Microorganism in Air.

The air almost always contains microorganisms, but their numbers vary depending on
weather, time of day, season and location in the outdoors, and on sources and activity
indoors.

Concentration of m’orgs outdoors seldom exceeds 105/m3 and only rarely reach 106/m3.

Indoor concentrations varies widely depending on local sources.

e.g. In relatively clean room environments <100 cfu’s/m3.

In farm buildings up to 2.9 x 109/m3 have been found when mouldy hay was being
handled.

There is no air microflora as such but microorganisms are released into the air from a
variety of sources and activities.

Sampling the atmosphere for the presence of particles, especially of biological origin,

may enable one to establish the cause and spread of infectious disease and other

environmental contaminants and is particularly important in hospitals, pharmaceutical

and health care industries, food processing, biotechnological and brewing industries.

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When planning a programme of air sampling
three measurements are of utmost importance:

a) Concentration and type of microorganisms in the air,

b) Size of the particles on which they are contained and,

c) Change in concentration with time.

1. Concentration & type.

Depends on location/source and activity.

Concentration may vary from extremely dilute to highly concentrated.

(In health care and pharmaceutical industries, specifications may require zero
to very low numbers of particles carrying m ‘orgs)

In agricultural environments the concentration may be very high.

Indoor occupied habitats tend to have more bacteria

suspended in the air whereas in agricultural
environments fungal spores would be more common.

Therefore it is essential to be able to collect a large volume of air and concentrate the
microorganisms for subsequent analysis especially in clean areas.

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In environments with high density of particulates a different approach to
collection is crucial as it is too easy to overwhelm the sampling device.

In such situations Impingement or centrifugal samplers may be more appropriate

as they allow dilution of the sample.

Sampling conditions can be designed to detect specific types of microorganisms.

With Impactors and some centrifugal types of samplers only one type of
microorganism may be sampled at a time e.g. Total Bacteria, Yeasts and
moulds, Staphylococcus.

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2 Particle size.

The size of the particles carrying the microorganisms is of importance in

predicting the penetration and retention in the respiratory tract as well as the
maintenance of viability during airborne travel.

Knowledge of the size of the particle is important in designing and selecting

samplers for use,
Some samplers are not efficient at collecting small particles, i.e. Biotest sampler.
Other samplers i.e. Anderson sampler are designed to collect particles down to
1m.

3 Changes in concentration with time.

The ability to detect changes in concentration or type of m’org, with time can be
very useful.

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The choice of air sampling device depends on the aims of the sampling
programme.

There are three principal groups of air sampling devices.

(i) Inertial collectors,

(ii) Filters,

(iii) Electrostatic and thermal precipitators.

Inertial Types
A. Impactors.

Cascade Impactor,

Slit air sampler ( Casella)

Anderson sampler (2 & 6 stage)

Hirst spore trap.

Burkard Spore trap for pollen and fungal spores.

B. Impingers
Porton Impinger, AGI, Millipore,
Multistage Liquid Impinger.

C Sedimentation (Settle plates)

D Filters

E Centrifugal.

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Inertial Collectors
In designing this type of sampler, use is made of the fact that particles moving in an air
stream have an individual inertia and may therefore be deflected from their original
path and be trapped onto a surface (IMPACTION) or into a liquid (IMPINGEMENT).

The inertia of the particle depends on the size and weight of the particle.
e.g.
a) Settle plates are best suited for still air conditions and selects for large particles
only, the greater the air movement the greater the particle size selected.

b) Impingers, have a jet, which when air is passed through, increases the velocity
of the air and hence the inertial velocity of any particles in the air stream.

The sampler contains liquid (impingement fluid) through which the air is forced
collecting the particles, which can then be assayed for m’orgs.

Many advantages with this method, including concentration of dilute aerosols,

collection of a wide range of m’orgs allowing subsequent dilution and selection of
different groups.

c) The Impactor is based on the same principle, but the particles are directed onto
a solid surface.

Main disadvantage in heavy aerosols leads to overloading of surface and

underestimation of microbial load in the air.

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 All Glass Liquid Impinger (AGI)
Porton Impinger.

It’s particle retention efficiency is high for particles down to 0.5m diam.

Adv.

Easy to use and easily cleaned and sterilised by autoclaving.

Because of the high impingement velocity some sensitive m’orgs may be killed
during collection.

Typically operates at 11 or 25 l air/min.

The jet is a short length of capillary tubing which acts as it’s own critical orifice.
When the pressure drop across the jet exceeds 1/2 atmosphere the air flow attains
sonic velocity.

Increasing the suction further will not result in any further increase in air velocity
through the jet.

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 Multistage Liquid Impinger.
This can be used to concentrate fairly dilute aerosols by prolonging the sampling
time. There is no great loss of viability as the impingement is gentle and there is
little evaporation.

Three sizes are available, the most useful samples 50 l/min. Easily transportable,
robust and sterilisable.

The cyclone separator is also suitable for concentrating dilute aerosols.

The device is fitted with a liquid spray which is thrown onto the wall of the
sampler and continually washes down the deposited particles.

Critical orifices are used to regulate the flow of air.

Critical orifices can be attached in line to the vacuum to control air flow.
It is possible to concentrate the particles in 500 l air into 1 ml liquid for
subsequent analysis.

Typical sampling 360 l/min and 75 l/min.

Air Centrifuges.
They remove particles from the air by centrifugal force usually onto the inner
surface of a rotating cone. This surface is usually rinsed down by a spray and the
biological particles collected in a small volume of diluent. Typically 500 l air
collected into 1 ml liquid sample.
Critical orifices are used to control the flow rate.
The suspension can be assayed for the number of microorganisms or the type of

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m’orgs.

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Filters
Filtration is one of the most commonly used means of particle sampling.

i) Membrane type filters retain particles by direct action when the particles are
larger than the pore size,

ii) In the fibrous type of filter, inertial filtration takes place owing to a combination
of impaction and electrostatic charges.

Among the disadvantages of using filters include:

the effects of excessive drying on vegetative cells on the surface of the membrane,
selecting for resistant cells e.g. bacterial spores, and

blinding the filter, blocking the pores resulting in the problem of high pressure
drop across the membrane. This can force the membrane to burst or th epressure
drop results in lowering the flow rate of the sample.

iii) Electrostatic and thermal precipitators.

Particles are given an electric charge and are attracted to electrodes of opposite
polarity. Not suitable for viable biological particles. Destructive.

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 Slit Air Sampler.
Casella Slit-Agar sampler

Air is sucked in through narrow slits and is impacted onto nutrient medium in
Petri dishes.

(Use a general purpose agar medium for Bacteria, need a different medium for
yeasts and moulds, and can use selective and differential media for other
microorganisms).

The plates are rotated on a turntable in the device so the microorganisms are
collected on a wide band. Slit width, distance to agar surface, rate of rotation and
volume of air sampled can be varied in some samplers.

The collection efficiencey from an aerosol of Staphylococcus aureus, sprayed from

distilled water as single cocci was found to 96%.

For spheres of 1m diam. and unit density, impaction efficiencies of 90 and 98%
were calculated for the 1 mm and 0.3 mm slits resp.

Device is used mainly in still air environments.

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 The Cascade Impactor
Is used to collect airborne particles on microscopic slides.

A size distribution of the sample can be determined.

The particles on the slides can be stained and examined directly under the
microscope or can be washed into nutrient medium for counting and
identification, only applies if the material is resistant to drying.

The sampling rate is 17.5 l per min.

Particle Φ m
Stage 1 6-20
Stage 2 2-6
Stage 3 1-3
Stage4 0.5 - 1.5

The first two stages simulate the upper respiratory tract where pollens and larger
fungal spores are deposited causing rhinitis and asthma.

The 3rd and 4th stages trap particles that are retained in the deepest part of the
lung.

The Cascade impactor is used as the reference instrument against which the
efficiencies of other air sampling devices are compared.

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 THE ANDERSON SAMPLER.
Six stage and two stage Sampler.
This is a size grading impactor.

Air is drawn in through a circular orifice and then through a succession of six
circular plates each perforate with 400 hundred holes through which the particles
are collected onto sterile medium in petri dishes.

The succession of plates have progressively smaller holes so that the largest
particles are impacted into the first dish and the smallest into the last.

Air is sampled at 28.3 l/min and efficiency of 100% is reported for single bacterial
cells.
The first stage samples for all large particles down to 12 m.

it has been shown that a large proportion of pathogenic microorganisms in

occupied environments are carried on particles with an aerodynamic diam.
>15 m, therefore caution must be exercised in choosing an appropriate
sampler.

This device has been used on studies of Anthrax and mouldy hay.

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A two stage sampler separates particles into non respirable and respirable on the
upper and lower dish.

In this device the plates each contain 200 perforations.

One drawback of the Anderson is the potential of overloading in high

concentration atmospheres.

With a fixed number of holes per stage, the chances of having more than one
particle per hole are large when most of the holes are already filled.

Under these conditions the plates have to examined under the microscope to count
the colonies before they overgrow one another.

Like all impaction devices the sampler is limited to counting particles containing
viable cells and not capable of determining the number of cells per particle.

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Another disadvantage is that prolonged sampling can cause dehydration of the
agar plates, altering the flow rates and causing evaporative stress to the
microorganisms.

The devices are fairly expensive per cost of sample.

The greatest advantage of the sampler is its ease of operation.

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 PARTICLE SIZE CARRYING
MICROORGANISMS.

e.g. At cotton mill sites, sewage plants and using skin fragment experiments the
following results were observed.

• In the cotton mill the majority of particles carrying viable G- bacteria were in
the size range 2 to 6  m.

• At the sewage plant 62% of the particles were 5 m or greater and 30% were
between 2 to 6  m.

* In the skin fragments 48% of the particles were 8.2 m or larger.

The numbers of bacteria per particle were found to be as follows:

13 bacteria per particle at the cotton mill;

24 bacteria per particle at the sewage plant;

147 bacteria per particle in the skin, fragment experiments.

Results were obtained using the Anderson

sampler.

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Skin scales have been shown to form rafts that range in size from <4 pm to >25 pm
and on average behave as if they were spherical particles of ~14 m diam.

These particles were shown to contain on average ~4 viable Staph. aureus cells.

In the outdoor urban and rural environments it has been shown that ~77% of the
bacteria are carried on particles ≥ 9.2 m and only 13% occur as single cells.

For the most part fungal spores exist as single propagules.

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In the case of the yeast Cryptococcus neoformans, which is pathogenic for humans
and occurs in quantity in pigeon droppings, 60% of the cells were <4.7 m diam.
and 4.4% were <1.1 m diam.

These cells can penetrate deep into the lungs.

In laboratory culture media these cells occur as heavily encapsulated cells of 4 to
20 m diam.

Information from laboratory studies may sometimes be misleading and do not

always extrapolate to the field situation.

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 General Points.

Depends on the aims of the sampling programme

i.e. is the sampling programme designed to search for a particular organism or
group of microorganisms or to measure all organisms in the atmosphere?

Are the concentrations likely to be high or low?

Are the numbers of cells or the numbers of particles of importance?

• If the concentrations are suspected to be low then a cyclone sampler or other

high volume sampler can be used.

• multistage liquid sampler is useful... . . . If the density of viable particles is high

then the sampling liquid can be diluted and used to inoculate a range of culture
media. Evaporation of the sampling fluid can occur during sampling periods
longer then 30 mins.

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* The type of. recovery media can be important
• The use of inhibitory or selective media can result in reduced recoveries as the
collection of airborne microbes causes stress damage to microbial cells.

* If the density of viable particles is high then the surface of solid media samplers
can become overcrowded . although multistage impactors and Anderson samplers
tend to operate at relatively low flow rates.

• It is possible for more than one particle to enter a hole to occur, thereby
underestimating the total particle count, also one cannot obtain the number of
viable cells in each particle since each particle gives rise to a visible colony (may
have to examine the plate surface microscopically) and excessive sampling
times can dry out the plate surface.

• Anderson sampler is designed to collect particles down to 1 m.

• The RCS is —100% efficient for collecting particles 15 m but virtually ignores
particles 1 m and less, efficiency increasing with increasing particle size. It has
—60% efficiency for particles 5 m.
• Filters, although efficient collectors of particles, may result in the recovery of
low numbers of viable cells.

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