Comparison of Two Biological Aerosol Sampling Methods

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1987, p. 99-104

Vol. 53, No. 1

0099-2240/87/010099-06$02.00/0 Copyright ©D 1987, American Society for Microbiology

Comparison of Two Biological Aerosol Sampling Methods NEIL J. ZIMMERMAN,'* PARKER C. REIST,2 AND ALVIS G. TURNER2 School of Health Sciences, Purdue University, West Lafayette, Indiana 47907,1 and Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 275142 Received 22 July 1986/Accepted 1 October 1986

Two biological aerosol samplers, the Andersen two-stage microbial impactor and the May three-stage glass impinger, were examined to determine the benefits and effectiveness of the May sampler compared with the Andersen sampler, one of the most widely accepted samplers. Side-by-side samples were collected during simulated wastewater spray irrigation dispersion studies. Escherichia coli colony counts and air concentrations were statistically treated to determine the dependability of the May results with respect to the Andersen results. After data pairs containing potentially overloaded Andersen counts were eliminated, a linear regression of the remaining data was performed. It indicates that although the May sampler reports 82% of the Andersen sampler value, the correlation between the two samplers is good with an r2 value of 0.84. This comparison indicates that although there are differences between the two samplers, they do give comparable results and that when both are used in a sampling program, they tend to complement each other.

Airborne bacteria, viruses and fungi are potentially a source of a wide variety of health hazards. In addition to ambient atmospheric levels of biological organisms, increased hazards are possible in hospitals, animal slaughter and processing facilities, and wastewater treatment facilities. Offshoots of the latter source are the land disposal of wastewater and spray irrigation with wastewater. A good deal of controversy has arisen over the possibilities of health hazards from wastewater spray (4-6). These and other situations require that levels of airborne biological concentrations be determined to provide a basis for health protec-

aerosolized bacteria. This procedure simulates the aerosolized portion of typical wastewater spray, which is generally less than 1% of the total spray volume (7). The microorganism selected as the tracer was the bacteria Escherichia coli, a widely used indicator organism. An E. coli spray concentration of approximately 6 x 106 CFU/ml was used since it represents an upper estimate of the total bacterial concentration in unchlorinated municipal wastewater secondary effluent (3). Experiments were conducted in an open field marked off in a radial grid pattern (12). This grid was used for positioning the sampling stations which circled the center spray point. After determining the downwind direction from the on-site meteorological tower, samplers were positioned generally 38 to 152 m (125 to 500 feet) downwind of the spray origin. For each datum pair of May and Andersen samplers, the samplers were located adjacent to each other with the assumption that they were sampling the same atmosphere. These pairs of data were collected under a wide range of meteorological conditions and extend over a wide range of air concentration values. For a typical run, the sampling pumps were started, and a 1-liter portion of pH 7.2 phosphate-buffered solution seeded with E. coli was sprayed in approximately 15 min. Calibrated critical orifices (14) were placed in the sampling trains which maintained a constant air flow rate of 55 liters/min for the May sampler and 28.3 liters/min for the Andersen sampler. After the samples were processed to determine the number of bacteria collected, the concentrations in air were calculated as follows:

tion. There are several methods available for the measurement of viable airborne particles. Many investigators have used the Andersen viable aerosol impactor (2, 8, 13) sampler or the AGI-30 all glass impinger (9), or both. An alternative to these samplers is the May three-stage glass impinger (10) which has some significant advantages despite its infrequent references in the literature. This paper presents a comparison of the May sampler versus the most widely accepted sampler, the Andersen impactor. MATERIALS AND METHODS

Experimental procedures. The comparison of the two biological aerosol samplers was performed with data collected in a field study (12) to determine the atmospheric dispersion of viable organisms from wastewater spray irri-

Concentration in air (CFU per cubic meter) = bacterial count in sampler x correction factors sampling rate (cubic meters

per

minute)

gation systems. A source of viable organisms was generated by spraying distilled water seeded with a known microorganism concentration through a high-pressure spray gun. The result was a totally aerosolized fine mist of droplets which was subject to rapid evaporation leaving mainly single *

x

sampling time (minutes)

(1)

Samplers. The selection of the proper biological sampling apparatus requires consideration of the organism to be collected, as well as other factors including viability, limits of detectability and instrument sensitivity, and physical and economic constraints. A number of inertial impaction samplers have been designed or adapted as viable samplers. These can be divided into two basic categories, those which

Corresponding author. 99

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FIG. 1. The Andersen (left) and May (right) samplers.

use a liquid collection medium and those which collect the sample on a solid surface (11). The two samplers used in this study (Fig. 1) are representative of those two groups. In a liquid-medium sampler (typically some variation of a glass impinger) the particle is drawn in and then projected into a liquid, usually with enough force to break up the particle if it contains more than one viable component. This liquid is then recovered, and a sample is plated into a nutrient agar growth medium. In this study, the liquid medium used was a pH 7.0 phosphate-buffered solution, and the plating medium used was Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.). After incubation, this method gives a total viable organism count in the air sample. The solid-surface sampler projects the particle directly onto the surface of a nutrient growth medium. The medium is then incubated, and the number of colonies is counted. Examples of this type include slit samplers, sieve samplers, and cascade impactors. In this study the May three-stage glass impinger was used. This is a liquid-medium sampler developed by K. R. May (10) and available through the British scientific-glass-blowing firm of A. W. Dixon (Anerley) Ltd. Although it is relatively inexpensive, at $50, it has apparently seen little application in the United States, as indicated by its absence from the scientific literature. It is of good design and workmanship with many improvements over other types of glass impingers. One of its major advantages is that it consists of three collection stages which approximate the particle size removal capabilities of the major portions of the human respiratory system: the nasopharyngeal, tracheobronchial, and the alveolar sections. The removal mechanism of the first two stages is by impaction on a wetted sintered disk, while the smallest particles in the third stage impact directly onto the wetted

glass side wall after exiting from a smooth curved glass jet. The cutoff diameters (defined as the diameter of particles 50% of which are removed by a stage), have been estimated by May (10) to be 6.0, 3.3, and 0.7 ,um, respectively, for a typical bacterial particle (estimated specific gravity of 1.5) and nominal flow rate of 55 liters/min. May points out that while the cutoff curves for retention of particles by particle diameter are not as steep as the optimal case, they are certainly as sharp as the parts of the respiratory system that the sampler is designed to simulate. The original Andersen microbial sampler is a solid-surface impactor (1) with six stages. Each stage has 200 or 400 precisely drilled holes with the hole diameter becoming progressively smaller from the top to the bottom stage. A petri dish containing an appropriate type of agar is positioned under each stage. As the air flows through the sampler at the standard rate of 28.3 liters/min a particle is deposited by impaction on the agar surface when the momentum imparted to the particle is too great to allow it to follow the airstream to the next stage. The six stages provide a good deal of information on the size distribution of the sampled aerosol cloud, with the top stage collecting basically nonrespirable particles, those greater than 7 pm, and the lower stages collecting those respirable particles capable of reaching the alveoli. Actual size ranges and efficiencies vary depending on the density of the particle, and some overlapping of stages does occur, but overall results from this sampler have been widely accepted. In this study a two-stage version of the Andersen sampler was used, with Trypticase soy agar (5% sheep blood) petri dishes as collection plates. The sampler is very similar in design and operation to the six-stage Andersen sampler except that only the second and fifth stages are used and each stage has only 200 holes. This arrangement provides a good separation between nonrespirable and respirable parti-

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COMPARISON OF BIOLOGICAL AEROSOL SAMPLING METHODS

cles. For the work reported here, two stages were adequate since approximately 90% of the aerosolized E. coli were estimated, by analysis of the research data, by literature search, and by Poisson probability calculations, to be single cells after initial evaporation (12). The typical oblong shape (1 by 3 ,um) of an E. coli cell allowed it to be efficiently trapped in the second stage of the two-stage sample or any of the bottom three stages of the six-stage sampler. Tests have shown very good comparison between the two- and six-stage samplers when they are exposed to a wide range of E. coli concentrations (12). The two- and six-stage Andersen samplers are readily available in the United States, but the cost, $800 and $1,400, respectively, is considerably higher than that of the May sampler. It should be noted that the seeded tracer spray was the only source of airborne E. coli. Control samples collected upwind of the spray or collected without any tracer being sprayed consistently showed zero E. coli colonies. Thus, the selection of Trypticase soy agar was felt to be the best E. coli growth medium. In fact, a medium more selective for E. coli may have actually inhibited the growth of the E. coli bacteria in their somewhat weakened state, owing to their airborne exposure to sunlight, possible moisture loss, and physical trauma. RESULTS

A total of 49 pairs of data were collected comparing the May and the two-stage Andersen samplers. Of these 49 cases, 8 were eliminated owing to excessive overloading of the Andersen sampler, a physical limitation which will be discussed later. Another six pairs were eliminated because of contamination of the liquid in the May sampler or the agar material in the Andersen sampler. Data for the remaining 35 cases are listed in Table 1 both as total bacteria collected in the sampler and as concentrations in air adjusted for air flow through the sampler. The total bacteria collected is an important parameter in terms of examining the physical collection capabilities of the samplers. The Andersen sampler total E. coli values in Table 1 represent the sum of the actual colony counts on the two petri dishes in the sampler. The May sampler total E. coli values were generated by: 3

Total E. coli in May sampler =

i=1

TABLE 1. Total E. coli collected and concentrations in air determined by samplers (n = 35) Date

(1979) 8/8 8/8 8/8 10/9 10/23 10/23 10/23 10/29 10/30 10/30 10/30 10/30 10/30 10/30a 10/31 10/31 10/31 10/31 11/7 11/7 11/7 11/7 11/7 11/7 11/7 11/ 11/9 11/9 11/9l 11/9l 11/9l 11/14a 11/14a 11/14a 12/14 a

Total E. coli count

Concn (CFU/m3)

May

Andersen

May

Andersen

7 0 26 11 105 12 12 176 213 32 18 91 85 245 170 101 394 584 86 24 75 106 14 0 64 159 20 144 213 504 395 406 440 735 91

10 6 15 7 42 12 9 111 95 28 19 70 80 163 107 47 151 152 68 8 13 64 9 13 37 80 19 100 128 169 146 151 120 174 82

6 0 38 17 136 16 16 213 408 61 34 174 103 297 238 111 551 644 104 29 105 133 18 0 80 199 30 210 194 764 359 509 552 722 165

17 17 42 22 106 30 23 261 353 104 71 260 188 384 291 101 410 326 160 19 35 156 22 32 90 195 56 272 226 498 258 368 292 332 290

These data pairs are eliminated in Fig. 3.

Andersen values generally above 120 total counts are more scattered and have a larger May-to-Andersen concentration ratio than the other points. This may be due to phenomenon

average of counts on duplicate liquid recovered plates for stage i (CFU per milliliter) xfrom stage i (milliliters) correction factor (0.8)

The correction factor includes adjustments for losses in the extraction process and losses during sampler operation. This factor was determined from recovery efficiency tests with the sampler (12). The data expressed as total collected counts, however, cannot be compared directly because the samplers were operated at different flow rates and for different sampling times. Converting these counts into concentrations in air by equation 1 makes the data directly comparable in terms of numbers of bacteria per cubic meter of air. The 35 air concentration pairs were plotted on a scatter diagram (Fig. 2). A linear least-squares line of best fit was calculated for the points. The simple linear regression line fit to the points, with the square of the correlation factor r2 = 0.76, does not appear to represent a very good fit (Fig. 2). A closer examination, however, indicates that the points with

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(2)

Andersen referred to as the positive hole theory (1) which states that as more and more spaces on the agar under the 200 holes of each stage are filled with bacteria, the probability that a new particle will enter an already occupied space increases. This particle would not be counted since only one visible (countable) colony results at each impaction site, regardless of the number of individual bacteria originally impacted. From other data collected in the field dispersion study, comparing a 200-hole-per-plate two-stage Andersen to a 400-hole-per-plate six-stage Andersen (12), the positive hole correction does not appear to be a significant factor in this study when the Andersen count is less than 120. Eliminating the nine datum pairs in the high-count region (.120 counts in the Andersen sampler), the remaining 26 points were replotted in Fig. 3. Their elimination from the new linear regression calculation seems justified, since these

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00

200 100

+

+

4

+

500 450 400 350 300 200 250 AN ENCNRATItN I FIG. 2. May versus Andersen sampler E. coli air concentrations, in number per cubic meter

0

50

150

100

nine datum pairs appear to be more affected by the physical limitations of the Andersen sampler. This plot indicates a different and better fit, suggesting that indeed there were two different groups of data in the original datum set. The simple and weighted linear regression lines for the 26 remaining datum pairs are almost identical, with the simple linear regression intercept estimator not significantly different from zero (P = 0.10). The weighted regression line, therefore, is drawn in Fig. 3. The slope of the best-fit line is 0.82 which is statistically significantly different from a slope of unity (P = 0.05). Thus, the samplers do not appear to be capable of reporting equal values for the same atmospheric conditions. The improved value of r2 = 0.84 and a visual examination of the fit, however, indicate that the two samplers are fairly

(all data).

well correlated in their relationship given by the equation of the line, with the May sampler reporting 82% of the Andersen sampler value. Applying a confidence interval band to the line gives an indication of the possible variations away from the predicted values given by the equation. A 90% confidence interval for a new (predicted) value of Y based on any given value of X is shown by the dots in Fig. 3. DISCUSSION

Each of the samplers has its advantages and disadvantages which must be considered in determining its applicability to a particular situation. For example, the procedures for preparation and sample extraction of the May sampler are

400 Y = 0.82 X R2 = 0.84

350

300

E 250

R200

350 150 200 250 300 NIES CtKENTATN FIG. 3. May versus Andersen sampler E. coli air concentrations, in number per cubic meter (eliminating data with Andersen counts .120). Dots indicate 90% confidence limits for best-fit line. 0

50

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COMPARISON OF BIOLOGICAL AEROSOL SAMPLING METHODS

VOL. 53, 1987

relatively tedious. Adherence to aseptic techniques in washing, loading, sterilizing, field use, extraction, measurement, and plating of the sample is very important to avoid possible contamination. Because of this lengthy preparation and recovery, it is difficult to use each sampler more than once per field visit. Once the sample has been collected, it should be refrigerated and processed as soon as possible to limit either die-off or cell growth which would give a result not representative of the collected sample. Because of liquid loss by evaporation the May sampler should not be run much longer then 30 min, and as air temperature increases and humidity decreases, even shorter times may be required. The sampler cannot be effectively used below 3°C since the air flow conditions expose the liquid to pressures less than atmospheric, causing the liquid to freeze. An advantage of the May sampler is that it cannot be overloaded at high concentrations. Once the liquid is extracted, one or two dilutions can be performed to allow any air concentration to be analyzed. For example, at a 10-min sampling period of 50,000 CFU/m3 (1,400 CFU/ft3), two serial 1/10th dilutions would reduce the colony count to no more than 50 per plate. A knowledge of the approximate concentrations in air expected may help determine more precisely the dilution level, but in an unfamiliar sampling situation, the additional dilutions and platings can be used so that the appropriate range will be measured. Another advantage of the May sampler is that, unlike the Andersen sampler which can only collect colony-forming samples, the May sampler can collect other material for independent analysis after the plating is completed. In this study, for example, an

inorganic fluorescent dye

tracer was

simultaneously

sprayed. After sufficient liquid was removed for the biological tests, the remaining liquid could be analyzed for dye concentration. The Andersen sampler encounters problems with overloading in high-concentration atmospheres. With a fixed number of holes per stage4 the chances of having more than one particle per hole are large when most of the holes are already filled. These additional counts will be lost unless the plate is examined microscopically soon after incubation, before separate colonies overgrow one another. This sampler is limited to counting particles containing viable organisms and cannot determine the number of viable cells contained in that particle (a more important number physiologically), since one visible colony will result per impaction site. Excessive sampling times, more than 30 to 45 min, can dry out the agar media in the plate, changing the flow characteristics of the sampler and causing evaporative stress to the microorganisms. A further limitation that was previously mentioned was the relatively high cost per sampler, an important consideration in field studies for which many samplers are required. Perhaps the greatest advantage of the Andersen sampler is its ease of operation. It can be assembled in the field in minutes, using prepared sterile petri dishes, and when the run is complete, the plates can be stored with no special provisions until it is convenient to incubate them. Temperatures below freezing will not affect its efficiency. Overloading can be overcome by reducing the sampling time sufficiently. For example, at a concentration of 20,000 CFU/m3 (570 CFU/ft3) a sampling time of 15 s could be used to reduce the number of colonies collected to approximately 140. In an unfamiliar sampling situation with an unknown concentration, the proper sampling time still must be found by trial and error. Sources of variability. To understand more fully the com-

103

parison of these two samplers, we examined the sources of sample variability. The variability between results from the May and Andersen samplers is in part a function of the reproducibility of the two individual samplers. In a separate study of the reproducibility of the Andersen two-stage samplers, 23 pairs of data comparing a metal and plastic version of the two-stage sampler were collected. Applying the correlation and linear regression model approach as the statistical analysis method, the two samplers appeared to function interchangeably, with an r2 = 0.96 and a weighted linear regression equation of y = 0.99x (12). A source of Andersen sampler variability is the uncertainty of the application of the positive hole correction factor to the Andersen counts. This factor is based on probability statistics with little or no experimental evidence to support it. At very low counts, the effect is minimal. At very high counts there is definitely a noticeable effect. Unfortunately, the most uncertain area occurs in the midrange of values, often the most useful range in practice. Variability when comparing adjacent May samplers could be as great as 10%. Another source of variability is the standard microbiological procedure of duplicate plating of the aliquots from the liquid sample. The average of the two plate counts was used for subsequent calculations, but the variation associated with this average could be as high as 30% and was typically between 10 and 15%. This source of variation could account for most, if not all, of the error observed. As mentioned earlier, recovery efficiency tests could also account for some of the variation observed in the May data. One area of comparison that may be a source of variability between samplers in some situations is the difference in inlet design and subsequent inlet sampling velocities. The Andersen sampler, with its lower flow rate as well as its greater total inlet area, has an inlet velocity approximately one-third that of the May sampler. For high wind speeds especially, the May sampler would be expected to exhibit a higher collection efficiency for larger, nonrespirable particles (>10,um). However, since the majority of microbial aerosols in this study, as is the case with many airborne microorganisms, were expected to be respirable, the collection efficiency should be relatively independent of inlet design and velocity. Conclusions and recommendations. Although there appears to be many areas of potential variability between the May and Andersen samplers, the May and Andersen microbial samplers appear to be fairly well correlated at least up to Andersen counts of 120 with an r2 = 0.84 for the May sampler reporting 82% of the Andersen value for the conditions of this study. Given the vagarjes of microbiological analytical work, especially at microorganism levels in the order of tens and hundreds instead of the usual ranges of thousands and millions, it seems remarkable to have achieved such good correlation between two 'samplers operating on different principles. Even though each sampler has its set of advantages and disadvant?iges, both can serve a useful function in airborne microbiological sampling and analysis. With a properly designed sampling strategy, the May and Andersen samplers can, in fact, complement one another, extending the airborne microbial sampling range available to the researcher. The Andersen sampler is a much easier instrument to operate, involving very little preparation and analysis time compared with the lengthy procedures required for the May sampler. The May sampler is very effective at high concentrations, and since the Andersen sampler is more easily

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overloaded under these conditions, the May sampler can offer added flexibility in an airborne microbial study. Adding to this flexibility, chemical or particulate as well as biological contaminants can be collected and analyzed with the May sampler. The May sampler, like the Andersen sampler, provides the benefits of particle size distribution, but in addition, it indicates total airborne viable organisms rather than just airborne particles containing viable organisms. For a general microbial sampling program both samplers could be used. The samplers can be operated side-by-side initially to determine the concentration, affording both high and low concentration capabilities and confirming the correlation factor. Because of their lower cost, additional May samplers could be used as well. The May samplers should be located closest to the emissions source or point of highest suspected concentration. Either or both can be used for expected intermediate and low concentrations. Preference for one over the other should be a matter of individual evaluation, taking into account the material being samnpled, time, budget, and technical support. ACKNOWLEDGMENTS

The work upon which this paper was based was supported by funds provided by Project no. B-104 NC from the Office of Water Research and Technology, U.S. Department of the Interior, through the Water Resources Research Institute of the University of North Carolina. We are grateful to Andersen Samplers, Inc., Atlanta, Ga., who provided the Andersen samplers for use in this project. LITERATURE CITED 1. Andersen, A. A. 1958. New sampler for the collection, sizing and enumeration of viable airborne particles. J. Bacteriol. 76:471-484. 2. Clark, C. S., G. L. Van Meer, A. B. Bjornson, C. R. Buncher, P. S. Gartside, C. C. Linnemann, and E. J. Cleary. 1978. A seroepidemiologic study of workers engaged in wastewater collection and treatment, p. 263-271. In Proccedings of the International Symposium on Land Treatment of Wastewater, vol. 2. University of Cincinnati, Cincinnati. 3. Crites, R. W., and A. Uiga. 1979. An approach for comparing

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health risks of wastewater treatment alternatives-a limited comparison of health risks between slow rate land treatment and activated sludge discharge. EPA 430/9-79-009. Office of Water Program Operations, U.S. Environmental Protection Agency, Washington, D.C. Feliciano, D. V. 1979. Wastewater aerosols and health risks. J. Water Pollut. Cntrol Fed. 51:2573-2579. Hickey, J. S., and P. C. Reist. 1975. Health significance of airborne microorganisms for wastewater treatment processes. Part I. Summary of investigations. J. Water Pollut. Control Fed. 47:2741-2757. Hickey, J. S., and P. C. Reist. 1975. Health significance of airborne microorganisms for wastewater treatment processes. Part II. Health significance and alternatives for action. J. Water Pollut. Control Fed. 47:2758-2773. D. E., Johnson, D. E. Camann, J. W. Register, R. E. Thomas, and C. A. Sorber. 1980. The evaluation of microbiological aerosols associated with the application of wastewater to land: Pleasanton, California. EPA 600/1-80-015. Office of Research and Developments, U.S. Environmental Protection, Agency, Washington, D.C. Katzenelson, E., and B. Teltch. 1976. Dispersion of enteric bacteria by spray irrigation. J. Water Pollut. Control Fed. 48:710-716. Lembke, L., R. Knisely, R. VanNostrand, and M. Hale. 1981. Precision of the all-glass impinger and the Andersen microbial impactor for air sampling in solid waste handling facilities. Appl. Environ. Microbiol. 42:222-225. May, K. R. 1966. Multistage liquid impinger. Bacteriol. Rev. 30:559-570. May, K. R. 1970. Assessment of viable airborne particles. In T. T. Mercer, P. E. Morrow, and W. Stoeber (ed.), Assessment of airborne particles: fundamentals, applications, and implications to inhalation toxicity. Charles C Thomas, Publisher, Springfield, Ill. Reist, P. C., N. J. Zimmerman, A. G. Turner, D. E. Francisco, P. Robinson, M. Overcash, and R. Sneed. 1980. Wastewater spray transport in land application. Report no. 152. Water Resources Research Institute, University of North Carolina, Raleigh. Sorber, C. A., H. T. Bausum, S. A. Schaub, and M. J. Small. 1976. A study of bacterial aerosols at a wastewater irrigation site. J. Water Pollut. Control Fed. 48:2367-2379. Zimmerman, N. J., and P. C. Reist. 1984. The critical orifice revisited: a novel low pressure drop critical orifice. Am. Ind. Hyg. Assoc. J. 45:340-344.