APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1978, p.

1095-1101
0099-2240/78/0035-1095$02.00/0
Copyright 1978 American Society for Microbiology

Vol. 35, No. 6

Printed in U.S.A.

Airborne Bacteria in an Urban Environment

ROCCO L. MANCINELLIt * AND WELLS A. SHULLS
Department of Environmental, Population, and Organismic Biology, University of Colorado, Boulder,
Colorado 80302

Received for publication 31 October 1977

The earth's atmosphere is teeming with airborne microorganisms. These organisms are
thought to exhibit correlations with air pollution
and weather. Most airborne bacteria originate
from natural sources such as the soil, lakes,
oceans, animals, and humans. Many "unnatural"
origins are also known, such as sewage treatment
(1, 21), animal rendering (23), fermentation processes (8), and agricultural activities which disturb the soil. Viable airborne microorganisms
are not air pollutants, but should be considered
as a factor affecting air quality (29). Several
early investigations were undertaken to attempt
to determine the relationship between the number of viable bacteria found in the air and various
meteorological parameters, such as temperature,
humidity, and wind speed. No relationships were
found (22). Since that time more sophisticated
techniques for measuring air pollutants and viable airborne microorganisms have been developed. These developments have led to more
accurate studies which show that correlations
do exist between viable microorganisms and air
pollutants (16, 29).
Lee et al. (16) demonstrated that correlations
exist between bacterial density and carbon monoxide, hydrocarbons, nitric oxide, nitrogen dioxt Present address: Tri-County District Health Department, Adams City, CO 80022.

ide, and sulfur dioxide. Lighthart and co-workers

(17, 18) investigated the effects of various concentrations of carbon monoxide and sulfur dioxide on various microorganisms in the laboratory
and showed that these agents reduce bacterial
density extensively in log-phase cultures and
only partially in stationary-phase cultures.
A wide variety of air sampling devices has
been developed for trapping viable airborne microorganisms. Some are complicated, such as
the Anderson sampler (3), which attempts both
to size and to count bacteria by passing the air
to be sampled through a series of pores of various
sizes. Buchanan et al. (6) used a liquid scrubber
to trap bacteria from an aerosol. May (20) used
a monolayer of soft agar and impinged bacteria
onto it. Timmons et al. (25) developed a sampler
in which the air is passed across a gelatin foam
via a vacuum pump. This apparatus was
mounted on a probe in front of an airplane and
connected to an anemometer. This hook-up allowed for changing the voltage to the pump,
thereby varying the air flow across the gelatin.
This system has various technical problems, including the foam trap which is not very accurate
due to the difficulty in attaining and maintaining
laminar flow through a system that is constantly
varying at the high wind velocities encountered
while flying. The Anderson sampler has a large

1095

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Samples were taken at random intervals over a 2-year period from urban air
and tested for viable bacteria. The number of bacteria in each sample was
determined, and each organism isolated was identified by its morphological and
biochemical characteristics. The number of bacteria found ranged from 0.013 to
1.88 organisms per liter of air sampled. Representatives of 19 different genera
were found in 21 samples. The most frequently isolated organisms and their
percent of occurence were Micrococcus (41%), Staphylococcus (11%), and Aerococcus (8%). The bacteria isolated were correlated with various weather and air
pollution parameters using the Pearson product-moment correlation coefficient
method. Statistically significant correlations were found between the number of
viable bacteria isolated and the concentrations of nitric oxide (-0.45), nitrogen
dioxide (+0.43), and suspended particulate pollutants (+0.56). Calculated individually, the total number of Micrococcus, Aerococcus, and Staphylococcus, number
of rods, and number of cocci isolated showed negative correlations with nitric
oxide and positive correlations with nitrogen dioxide and particulates. Statistically
significant positive correlations were found between the total number of rods
isolated and the concentration of nitrogen dioxide (+0.54) and the percent relative
humidity (+0.43). The other parameters tested, sulfur dioxide, hydrocarbons, and
temperature, showed no significant correlations.

1096

MANCINELLI AND SHULLS

rugged, and easy to handle in the field and would

exhibit laminar flow. The sampler that was developed
is a Millipore Swinnex-47 filter holder (Millipore
Corp., Bedford, Mass.), for a 47-mm filter, with the top
cut out along the inside edge of the gasket wail exposing the surface of the filter. This holds a white gridded
47-mm 0.45-,um membrane filter. A specially constructed glass housing was fitted over the filter holder
and filter. This construction allows the air entering
the system, along with any particles in the air, to be
evenly distributed on the surface of the filter (Fig. 1).
The even distribution of air and particles is possible
because this apparatus is designed so that the air flow
is laminar. This was demonstrated in the laboratory
by setting up the apparatus and placing a source of
smoke in front of the glass housing. The smoke progressed through the tube evenly in a straight line
toward the filter. A vacuum pump was connected to
the filter holder via a 10-foot (ca. 3.05 m) vacuum
hose, and 0.5 atmosphere of vacuum was applied to
the filter. The filter holder was held in an upright
vertical position with a three-pronged clamp and ringstand. The vacuum pump was placed 10 feet (ca. 3.05
m) away from the sampler and isolated from it so as
not to cause any false perturbations in the area from
the pump exhaust.
A Hastings hot-wire anemometer was used to de-

FIG. 1. Glass housing and modified Millipore

Swinnex-47 filter holder.

termine that the rate of air flow through the system

was 30 liters/min. This measurement was then used
to calculate the Reynolds number of the system. It is
generally accepted that when the Reynolds number is
less than 2,000 the flow is laminar (15). Using standard
conditions of temperature and pressure, p = 1.2 x 10-3
g/cm3; V = 30.48 cm/s; D = 4.7 cm; and vq = 1.9 x 10-4
p. The Reynolds number for this system is 904.8. Since
this is less than 2,000, it is accepted that this apparatus
exhibits laminar flow. The variance in testing conditions from the standards above did not show any
significant change in the Reynolds number.
The sampler developed for this study was effective
for sampling viable microorganisms at times of little
or no wind. Difficulty arose when sampling during
windy conditions. It has been proven that the efficient
sampling of moving air must be done isokinetically
(19). If V is the velocity of air moving inside a tube
and V. is the velocity of air outside the tube, then,
when V > V., the concentration of particles inside the
tube is less than the concentration of particles outside
the tube. The reverse is true if V < V.. If there is no
wind, or a negligible amount of wind, then V. = 0 or
very near zero, and the air flow is pulled in evenly
from all directions at the end of the tube. The difficulty
is due to the inconsistency of wind velocity. For ideal
isokinetic sampling, the sampler must be facing into
the wind, and the air flow through the sampler must
be varied with any change in wind velocity.
The efficiency of the sampler for this study was
determined by constructing an atomizer and an aerosol
chamber. The aerosol chamber consists of a glass
conduit 25 cm in diameter and 1 m in length, tapering
to a ground glass joint at one end. Affixed to the
ground glass joint is a specially made atomizer. Placed
in the glass conduit is a movable Teflon piston-like
sampler housing holder. This holder has a neoprene
gasket along the outer edge and a neoprene 0-ring
surrounding its center hole, giving an airtight seal
around the filter holder glass housing (Fig. 2). The
movable Teflon piston allows one to position the air
sampler at various distances from the atomizer along
the glass conduit. After several trials, it was determined that the optimum position for the sampler was
at a point approximately 2 cm in front of the aerosol
cloud. Since all of the flow determination tests were
carried out with the sampler in a vertical position, a
1% solution of bromocresol purple was atomized into
the chamber and sucked onto the filter to determine
the effects, if any, of gravity on the system when in a

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surface area which lets bacteria collect on the

sampler walls, allowing them to go undetected,
and does not exhibit laminar flow in the system.
This means that eddy currents form which cause
the bacteria to collect in groups and allow the
development of a colony from more than one
organism, yielding erroneous data for the calculation of the total number of bacteria. Buchanan's liquid scrubber and May's agar gel
have similar problems. Millipore samplers correct most of the problems associated with the
above-mentioned devices. However, the best filter holder made by Millipore for aerosol sampling has a narrow orifice, making it difficult to
attain laminar flow while sampling 900 liters of
air in 30 min, as was done in this study.
A filter holder exhibiting laminar flow was
developed to sample viable airborne microorganisms. This apparatus also separates individual
organisms so that each colony that develops can
be attributed to one single microorganism.
The purpose of this study was not only to
determine the existence of an interrelationship
between the number of viable bacteria and air
pollutants, as well as between the number of
viable bacteria and weather, but also to identify
the genera of bacteria found in the air and to
correlate this information with air pollution and
weather parameters.
(This paper is in partial fulfillment by R.L.M.
of the requirements for the M.A. degree at the
University of Colorado, Boulder.)
MATERIALS AND METHODS
An air sampler was designed that would be simple,

APPL. ENVIRON. MICROBIOL.

AIRBORNE BACTERIA IN AN URBAN ENVIRONMENT

VOL. 35, 1978

1097

I I

horizontal position. Upon drying, there was a light

color evenly distributed on the surface of the
filter, indicating that the horizontal position had a
negligible effect on the sampler. Several tests were
then made by placing into the atomizer 25 ml of a 10'
dilution of a 1:1 mixture of 24-h-old cultures of Serratia marcescens and Escherichia coli K-12 in nutrient broth. The 10' dilution was made from a 1:1
mixture of the bacteria that had been first diluted with
nutrient broth in a Spectronic-70 spectrophotometer
until the percent transmittance reached 75% at a wave
length of 555 nm. Five 1-ml samples of the 10-5 dilution
were plated onto petri dishes containing nutrient agar
as a control to determine the number of bacteria
placed into the atomizer. The bacterial nutrient broth
solution was then made into an aerosol over a period
of 10 min. The amount of nutrient broth remaining in
the flask was measured and subtracted from the total
of 25 ml added, yielding the amount that had been
atomized. The ifiter was aseptically removed and
placed in a 47-mm petri dish on top of an absorbent
pad with 3 ml of nutrient broth and incubated at 25C.
After 48 h, the colonies that developed on the plates
green

counted. This total was then calculated as a

percent of the total number of bacteria atomized,
which for 10 trials resulted in an average efficiency of
85% for the sampler. The filter showed an even dispersal of the quite distinct red and white colonies. To
determine whether the number of viable bacteria per
milliliter of fluid atomized varied from that of fluid not
atomized, three 10-ml samples of a mixture of S.
marcescens and E. coli K-12, prepared as described
above, were atomized into three sterile flasks. A 1-ml
sample from each flask was plated out into petri dishes
containing nutrient agar and incubated at 24C for 48
h. Three 1-ml samples of the unatomized fluid were
placed into three petri dishes containing nutrient agar
and incubated at 25C for 48 h. The results showed
only a 3% variance in the number of viable organisms
in the two sets of fluid plated.
Two samples of S. marcescens were atomized and
trapped onto filters. These filters were then criticalpoint dried, mounted onto metal studs, and coated
with gold in preparation for scanning electron microscopy. Scanning electron micrographs showed no broken cells and no organisms that had impinged at the
same locus.
The sampler was then tested under experimental
conditions atop the laboratory building 10 m above

were

ground level and 304.8 m from a street whose average

traffic density is 20 automobiles per min. These samples were taken randomly during the day from 10:00
a.m. to 3:00 p.m. under both cloudy and clear skies,
with the wind velocity ranging from 0 to 4 miles per
hour (0 to 6.4 km/h). The apparatus was set up as
previously described with the filter facing upwards
perpendicular to the ground and the vacuum pump
isolated inside the building. Each sample was run for
30 min, resulting in 900 liters of air being filtered per
sample. After each sample was taken, the filter was
aseptically removed and placed in a 47-mm petri dish
on top of an absorbent pad containing 3 ml of nutrient
broth and incubated at 250C. After 48 h, the colonies
that developed on the plate were counted and transferred to nutrient agar slants. Each organism isolated
was then identified by use of a flow chart based on the
taxonomic criteria outlined in Bergey's Manual of
Detenninative Bacteriology, 8th edition (7). Plates
were also incubated at 37 and 15C. These plates
showed no significant difference in the numbers and
types of bacteria isolated. Plates were also incubated
for 72 h, 96 h, and 10 days, with no increase in the
number of colonies formed after the initial 48 h.
The procedure used in this study is not favorable
for the isolation of Actinomycetes, which require acidified nutrient broth enriched with 5% glucose, nor is
this procedure recommended for the isolation of fungi
or any eucaryotic cells.
The Environmental Protection Agency and the Colorado Department of Health furnished the air pollution data. The National Oceanographic and Atmospheric Administration furnished the weather data.
The correlation coefficients (r) were calculated using the Pearson product-moment method. For a sample number of 21 and a 5% level of significance, the
critical value for r is 0.43 (30).

RESULTS
A summary of the pollutant concentrations,
number of viable bacteria found, and meteorological parameters measured, is given in Table
1. The incomplete data in this table were due to
lack of recorded data from the Environmental
Protection Agency, the Colorado Department of
Health, and the National Oceanographic and
Atmospheric Administration.

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FIG. 2. Atomizer and aerosol chamber. (A) Airpressure inlet and atomizer; (B) glass housing for filter; (C)
outer Teflon piston gasket; (D) front view of Teflon piston; (E) center hole for filter glass housing; (F) hex
screw for tightening outer gasket; (G) Erlenmeyer flask.

1098

APPL. ENVIRON. MICROBIOL.

MANCINELLI AND SHULLS

airborne bacteria isolated in the samples and

their percentage of occurrence in the urban air.
DISCUSSION
Several investigations have been conducted to
try to determine the factors that affect the survival of airborne bacteria (16-18, 26). Some of
the most frequently mentioned factors that influence bacterial viability are relative humidity,
temperature, and radiation. Most of these studies have been done with artificially generated
aerosols and have been difficult to compare,
since different methods were used to collect and
in some cases to generate the bacterial aerosols.
Also the relationship of laboratory bacteriological aerosols to bacteria found in urban air has
not been clearly established.
Table 2 presents a summary of correlations
between all the parameters measured in this
study. The correlations were determined by the
Pearson product-moment method. For a sample
number of 21 and a 5% level of significance, the
correlation coefficient must be equal to or
greater than 0.43 (30). Suspended particulate
matter was associated with automotive pollutants including nitric oxide (NO), nitrogen dioxide (NO2), and total hydrocarbons (16). Meteorological parameters were associated with air
pollutants. Significant negative correlations existed between temperature and nitric oxide, nitrogen dioxide, hydrocarbons, and carbon mon-

TABLE 1. Number of viable bacteria per liter, air pollutant concentrations, and weather conditions that
existed at the time of sampling
Sample
no.

No. of bacteria per liter

0.320
0.187
0.573
0.960
0.813
0.800
0.253
0.120
0.013
0.120
0.120
0.266
0.053
0.026
0.573
1.800
1.880
1.810
1.173
0.330
0.424
'NA, Not available.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21

Particu-

0 19Ml
N2(LM)
ug
in')/l
ae

0.08
0.08
0.08
NAa

NA
NA
0.171
0.100
0.070
0.014
0.015
0.015
0.205
0.140
0.125
0.011
0.014
0.014
0.069
0.058
0.118

0.041
0.040
0.042
0.040
0.040
0.040
0.087
0.075
0.055
0.058
0.058
0.058
0.066
0.082
0.085
0.094
0.094
0.094
0.069
0.037
0.069

0.002
0.004
0.003
0.000
0.010
0.010
0.031
0.021
0.008
0.007
0.005
0.005
0.008
0.034
0.034
0.010
0.008
0.008
0.010
0.004
0.005

95
100
100
35
35
35
55
55
55
30
30
30
65
65
65

120
120
120
56
NA
NA

Hydrocar- % Relative Tm
os(g humidity
12
13
13
35
35
35
2.8
2.8
2.8
2.2
2.2
2.2
4.0
4.0
4.0
3.2
3.2
3.2
1.6
NA
NA

16
16
16
20
20
20
40
35
25
15
15
15
30
25
23
27
27
27
25
31
NA

'

13.89
9.44
9.44
22.22
22.22
22.22
14.44
14.44
14.44
10.00
10.00
0.00
0.00
0.00
12.78
8.89
8.89
8.89
14.44
10.00
18.33

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The average number of viable airborne bacteria found was in reasonable agreement with
the findings of other investigators (1, 16, 27, 29).
In this investigation, the number of viable bacteria isolated ranged from 0.013 to 1.88 bacteria
per liter (Table 1).
Table 2 lists the correlation coefficients for
the prevalent bacterial genera and the total
numbers of rods, cocci, and viable bacteria
found. The data in Table 2 were consistent for
all parameters tested. Nitric oxide (NO) exhibited a negative correlation, -0.25 to -0.45,
with each of the bacterial parameters tested.
Nitrogen dioxide (NO2) and airborne particulates each exhibited positive correlations with
the bacterial parameters. Nitrogen dioxide
(NO2) statistically had a significant positive correlation with the total number of rods isolated,
+0.54. Airborne particulates exhibited a statistically significant correlation with the number of
Micrococcus isolated, + 0.45, and with the total
number of viable bacteria isolated, +0.56. Sulfur
dioxide (SO2), hydrocarbons, and temperature
did not show any statistically significant correlations. Relative humidity was only statistically
significant correlations. Relative humidity was
only statistically significantly correlated with
the number of rods, with a correlation coefficient
of +0.43, and concurred with data presented by
Lighthart et al. (18).
Table 3 gives the genera of all the viable

AIRBORNE BACTERIA IN AN URBAN ENVIRONMENT

VOL. 35, 1978

1099

TABLE 2. Correlation coefficients of bacteria andpollutantsa

Bacterial parameter

NO

NO2

SO2

Particulates

Micrococcus
Aerococcus
Staphylococcus
Total no. of rods isolated
Total no of cocci isolated
Total no. of viable bacteria
isolated

-0.37
-0.25
-0.31
-0.34

_0.45b

+0.43b

-0.12
-0.02
-0.03
-0.01
-.011
-0.03

+0.45b

-0.30

+0.29
+0.27
+0.35
+0.54b
+0.29

HydrocarRelative
bons
humidity

Temp

+0.31
+0.38
+0.41
+0.37

+0.00
+0.01
-0.03
-0.05
+0.04

+0.04
+0.06
+0.12
+0.43b
+0.07

-0.02
-0.05
-0.01
+0.01
+0.04

+0.56b

+0.14

+0.12

+0.17

TABLE 3. Percentage of occurrence of each genus

isolated
Genus

% Occurrence

Micrococcus ................... 41
8
Aerococcus ...........
11
Staphylococcus ..........
Peptococcus ....
......
3
Peptostreptococcus ............. 4
Neisseria ...............
3

Streptococcus .................. 3

Paracoccus .... 5
Pediococcus ...........
2
BaciUus ....8...............8
Sarcina ............. 4
SporolactobaciUus ............. <1
Clostridium ..............
<1
Sporosarcina ............ <1
Serratia
............. 3
Pseudomonas
.... 2
Leuconostoc .<1
Xanthomonas ............... <1
Lactobacillus ....... ..... <1

oxide (16). A significant positive correlation has

been shown to exist between relative humidity
and carbon monoxide. A significant negative correlation exists between relative humidity and
sulfur dioxide (16). These observations are important because they give some idea of the complex dynamic interactions that are occurring in
the atmosphere.
There is a definite effect of air pollutants on
airborne bacteria viability. As shown in Table 2,
there was a statistically significant correlation
between the total number of viable bacteria
isolated and the concentrations of suspended
particulate matter (+0.56). The depth of this
effect is reinforced by the data shown in Table
3 for Micrococcus, Aerococcus, and Staphylococcus, the total number of rods isolated, and
the total number of cocci isolated. It is reasonable to assume that bacteria in the air are protected from harsh environmental conditions,

such as drying, by some of the suspended particulates. It was also felt that a 30-min sampling
period would not produce a significant drying
effect because any bacteria which were trapped
were viable, since the effect of drying had already occurred in the air before sampling began.
This can account for the significant correlation
coefficient observed for this parameter. Empirical observation of the filters showed that whenever there was a large amount of particulate
matter on the filter there was also a large number of viable bacteria on the filter.
Statistically significant correlations were
found between the number of viable bacteria
and nitric oxide (-0.45) and nitrogen dioxide
(+0.43). There were no correlations found between the number of viable bacteria and sulfur
dioxide, hydrocarbons, relative humidity, and
temperature. The same type of results were obtained for Micrococcus, Aerococcus, Staphylococcus, and the total number of cocci isolated. A
significant correlation was found between the
percent relative humidity and the total number
of rods isolated (+0.43). This may be due to the
protection of the rods from bactericidal air pollutants by humidity in the air (16).
From Table 3 it can be seen that all of the
microorganisms found are known soil inhabitants. The most prevalent organism found was
Micrococcus (41%), followed closely by Staphylococcus (11%) and Aerococcus (8%). The results
of this investigation show that gram-positive
cocci far outnumber gram-negative organisms of
any kind in the urban air. This was not necessarily the case in the soil, where there can be
found numerous rods such as Bacillus and Clostridium and more gram-negative organisms (2).
As previously stated, bacteria enter and are
dispersed into the air from various sources. The
survival of these airborne microorganisms is a
function of the weather and of the chemical
composition of the atmosphere. Certain air-pollutant gases, such as formaldehyde, acrolein,

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"Pearson product-moment correlation coefficients are given for each bacterial and air pollution pair.
Micrococcus is signficantly positively correlated with airborne particulates. The total number of rods isolated
is significantly positively correlated with N02 and relative humidity. The total number of viable bacteria isolated
is significantly correlated with NO, N02, and airborne particulates.
b Statistically significant for a sample number of 21 and a 5% level of significance.

ACKNOWLEDGMENTS
Expert technical assistance by Mary Mancinelli and Jane
Ripperger and glassblowing by Eugene Lutter are gratefully
acknowledged.

APPI,. ENVIRON. MICROBIOL.

This work was supported by a Grant-in-Aid from the Graduate School, University of Colorado, and by a research grant
from Sigma Xi.

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1100 MANCINELLI AND SHULLS

ozone, and sulfur dioxide, have been used as
disinfectants, indicating that these pollutants
definitely have a negative effect on bacterial
viability (13, 24, 28).
Lighthart et al. (18) studied the survival of S.
marcescens, a gram-negative rod, at a mid- to
high-relative-humidity range in urban concentrations of sulfur dioxide and concluded that the
death rate of these bacteria increases as the gas
concentration increases and the relative humidity decreases.
A death mechanism has been postulated to
exist for airborne vegetative gram-negative bacteria (3, 5, 17). This mechanism is related to
relative humidity and oxygen concentration
(9-12, 14, 26, 31). According to this postulated
mechanism, a gas pollutant blocks the cytochrome system. The data presented in this study
neither support nor disprove the concept of such
a death mechanism. The studies mentioned
above used prepared aerosols with prepared concentrations of the air pollutants and have a
constant control of relative humidity. This
study, however, concerns only those bacteria
actually found in the urban air, which is a much
more complex system than any of those used in
the laboratory.
Micrococcus and Staphylococcus were found
in greater numbers than most other bacteria
isolated in the samples. These bacteria contain
carotene pigments which enable them to survive
exposure to the sun's radiation better than the
other organisms isolated. This is one of the
reasons for finding a greater abundance of pigmented bacteria than of other types. Also these
organisms are known to be associated with humans, and since these samples were taken from
a populated area, it follows that a substantial
number of these organisms would be isolated
from the air.
This study has shown that there is a definite
relationship between viable airborne microorganisms and air pollution. Some of these relationships are positive, i.e., encourage bacterial
viability, and some are negative, i.e., inhibit bacterial viability. The dynamic chemical and biological interactions occurring in the atmosphere
are much more complex than has been previously realized. This study barely scratches the
surface in exposing these complex chemical and
biological interactions. Additional studies in the
laboratory, using prepared aerosols, and air sampling from urban and isolated rural areas must
be done to determine what is actually happening
in the atmosphere.

VOL. 35, 1978

AIRBORNE BACTERIA IN AN URBAN ENVIRONMENT

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27. Wolf, H. W., P. Skaliy, L B. Hall, M. M. Harris, H. M.
Decker, L. M. Buchanan, and C. M. Dahlgren. 1959.
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28. Won, E., and H. Ross. 1969. Reaction of Airborne Rhizobium meliloti to some environmental factors. Appl.
Microbiol. 18:556-557.
29. Wright, J. J., V. W. Greene, and H. J. Panlus. 1969.
Viable microorganisms in an urban atmosphere. J. Air
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