Aero Biology

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AEROBIOLOGY: SAMPLING OF

AIRBORNE MICROORGANISMS

23.1. OVERVIEW Objective: To collect air samples for subsequent microbial analysis.

• Collect bioaerosol samples with a glass impinger and filtration apparatus


• Assay samples for airborne bacteria and fungi by the spread plate and membrane filtration
methods
• Determine the concentration of bacteria and fungi per cubic meter of air

23.2. THEORY AND SIGNIFICANCE

Aerobiology has been defined as the study of aerosolization, aerial transmission, and deposition of
biological materials. A collection of airborne biological particles is called a bioaerosol. Bioaerosols
are generated by a wide variety of natural and human-made processes including coughing,
sneezing, wave action, splashes, wind, cooling towers, ventilation systems, etc. Inhalation,
ingestion, and dermal contact are routes of human exposure to airborne microorganisms, but
inhalation is the predominant route that results in adverse human health effects. Airborne
Legionella pneumophila, Mycobacterium tuberculosis, and some pathogenic viruses are known to
be transmitted by aerosols. Asthma, hypersensitivity pneumonitis and other respiratory illnesses
are also associated with exposure to bioaerosols.

Deterioration of building materials, offensive odors, and adverse human health effects are
associated with microbial contamination of indoor environments, such as residences, offices,
schools, health care facilities, enclosed agricultural structures (barns and crop storage areas)
industrial facilities and recycling facilities (Stetzenbach, 2003). Sources and reservoirs of
microorganisms are present within these settings, including building materials and furnishings,
pets, plants, and air-conditioning systems. Fungi, which can colonize drier surfaces than bacteria,
tend to grow in a wide variety of building materials, such as wallboard, ceiling tiles, carpeting and
vinyl flooring.

Temperature and relative humidity are the two most important factors affecting the survival of
microorganisms in the airborne state. For this reason these are usually recorded during the
collection of bioaerosols. In general, bacteria and fungi are more stressed as the rate of
evaporation increases, which occurs as relative humidity deceases and temperature increases.
Thus,
Figure 23-1 37 mm polystyrene 3-piece monitoring cassette. (Photo courtesy Millipore
Corporation.)

Increased survival is favored at higher relative humidity and lower temperatures. The effect of
relative humidity varies more with virus type with some surviving better at high relative humidity
and others better at low relative humidity.

There are three principal methods used to quantify microorganisms in the air.

• Impingement is the trapping of airborne practices in a liquid matrix

• Impaction is the forced deposition of airborne particles on a solid surface

• Filtration is the trapping of airborne particles by size exclusion Gravity is a non-quantitative


method used in which agar medium is exposed to the environment and airborne microorganisms
are collected primarily by settling. This method is often used because it is inexpensive and easily
performed.

A commonly used liquid impinger is the AGI-30 (ACE Glass, Vineland, NJ). The AGI-30 operates by
drawing air through the inlet and into a liquid. Any particles in the air become trapped in the
liquid, which can then be assayed for the presence of microorganisms. The AGI-30 is usually
operated at a flow rate of 12.5 liters per minutes. The AGI-30 is easy to use, inexpensive, portable,
reliable, easily sterilized, and has high biological sampling efficiency in comparison with many
other sampling devices. The usual collection volume is 20 ml, and the typical sampling time is
about 20 minutes. Longer sampling times result in too much evaporation of the liquid in the
impinger, and the inactivation or death of microorganisms in the liquid.
The impaction method separates particles from the air by utilizing the inertia of the particles to
force their deposition onto a solid or semi-solid surface. The collection surface is usually an agar
medium. The Anderson sixstage impact or sampler (Anderson Instruments Inc., Smyra, GA) consist
of six stages with decreasing nozzle diameters, so that successive stages collect progressively
smaller particles. Thus the six-stage sampler measures the cultivable bioaerosol concentrations in
specific particle size ranges.

Filtration techniques are used largely for the collection of fungi and bacterial spores because they
are desiccation resistant. Filters are usually held in disposable (although they may be reused)
plastic filter cassettes during bioaerosol sampling. Membrane filters used for sampling are usually
supplied as disks of 37- or 47 mm diameter. Because the pressure drop across a filter increases
with air velocity through the filter, the use of larger filters results in a lower pressure drop for a
given volumetric flow rate. The use of the smaller (37-mm) filter concentrates the organisms onto
a smaller total area, thus increasing the density of particles per unit area of the filter. This may be
helpful for direct microscopic examination of low concentrations of organisms. In areas of high
concentration, the organisms may have to be eluted, diluted, and then refiltered for microscopic
examination or assay. For a better quantitative measure of the air volume sampled, a limiting
orifice may be placed between the cassette and the vacuum source.

Figure 23-2 Liquid impingement device for collecting biological aerosols.


23.3. PROCEDURE

First Period

Materials

All glass impinger AGI-30

1 37-mm air monitoring cassette

20 ml of 0.1% peptone solution

1 500- or 1000-ml Erlenmeyer flask

rubber or plastic tubing for connecting the impinger and cassette to the vacuum source

1 vacuum pump or vacuum source

1 100 ml sterile graduated cylinder

1 dilution blank with 0.1% peptone or phosphate buffered saline

2 1-ml pipettes

1 10-ml pipette

4 sterile 0.45m pore, 47-mm diameter membrane filters

1 filter unit (same as in Experiment 10)

2 sterile 37 mm 0.45mm pore filters

vacuum pump or other source

fórceps

gas burner

pipette bulb

vortex mixer

4 nutrient agar (NA)1 or trypticase soy agar (TSA)1 plates

4 Sabouraud dextrose agar (SDA)1 plates


AIR SAMPLING BY IMPINGEMENT

1. Set up the AGI-30 all glass impinger as shown (Figure 23-2).

2. Add 20 ml of 0.1% peptone to the reservoir followed by 0.1 ml of anti-foam.

___________

1 Anti-foam B (Sigma Chemical Company, St. Louis, Missouri).

3. Add 0.1 ml of anti-foam agent.

4. Turn the vacuum source on for 10 minutes.

5. With a 1-ml pipette remove 0.5 ml of fluid from the reservoir and place 0.1 ml each on one agar
plate of either NA or TSA and spread plate the samples as described in Experiment 5. Place
another 0.1 ml on a plate of Sabouraud dextrose agar for detection of fungi.

6. With a 10 ml pipette remove 6 ml of liquid from the reservoir and pass 5 ml through a 0.45mm
membrane filter as described in Experiment 10. Place the filter on an NA or TSA plate. Repeat the
procedure, but place the membrane filter on Sabouraud dextrose agar.

7. Incubate the NA or TSA plates at 35°C for 24–48 hours.

8. Incubate the SDA for 2 to 7 days.

AIR SAMPLING BY FILTRATION

9. Connect the air-sampling cassette to the vacuum source.

10. Turn on the vacuum source for 10 minutes.


Figure 23-3 Sieve-type sampler (Andersen sampler) for biological aerosols.

11. Remove the membrane filter from the cassette with a pair of flamed forceps (see Experiment
10). And place on either a plate of NA or TSA.

12. Repeat the same procedure placing the membrane filter on plate of SDA.

13. Incubate the plates as described under impingement.


SECOND PERIOD

Materials

Incubated plates from the previous Period 1

1. Examine the agar plates and count the umber of bacteria (NA or TSA) and fungi (SDA)
colonies.

23.4. TRICKS OF THE TRADE

DO NOT:

• Leave the vacuum on for more than 10 minutes as the fluid in the AGI- 30 will decrease
significantly in volume and the bacteria in the filtration cassettes will desiccate.

23.5. CALCULATIONS

Calculate the number of bacteria and fungi per cubic meter.The AGI-30 limiting orifice at the end
of the glass tube, which is submerged into the collection liquid, limits the amount of air passing
through the liquid to 12.5 liters per minute. The concentration of microorganisms is usually
reported as numbers per cubic meter of air, which is calculated as follows.

A) Volume of air (L) = Sample time (min) x 12.5 L/min


B) Number of organisms collected by impinger = Number of organisms in volume assayed
(CFU/ml) x ml remaining in impinger after operation*
C) Number of organisms per L of air (CFU)= Number of organisms collected by
impinger/Volume of air

23.6. QUESTIONS AND PROBLEMS

1. What are the limitations of the different methods for detection of microorganisms in air?

-------------------

*Remember the volume of the liquid in the impinger may decrease during operation because of
evaporation.
2. Why should you not collect an air sample for more than 20 minutes with an AGI-30?

3. Why are fungi of concern in the air?

4. Why are bacterial spores and fungi more likely to be detected with a filtration cassette than
vegetative bacterial cells?

5. What types of environments would you expect to have high concentrations of bacteria in the
air? Low concentrations?

6. How does relative humidity effect the survival of airborne microorganisms?

23.7. REFERENCES

Buttner, M.P., Willeke, K., and Grinshpun, S.A. (2003) Sampling and analysis of airborne
microorganisms. In: Manual of Environmental Microbiology. C.J. Hurst, R.L. Crawford, G.R.
Knudsen, M.J. McInerney, and L.D. Stetzenbach, eds., pp. 814–826. ASM Press. Washington, DC.

Dowd, S.C., and Maier, R.M. (2000) Aerobiology. In: Environmental Microbiology. R.M. Maier, I.L.
Pepper, and C.P. Gerba, eds. pp. 91–122. Academic Press. San Diego, CA.

Durand, K.H., Muilenberg, M.L., Burge, H.A., and Seixas, N.S. (2002) Effect of sampling time on the
culturability of airborne fungi and bacteria sampled by filtration. American Occupational Hygiene
46, 113–118.

Lundholm, I.M. (1982) Comparison of methods for quantitative determination of airborne bacteria
and evaluation of total viable counts. Applied and Environmental Microbiology 44, 179–183.

Stetzenbach, L.D. (2003) Introduction to Aerobiology. In: Manual of Environmental Microbiology.


C.J. Hurst, R.L. Crawford, G.R. Knudsen, M.J. McInerney, and L.D. Stetzenbach, eds., pp. 801–813.
ASM Press. Washington, DC.

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AGI-30 can be obtained from Ace Glass Inc., Vineland, NJ. www.aceglass.com. Air sampling
cassettes, 37 mm filters, and vacuum pumps can be obtained from Pall Gelman Laboratory, Ann
Arbor, MI. www.pall.com/gelman and Millipore Corporation, Bedford, MA. www.millipore.com.

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