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
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. 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
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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, 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-
APPL. ENVIRON. MICROBIOL.
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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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. 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 25°C. 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 24°C for 48 h. Three 1-ml samples of the unatomized fluid were placed into three petri dishes containing nutrient agar and incubated at 25°C 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 15°C. 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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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 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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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
"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. 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 .............
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
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. 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
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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, 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-
APPL. ENVIRON. MICROBIOL.
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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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. 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 25°C. 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 24°C for 48 h. Three 1-ml samples of the unatomized fluid were placed into three petri dishes containing nutrient agar and incubated at 25°C 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 15°C. 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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APPL. ENVIRON. MICROBIOL.
MANCINELLI AND SHULLS
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 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
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
"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. 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 .............
