very small opening or a capillary tube and bubbled through
the liquid. The organisms get trapped in the liquid medium. Aliquots of the liquid are then plated to
determine its microbial content. Aliquots of the broth are then plated to determine microbial content e.g.
Bead-bubbler device

Impingement on to Liquid is divided into three types, they are following:

(i) Raised Impinger

(ii) Bead- Buffer Device

(iii) Lemon Sampler

Raised Impinger - In this type of sampler impingement is made within bulk fluid by a jet of air.

Bead Bubbler Device -It is also an oldest device for sampling air. It consists of a 250ml suction flask
which has an outlet on the side connected to suction pump. A glass bubbler, which is nothing but a glass
tube with minute openings at the bottom, is kept in place inside the flask by a rubber stopper. Glass beads
of size about 5mm in diameter are kept around the glass bubbler.

In addition the flask contains known volume of broth. Air is drawn into the flask through the glass
bubbler when the flask is continuously shaken. The incoming air escapes into the broth in the form of
bubbles through the holes at the bottom of glass bubbler.
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The shaking action of the flask and hence the glass beads facilitate the formation of bubbles. Thus,
microorganisms in the air are dispersed in the broth and after sampling, an aliquot from the broth is
plated for count.

Lemon Sampler - It consists of a glass Folin aeration tube with a perforated bulb with six holes at one
end. The bulb end of aeration tube is contained within a test tube by a two hole rubber stopper. The bulb
is actually centered near the bottom of the tube and is immersed in 20ml of broth. Two or three drops of
olive oil is added to the broth to prevent foaming.

A kjeldahl trap with square glass baffle is shortened at both ends for convenience. The intake end is
slightly bent and inserted into the other hole of the stopper. A flow meter measures the rate of airflow
entering the upper open end of the Folin tube. An air pump is attached to the exhaust end of the kjeldahl
trap.

The entire bubbler should be sterilized by autoclaving. Alternatively it can be sterilized by rinsing with
70% alcohol and dried afterwards. Air is drawn at the rate of 25-30 liters per minute and dispersed
through the broth.

An impinger (Fig. 10.4) operates by drawing air through an inlet that is similar in shape to the human
nasal passage. The air is transmitted through a liquid medium where the air particles become associated
with the fluid and are subsequently trapped. The AGI-30 impinger (All Glass Impinger) is a liquid-filled
cylinder which collects particles by their impingement into a fluid. The capillary tip of the inlet tube
inside the cylinder is located 30 mm from the impinger bottom, thus the nomenclature. AGI - 30 is easy to
use, inexpensive, portable, reliable, easily sterilised and has high biological sampling efficiency in
comparison to many other sampling devices but there may be loss in viability of the collected biological
sample due to sheer force used to collect the air.

The usual volume of collection medium is 20 m1 and the typical sampling duration is approximately 20
minutes which prevents evaporation during the sampling of warm climates or freezing of the liquid
medium when sampling at lower temperatures. The liquid and suspended microorganisms can be
concentrated or diluted by using this method of impingement.

A simple medium is 0.85% sodium chloride which is an osmotically balanced, sampling medium used to
prevent osmotic shock of recovered organisms. Another medium in use is peptone (1%) which is used as
a medium for stressed organisms. Finally, enrichment medium can be used to sample selectively for
certain types of organisms.

 A major drawback when using an impinger is that there is no particle size

discrimination which prevents accurate characterisation of the sizes of the
airborne particles that are collected.
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Impaction
The Anderson six-stage impaction sampler (Fig. 10.5) provides accurate particle size discrimination in
contrast to the impinger. This device was developed by Anderson in 1958 and the general operating
principle is that air is sucked through the sampling port and strikes agar plates. Impaction procedure
depends upon the internal properties of the particle (size, density), on the physical parameters of the
impactors (inlet nozzle dimensions) and the airflow pathway.

The principle that underlies this sampling device is simple and ingenious. Air impinging onto the lop
agar plate is travelling at relatively low speed, and is deflected around the agar plate. Only the larger
(heavier) airborne particles will have sufficient momentum (defined as mass x velocity) to break free from
this air current and impact onto the top agar surface. But then the same volume of air is sucked through a
series of small holes, so its velocity is increased and this enables smaller particles to impact onto the
second agar plate and so on, down the series of plates with increasingly smaller holes, so that the
momentum of the airborne particles is increased at each stage.

The result is a size (mass) separation of the

airborne particles, which is remarkably similar to
that which occurs in the human respiratory tract
(Fig. 10.5c), as explained later. Large spores such as
those that impact onto a rotorod tape (greater than
7-10 micrometres) will impact onto the topmost
agar plate. Smaller spores (3-7 micrometres) will
impact on the middle agar plates, and even very
small spores (e.g. the spores of actinomycetes, 1-2
micrometres diameter) will impact on the lowest
agar plates.

When the apparatus is running , the incoming air

impinges onto the topmost agar plate, where
airborne particles can impact on the agar surface. Then the air is drawn round this first agar plate, and
through the first set of perforations, so that particles can impact on the second agar plate, and so on down
the stack.

Larger particles are collected on the first layer, and each successive stage collects smaller and smaller
particles by increasing the flow velocity and consequently the impaction potential. The particle size
distribution of the air particles can be directly related to the particle size distribution that occurs naturally
in the lungs of animals.
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The lower stages correspond to the alveoli and the upper stages to the upper respiratory tract. The
biological sampling efficiency is somewhat lower because of the method of collection which is impaction
on an agar surface (on a solid surface).

After this apparatus has run for some time (a few

minutes or several hours, depending on the likely
spore load in the air), the agar plates are removed and incubated, to identify the organisms that grow on
them. Fig. 10.7a shows a plate from the lowest part of the Anderson sampler. The colonies are of
thermophilic actinomycetes (Faenia rectivirgula or Thermoactinomyces vulgaris) that are common causes
of Farmer's lung disease.

Figures above shows agar plates from the middle

part of the Anderson sampler, where several
species of Aspergillus and Penicillium have
developed from spores about 3-5 micrometres
diameter.

Figures L, M. The Anderson air sampler, shown in a

laboratory (L) and mounted with a wind-vane in a field site (M).

The circular orifice at the top sucks air at flow rate of 28.3 l/min. passing it through the sieve arranged in
gradually decreasing order of pore size. Air impacting on last Petri dish goes out. Particles of similar size
impact on a given Petri dish only. In this way spores/pollens of six dimension orders are impacted on
agar surface of individual Petri dish.

some sophisticated samplers based on impaction and

suction devices (battery - as well as power-operated)
have now been developed. These provide more
accurate data on species-composition as well as
density of aeroallergens. Based on suction device
with definite amount of air sucked in and impacting
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the bio particles on the adhesive material on the sampler is the most acceptable device world over.The
Burkard volumetric traps and Rotorod aeroallergen models are such samplers. Burkard personal slide
sampler (suction), battery operated, suitable for indoor environment is 10 cm high having a rectangular
orifice at the top. The micro slide is coated with glycerine jelly. The sampler sucks air at flow rate of
10l/minute through the orifice.

The particles get impacted on the slide forming a band. Slide is mounted and scanned. Burkard personal
Petri dish sampler is similar to the slide sampler except that it has a stage to hold a Petri dish and a sieve
to cover it. The orifice is circular. Petri dish containing suitable growth medium become exposed to a
definite volume of air through the sieve on to the Petri dishes. These two samplers arc suitable for indoor
environment. Burkard continuous 7 day trap (continuous impaction) uses a glycerine jelly coated tape
mounted on a rotating drum.

Figures J-K. The Burkard spore sampling device, shown in assembled form (J) and during preparation (K).

The drum is connected to a timer and rotates at a constant speed. Tape exposed for a week is cut into
seven strips, one for each day, mounted on a slide in glycerine jelly and scanned. Each strip can also be
divided into 24 vertical bands, each band representing the particular hour of the day.

Rotorod Aeroallergen Intermitted Sampler - This sampler has an intermittent rotating impactor, is
power operated and ideal for both indoor and outdoor studies. It is light weight, portable sampler. It has
an arm that holds two cubical rods coated with silicon grease. Unexposed rods remain folded. Arm has a
shield at top for protection from rains. Exposure time can De regulated. The rods are mounted on a
grooved slide in a basic Fuschin stain and scanned.Figure C: Rotorod sampler. Figures D, E: Pieces of sticky
tape on which spores and other particles were impacted. The identifiable particles include: in D, an ascospore (as), a
hyaline (colourless) fungal spore (h) and a conidium of the common leaf-surface fungus Cladosporium (c); in E,
multicellular conidia (resembling snowshoes) of a common leaf-surface fungus Alternaria (a), conidia (c) and
hyphal fragments (ch) of Cladosporium, and a large hyaline spore of a powdery mildew fungus (m).

Settle Plate Method - The principle behind this method is that the bacteria carrying particles are allowed
to settle onto the medium for a given period of time and incubated at the required temperature. A count
of colonies formed shows the number of settled bacteria containing particles.

In this method petridishes containing an agar medium of known surface area are selected so that the agar
surface is dry without any moisture. Choice of the medium depends upon the kind of microorganisms to
be enumerated. For an overall count of pathogenic, commensal and saprophytic bacteria in air blood agar
can be used.

For detecting a particular pathogen which may be present in only small numbers, an appropriate selective
medium may be used. Malt extract agar can be used for molds. The plates are labelled appropriately
about the place and time of sampling, duration of exposure etc. Then the plates are uncovered in the
selected position for the required period of time.
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The optimal duration of exposure should give a significant and readily countable number of well isolated
colonies, for example about 30-100 colonies.

Usually it depends on the dustiness of air being sampled. In occupied rooms and hospital wards the time

would generally be between 10 to 60 'minutes.

During sampling it is better to keep the plates about I metre above the ground. Immediately after
exposure for the given period of time, the plates are closed with the lids. Then the plates are incubated for
24 hrs at 37°C for aerobic bacteria and for 3 days at 22°C for saprophytic bacteria.

For molds incubation temperature varies from 1O-50°C for 1-2 weeks. After incubation the colonies on
each plate are counted and recorded as the number of bacteria carrying particles settling on a given area
in a given period of time.

Though the method has the advantage of simplicity, it has certain limits. In this method only the rate of
deposition of large particles from the air, not the total number of bacteria carrying particles per volume, is
measured.

Growth of bacteria in the settled particles may be affected by the medium used since not all
microorganisms are growing well on all media. Moreover since air currents and any temporary
disturbances in the sampling area can affect the count, many plates have to be used.

Impingement on solids: In this method, the microorganisms are collected, or impinged directly on the
solid surface of agar medium. Colonies develop on the medium where the organism impinges.

Several devices are used, of which the settling-plate technique is the simplest, In this method the cover of
the pertridish containing an. agar medium is removed, and the agar surface is exposed to the air for
several minutes. A certain number of colonies develop on incubation of the petridish.

Each colony represents particle carrying microorganisms. Since the technique does not record the volume
of air actually sampled, it gives only a rough estimate. However, it does give information about the kind
of microorganisms in a particular area. Techniques wherein a measured. Volume of air is sampled have
also been developed. These are sieve and slit type devices. A sieve device has a large number of small
holes in a metal cover, under which is located a petridish containing an agar medium.

A measured volume of air is drawn, through these small holes. Airborne particles impinge upon the agar
surface. The plates are incubated and the colonies counted. In a slit device the air is drawn through a very
narrow slit onto a petridish containing agar medium. The slit is approximately the length of the petridish.
The petridish is rotated at a particular speed under the slit One complete turn is made during the
sampling operation.
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Hollaender and Dalla Valle Sampler - This sampler consists of a brass container with removable bottom.
The container is fitted with an inverted glass funnel. In the lower portion of container a petridish base
with medium is placed which can be screwed tightly against the gasket during sampling.

The funnel is kept just above the petri dish without touching it. The inside of the funnel and rim are
swabbed with alcohol before use. The air sample passes through the funnel stem and the airborne
microorganisms are impinged upon the agar medium.

The air is drawn by means of a pump in series with a flow meter. Effective sampling rate is found to be 28
liters per minute. The method is simple, portable and efficient.

Centrifugation
Centrifugal samplers use circular flow patterns to increase the
gravitational pull within the sampling device in order to deposit
particles. The most common is the Reuter's air sampler which is based
on the centrifugal force. Air is sucked in through the propeller blades
and as it traverses through the body of the sampler, it gets deposited
onto the thin agar media which lines the inner wall of the sampler.
After a known period of time the media is taken out and incubated for
further studies.

A - Air is Drawn into the Sampler

B - Return air Flow

There is another device based on this principle, the cyclone which is a

tangential inlet and return flow sampling device. These samplers are
able to sample a wide range of air volumes (1-400 L/ min), depending
on the size of the unit. The unit operates by applying suction to the
outlet tube, which causes air to enter the upper chamber of the unit at
an angle. The flow of air falls into a characteristic tangential flow
pattern which effectively circulates air around and down along the inner surface of the conical glass
housing. As a result of the increased centrifugal forces imposed on particles in the air stream, the particles
are sedimented out. Analysis is performed by rinsing the sample with an appropriate liquid medium,
collection of the medium and subsequent assay by standard methodologies.

Filtration and Deposition

Both the methods (Fig. 10.10) are widely used for microbial sampling for cost
and portability reasons. Filter sampling requires a vacuum source and involves passage of air through a
filter, where the particles are trapped. After collection, the
filter is washed to remove the organisms before analysis.
Usually membrane filters are used for the purpose with
varied pore sizes.
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Deposition sampling is by far the easiest and most cost-effective method of sampling. Deposition
sampling can be accomplished merely by opening an agar plate and exposing it to the wind, which
results in direct impaction, gravity settling, and other depositional forces.

There are problems associated with this method of sampling. They have
low overall sampling efficiency because it relies on natural deposition. It has no defined sampling rates or
particle sizing, and poses an intrinsic difficulty in testing for multiple microorganisms with varied growth
conditions.

Analysis of microorganisms collected by depositional sampling is similar to impaction sample analysis.

There are other simple sampling methods that may be used to supplement volumetric air sampling.
Surface samples are taken by tape lift imprint, by swabbing the suspect surface with a culture swab, or by
submitting a bulk sample of the suspect surface.

For tape samples, a piece of absolutely clear (not frosted) tape that is one or two inches in length is used.
It is handled by the ends only. The adhesive side of the tape is positioned over the suspect area and the
tape is gently and firmly pressed. Care is taken that the tape is not rubbed back and forth. The tape is
removed from the surface and placed on a clean microscope slide, which is put into a slide box or into
plastic bags and further processed.

A direct microscopic examination is performed on surface samples. While culturing, a surface sample
may help resolve a specific identification problem. Used alone, such a culture may result in an inaccurate
characterisation of the surface sampled.

A direct microscopic examination of a surface shows exactly what is there, without any skewing by
laboratory procedures. Surface sampling is inexpensive and (for a direct examination) may be analysed
immediately. Surface sampling may also reveal indoor reservoirs of spores which have not yet become
airborne.

The primary purpose of a direct microscopic examination of a surface is to determine whether or not
mold is growing on the surface sampled, and if so, what kinds of molds are present. Secondarily, most
surfaces collect a mix of spores which are normally present in the environment. At times it is possible to
note a skewing of the normal distribution of spore types, and also to note 'marker' genera which may
indicate indoor mold growth.

In addition, when mold growth is present indoors, many more spores of a particular type will be found
trapped on surfaces. These spores may be ill forms which indicate recent spore release (close proximity),
such as spores in chains or clumps. Marker genera are those spore types which are present normally in
very small numbers, but which multiply indoors when conditions are favourable for growth. These
would include cellulose digestors such as Chaetomium, Stachybotrys, and Torula.

While a single Stachybotrys spore is occasionally seen as part of the nor al outdoor flora, finding 5 or 6 of
these spores on a single scotch tape slide of a duct surface is an indicator that Stachybotrys may be
growing indoors.
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But the presence of biological materials on a particular surface is not a direct indication of what may be in
the air. Health problems related to indoor microbial growth are generally caused by the inhalation of a
substantial number of airborne spores, sometimes over a substantial period of time (exceptions being, for
example, situations involving small children or immuno-compromised individuals).

The various sampling methods and their characteristics are summarised in Table 10.1.

 Sampling methods and their characteristics

Sampler type Principle Flow rate(1 min- Cut-off 110 (/m)

l) diameter(m50)

Sieve Impactor
impaction onto agar plate 28.3 0.65-7.0
(Anderson)Slit

Sampler (e.g. Casella) impaction onto rotating

30-700 ~0.5
agar plate

Centrifugal Impactor(RCS) impaction due to

40 4.0
centrifugal acceleration

impingement into
Impingers (e.g. AGI) 12.5 0.3
liquid

PBI SAS Sampler onto agar plate

90/180 2.0
(single-stage impaction) impaction

Settle plates gravity non-volumetric N/A

Contact plates surface sampling non-volumetric N/A

However,

Slit Sampler - Slit sampler is an efficient and

convenient device for the enumeration of bacteria
carrying particles in a unit volume of air. It was
introduced by Bourdillon et al. in 1941. It works on
the principle that when air is drawn from the
environment at a fixed rate and the suspended
particles are allowed to impinge on the surface of an
agar plate, on incubation each particle will form a
colony.
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The equipment consists of a box closed by an air tight door.  There is a slit of 0.33 mm width and 27.5 mm
length with vertical parallel sides of about 3mm deep at the top through which the sampled air enters.
The box is connected to a suction pump which maintains it at a negative pressure of 22.6 mm mercury. At
the correct negative pressure air will enter through a slit of above dimension at the rate of one cubic foot
(28.3 lit.) per minute.

Inside the box at the bottom there is a rotating circular platform for keeping the agar plate. The platform
is usually covered with adhesive or gripping material to ensure the agar plate being rotated with the
platform and is not slipping out of position while rotating. When the agar plate is correctly positioned on
the platform the slit will be exactly 2mm above and along the radius of the plate. Thus when the plate is
rotated along with platform

Slit Sampling Procedure - Agar plates with dry, even surface are selected and marked to record the
sampling area, time of sampling, duration of exposure, volume of sampled air etc. The slit should be
unblocked and free from dust and if necessary may be cleaned with alcohol and by inserting the edge of a
stiff paper. The door of the box is opened and the plate is placed at the centre on the platform.

The distance between the agar surface and the slit is adjusted to be 2mm. At the correct time, the motor
that rotates the plate and the suction pump that evacuates the sampler are switched on and the negative
pressure is maintained at -22.6 mm mercury. After sampling for the time necessary to collect the required
volume of air the suction pump and the rotor are switched off.

The door is opened and the plate is taken out. The plate is covered with the lid immediately and
incubated at 37°C for 24 - 48 hours. After incubation the colonies are counted and the result is expressed
as the number of bacteria carrying particles per given volume of air.

Limitations of Slit Sampling:

 When sitting and operating the sampler inside a room major source of  bacterial contamination in the air
is the dust from the skin and clothingsof the operating person liberated by the body, movements.
Thereforeunnecessary movements should be avoided while sampling

Advantages of Slit Sampler:

Highly efficient device and can collect upto about 95% of the water droplet particles sprayed into air.
Even respiratory secretion dropletnuclei of 0.2µm diameter can be collected in large numbers

Disadvantages of Slit Sampler:

Slit sampler is cumbersome and noisy equipment. When the vacuum pump is enclosed with acoustic
insulation noise can be reduced to someextent. Quieter and portable samplers are also available but they
areless efficient in collecting sample particles

Sieve Sampler:
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This is a mechanically simpler form of impinger. The instrument ismore or less similar to that of slit
sampler with an enclosed chamber.The particles containing microorganisms are distributed over the
plateas separate air jets through several holes. Upon incubation these particles form colonies which can be
counted.For more efficient sampling and size grading of particles Andersondeveloped a multistage sieve
device in which several impingers withholes of different sizes are arranged in series.

Electrostatic Precipitation:

Electrostatic precipitation is an efficient method of removing particlesfrom air. In Litton large volume air
sampler the air is allowed to passthrough the electrodes.The charged particles fall on a rotating disc
which is fed with collectingfluid at a rate of 10ml per, minute. Air is sucked into the chamber by arotating
fan at the bottom. The low resistance of the system enableshigh rates of air flow. They are suitable for
large volumes of air.Luckiesh et al. devised a sampler which contains two removablecovers. Each unit has
one upper electrode and one lower electrode. Inone unit the upper electrode is negative and the lower
electrode is positive and in the other unit the electrical condition is reversed. Air isdrawn at equal rates in
both the units. Charged microorganisms arecollected in the petridishes placed on opposite electrodes.

Hazardous Procedures in the Lab

Care should be taken while utiIising the procedures given below:

Syringe and needle

'Needle stick' injury is common during use or disassembly.
Aerosol may be liberated from a vibrating needle on withdrawal from a vein or a culture.
Splashing or spraying may be caused by the forceful ejection of contents.
Skin, clothing or bench may be contaminated by leakage from syringe.

Pipetting
Infective material may be ingested during mouth pipetting.
Careless handling of the pipette may result in aerosol formation while transferring inoculum (when the
broth is dropped into the tubes).

Inoculating loop
Vibration of an inoculation loop especially if longer than 4 cm can cause splashing and aerosol
production.

Flaming of a wet loop or cooling a hot loop in an agar plate, mixing a slide agglutination test or spreading
a film may also produce aerosol.

Petridishes
Water of condensation on the agar or in the lid may become contaminated and spill onto fingers and
bench.
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Shaking/mixing
Shaking produces an aerosol even in a closed container and the aerosol may be released on opening the
container.

Centrifugation

Vibration can generate aerosol within the container used for centrifugation.

Careless loading or unloading, breaking during centrifugation or premature opening after breakage can
lead to dissemination of culture fluids.

Freeze drying
Opening of sealed ampoules is hazardous if done in the open occupied room. It should be done only
inside the safety cabinet since it can generate aerosols.

Stoppering tubes
As previously discussed, opening tightly sealed cork stoppers, screw capped tubes or even cotton wool
plugs can cause splashing, leading to aerosol formation.

Microbiological safety cabinets

Unless properly installed, these cabinets can release infection into the air of the room or exhaust duct.

Animal procedures
Working with animals like during inoculation, collection of samples or performing necropsy (autopsy)
can account for lot of injury.

Bedding contamination with excreta or urine can liberate infected dust.

Transport of specimen
Improperly closed and packaged samples may leak onto the wrapping and contaminate the
surroundings.

Outside of the container may get contaminated during specimen collection.

Disposal of cultures
Contamination may occur during decontamination of discarded cultures, specimen containers and used
equipments (scalpels, needles).
Laboratory associated infections are quite serious and there is considerable rate of mortality. Safety in the
lab is the responsibility of all those working in it.