M. Hodgson, D. Vaughn Petersen, J. Ramsey, S. Donham, Packer Environmental Consultants, Freehold, NJ.
Evaluation of bioaerosol data is frequently based on a comparison of biodiversity and rank order of taxa between indoor and outdoor air samples. This study shows that a large variance of both rank order and biodiversity is to be anticipated between indoor and outdoor bioaerosol data sets, even where a building is not impacted by moisture or fungal amplification. Data is presented from triplicate samples collected concurrently, showing a wide divergence in taxa present and the prevalence of each taxa from concurrent sample sets collected at the same location. The data is analyzed using commonly applied statistical methods to demonstrate that samples collected from the same location at the same time will have sufficient variance to make comparison to samples collected at remote locations challenging without a sufficiently large data set to account for the variance in the sample collection and analysis methods. Data sets are presented that indicate a potential for both false positive results and false negative results that an investigator may obtain by overreliance on air sampling data. Data from spore trap analysis will be discussed in this presentation. The number of samples required to provide meaningful data using these methods are discussed.
L. Rogers, Sceptor Industries, Tampa, FL.
Defense of the current practice of evaluating bioaerosols with spore traps is challenging due to the widely variable results of even colocated samples, small sample volumes that are not representative of the environment, and the lack of quality control references. To overcome these challenges, large numbers of samples are necessary to collect representative sample volumes and to achieve statistical certainty of the results. Often, budget constraints severely limit the data collected and thus introduce significant questions as to the reliability of the results as well as the corrective action decisions. When the Department of Homeland Security needed rapid, accurate field technology for biothreat detection, new technologies had to be developed to overcome these challenges to reliable data collection. These new technologies employ high-volume air sampling devices combined with field detection screening techniques such as polymerase chain reaction (PCR) tubes, immunoassays, and fluoroassays that are directly applicable to indoor environmental quality. To evaluate the efficacy of two field detection techniques (immunoassay and fluoroassay), studies were performed under controlled conditions (chamber studies) and uncontrolled conditions (in the field). The air samples collected were analyzed in the laboratory by direct microscopy in addition to the field detection methods. The studies performed in the field were also run side by side with a statistically significant number of spore trap samples for comparison. This presentation will (1) discuss the advantages and disadvantages of the new technologies; (2) explain how they compare with currently accepted methods; (3) provide statistical and variability data; (4) suggest best practice application; (5) identify the limitations and the interpretation challenges presented by these new evaluation tools; and (6) explore the next evolution in bioaerosol evaluation and how it could revolutionize the way we interpret our environments.
W. Chen, P&K Microbiology Services, Cherry Hill, NJ.
Using the correct growth medium is crucial to the quality of fungal culturable analysis in an indoor air quality investigation. It is not uncommon for labs to receive samples collected on an inappropriate medium, such as DG18 (dichloran glycerol) only, rather than MEA (malt extract agar). There is no single standard medium in which all fungi grow. Each mold has its own particular moisture requirement. Its growth depends on enough free water being available. According to a widely accepted scheme, environmental fungi are classified based on their water activity (aw) as extremely xerophilic (aw< 0.75), moderately xerophilic (aw 0.75-0.79), slightly xerophilic (aw 0.80-0.89), and hydrophilic (aw > 0.90). Water activity for a specific fungal species is generally known as a range and therefore can be recovered on media with a range of water activities (general purpose media), such as MEA. Extremely xerophilic fungus has water activity below 0.75 and only can be isolated on DG18. Investigators have to choose the growth medium for culturable samples before air sampling at the site or before submitting surface samples to the lab for culturable analysis. There are many fungal media available for the analysis, either commercially or made by the lab. Theoretically, multiple media with different water activities should be chosen to optimize an investigator’s ability to recover fungi with different water activity requirement for growth. For this reason, use of at least two media is recommended: MEA and DG18. DG18 optimizes the collection of xerophilic fungi, and MEA optimizes the isolation of a broader range of environmental fungi with broader range of water activity requirement. When hydrophilic fungi are of specific concern, such as Chaetomium globosum or Stachybotrys chartarum, corn meal agar is recommended as a supplement to MEA for its ability to recover fungi with a higher water activity.
W. Crawford, SEA Limited, Jacksonville, FL.
Twenty-four viable microbial samples and 24 nonviable microbial samples were taken in six residences in four Florida counties, in January 2006 for one residence and in July 2006 for the remaining five residences. The 24 viable microbial samples and 24 nonviable microbial samples were taken sequentially in the same sample locations; half were taken outside each residence and half were taken insidee. Viable microbial samples were collected onto a malt extract agar (MEA) plate using an Aerotek 6 viable microbial particle sampler and pump calibrated to 28.3 L/min for 3 min. Nonviable microbial samples were collected using spore trap cassettes and a pump calibrated to 15 L/min for 10 min. A blank MEA plate and a blank spore trap cassette were submitted to the laboratory for each residence evaluated. Results indicate the indoor/outdoor ratio using spore trap cassettes were 4 to 29x lower than viable microbial samples in four out of the six residences. The remaining two residences had higher indoor/outdoor ratios using spore trap cassettes by a factor of less than 1 over viable sampling methods. Outdoor nonviable particles appeared to significantly impact indoor/outdoor ratios on spore trap cassettes. Nonviable spore trap analysis limited comparisons between spore/particle group identifications and species of organisms identified in viable sampling methods. Conducting indoor/outdoor comparisons of microbial air samples was not consistent between viable and nonviable sampling methods. Viable air sampling methods should be part of an overall fungal assessment of the structure to include surface swab samples; moisture measurements and/or infrared photography; visual inspection; photography; and temperature and relative humidity measurements. A bacterial assessment to include air samples and surface swab samples should also be performed as part of a microbial evaluation.
G. Mainelis, Rutgers University, New Brunswick, NJ; M. Yao, Yale University, New Haven, CT.
Exposure to airborne bacteria and fungi has been linked to various negative health effects. Currently, portable microbial samplers are becoming more popular for monitoring presence of viable bioaerosols; however, little data are available about their performance. This study investigated the overall performances of SMA MicroPortable, BioCulture, Microflow, Microbiological Air Sampler (MAS-100), Millipore Air Tester, SAS Super 180, and RCS High Flow portable microbial samplers when collecting bacteria and fungi both indoors and outdoors. The performance of these samplers was compared against that of the BioStage impactor. We also investigated the effects of the impactors’ physical parameters on their performance in field conditions. The results showed that in an indoor environment the BioStage impactor recovered the highest concentrations of both bacteria and fungi, while in an outdoor environment the RCS High Flow and the MAS-100 recovered microorganism concentrations equal to or higher than those of BioStage. Data analysis using analysis of variance between groups (ANOVA) from all environments and for both bacteria and fungi indicated that relative performance of all samplers was statistically different (lower) compared to the BioStage, except for the RCS High Flow and the MAS-100. The latter sampler also had statistically higher performance compared with other portable samplers, except the RCS High Flow. The Millipore Air Tester and the SMA had the lowest performances among the investigated samplers. In addition, for the first time, the relative performance of the investigated impactors was successfully described using a multiple linear regression model (R2 = 0.83), where the roles of samplers’ cutoff sizes and jet-to-plate distances as predictor variables were statistically significant. The data presented in this study will help professionals in selecting bioaerosol samplers for field studies, while the developed empirical formula describing the overall performance of bioaerosol impactors should assist in sampler design.
S. Seo, T. Reponen, C. Crawford, Y. Iossifova, T. Lee, S. Grinshpun, University of Cincinnati, Cincinnati, OH; F. Grimsley, Tulane University, New Orleans, LA; D. Schmechel, NIOSH, Morgantown, WV; C. Rao, CDC, Atlanta, GA.
A field-compatible collection system was developed and tested for the collection and analysis of fungal fragments. The new collection system consists of two types of Sharp-Cut cyclones (PM2.5 and PM1.0) and an afterfilter. Fungal particles are collected into three size fractions: (1) spores (>2.5 μm); (2) a fragment-spore mixture (1.0-2.5 μm); and (3) submicrometer sized fragments (<1.0 μm). The system was laboratory tested using polystyrene latex (PSL) particles and particles aerosolized from in vitro cultures of Aspergillus versicolor or Stachybotrys chartarum. In addition to the particle count, the (1→3)-β-D-glucan content in each size fraction was determined with the Limulus amebocyte lysate (LAL) assay. Finally, the new methodology was field-tested in flood-damaged homes in New Orleans. Experiments conducted with PSL particles showed that the 50% cutoff values of the two cyclones under the test conditions were 2.25 µm and 1.05 µm, respectively. No particle bounce onto the after-filter was observed when the total number of particles collected into the PM1.0 cyclone was kept below 108. The (1→3)-β-D-glucan assay of samples aerosolized in the laboratory from A. versicolor and S. chartarum suggested that surface area is an important factor for determining the (1→3)-β-D-glucan content for the entire size range of fungal particles. Field sampling showed that no particle bounce occurred onto the afterfilter during nine repeated tests, and that the (1→3)-β-D-glucan concentrations in the three different size fractions were well above the lower detection limit. Furthermore, the average ratios of the (1→3)-β-D-glucan concentrations in fragment and spore determined in the laboratory and field testing were comparable with each other: 0.031 and 0.023, respectively.
In conclusion, the new methodology is a promising tool for collecting and analyzing pure fungal fragment samples.
T. Bates, L. Grimsley, J. Edgar, Tulane University, New Orleans, LA; L. Relle, Relle IAQ Solutions, LLC, Belle Chasse, LA.
Collection efficiencies of four spore capture devices and collection media were analyzed and compared. The collection media included (1) a 25-mm, 0.8-μm mixed cellulous ester (MCE) filter with two traces, each with an area of 9.35mm2; (2) a 37-mm, 0.8-μm filter with a single trace in three different sizes — 20 mm2, 30 mm2, and 40 mm2; and (3) two spore trap sampling cassettes, both with an area of approximately 15.95 mm2. The filter cassette devices included the Bi-Air filter sampling cassette and the Smart Sampler (prototype 1). The spore trap cassette devices include the Air-O-Cell and Allergenco-D. The four samplers, placed side by side inside a laboratory controlled settling chamber, were introduced to 4-5 μm mean aerodynamic diameter Aspergillus/Penicillun-like spores. Each pump was calibrated and cassette challenged according to individual protocols. Microscopic analysis was used to enumerate the airborne fungal spores, and thus determine the relative collection efficiency of each sampler. Lactophenol cotton blue staining technique was used for the direct wet mounts of all four sampling media. The total enumeration of Aspergillus/Penicillun-like spores was highest in the Smart Sampler. The Smart Sampler, a filter sampler, was 1.73x greater when compared to Air-O-Cell, a spore trap sampler. Followed by the Bi-Air, filter cassette that represented a 1.6x greater collection efficiency than the spore trap. Particle distribution was most uniform in the Bi-Air, due to the design of the inlet. The uniformity of spore distribution on both filter cassettes was higher than that of the spore traps.
J. Edgar, L. Grimsley, T. Bates, Tulane University, New Orleans, LA; L. Relle, Relle IAQ Solutions, LLC, Belle Chasse, LA.
A new sampling cassette (prototype 3) inlet dispersion nozzle with a 34-mm diameter mixing chamber/funnel was analyzed to determine any bias in spore deposition when sampling for airborne fungal spores. The filter cassette was designed to collect an air sample through a 5/16-in inner diameter hose, passing through a diffusion nozzle into the mixing chamber and subsequently being deposited on the filter trace through a funnel assembly. The trace was a 2 x 22 mm rectangular opening. The trace outline was visible on the filter due to it being pressed against a 37-mm diameter mixed cellulose ester (MCE) filter by the cassette. The filter cassette had six raised pins, which left impressions in the filter, allowing it to be cut into either two or four equidistant 2-mm wide sections. Air samples were collected in a controlled environment where 4-5 µm Aspergillus spores were injected into a 2600-L hexagonal settling chamber and collected on MCE filters. Microscopic analysis was used to enumerate the airborne fungal spores deposited at each end and the middle of each sample. MycoPerm Red staining technique was used for the direct wet mounts of all samples. Analysis of the fungal spore concentration at both ends of the filter trace was compared to the fungal spore concentration in the center of the trace. The process of turbulent mixing and then subsequent deposition of fungal spores on the filter trace gave excellent results: a geometric mean difference of <3% between the middle section and end sections. The cassette design allows microscopic analysis of a portion of the filter and plating/culturing of the remaining portion of the filter with confidence that each filter section is statistically representative of the air sampled.
W. Tang, QLAB, Cherry Hill, NJ.
Impaction-type spore traps collect spores by impacting them onto a sticky slide. The collection efficiencies are different for particles in different aerodynamic sizes. The collection efficiency for particles with aerodynamic sizes at the cutoff size (d50) is only 50%. Smaller particles will have even lower collection efficiencies. The d50 for Air-O-Cell, Allergenco-D, and Micro-5 are 2.6 µm, 1.7 µm, and 0.8 µm, respectively. Many Aspergillus and Penicillium spores have physical sizes of 2-2.5 µm. Measured aerodynamic sizes of some fungal spores in low humidity can be as low as 1.7 µm. Many other factors also affect the collection efficiencies of impaction-type spore traps — e.g., slide overloaded with debris, low temperature, and device quality. The impact of each factor on collection efficiency will be presented and discussed. Filter membrane cassettes have been long employed as the standard devices in many air sampling procedures. The collection efficiency is near 100% for particles larger than the pores sizes of membrane, e.g., 0.4 µm and 0.8 µm. They were not suitable for fungal spore analysis because of the collection area is too large for direct microscopic examination at high magnification. Bi-Air and Laro-100 cassettes restrict airflow onto a small area on the filter membrane in order to be examined under microscope, which results in low maximum sampling flow rate (2 to 4 liters per minute [LPM]) due to small collection area. MoldSense QTrap is a filter cassette that can be used to collect airborne fungal spores at low to high flow rate (0.5 to 15 LPM) and analyzed them under microscope after extraction and concentration. Its performance is not affected by debris loading, temperature, or humidity. Its high collection efficiency makes it an excellent spore trap for indoor air sampling, especially for air with a high amount of debris.
J. Edgar, L. Grimsley, T. Bates, Tulane University, New Orleans, LA; L. Relle, Relle IAQ Solutions, LLC, Belle Chasse, LA.
Sampling collection efficiencies of the Air-O-Cell spore trap cassette and a new filter cassette (cassette prototype 2) were analyzed within a heating, ventilation, and air conditioning (HVAC) system. Air samples were collected by using sample flow rates of 15 liters per minute (LPM] for the Air-O-Cell spore trap and flow rates of isokenetic plus 15% for the filter cassette. The filter cassette was loaded with a 0.8-μm mixed cellulose ester (MCE) filter. Microscopic analysis was used to enumerate the airborne fungal spores and thus determine the relative collection efficiency of each sampler. MycoPerm Red staining technique was used for the direct wet mounts of all samples. The study environment was a single room commercial property with a simple HVAC system. Visible fungal growth was noted external to the HVAC system throughout the room on the walls and ceiling. Upon inspection, there was no visible fungal growth within the return air plenum, assessable coils, or assessable distribution ducts. To simulate fungal growth within the HVAC coils, nonviable Aspergillus/penicillum-like fungal spores were injected into the ducts just downstream of the first supply air grille, which was 20 ft upstream of the first sample point. Testing was conducted using the filter cassette for 2-, 4-, and 24-hr periods, with 5-min and 10-min Air-O-Cell spore trap samples being taken side by side with the filter cassette every 2 hr. A comparison using 2-4.5 µm Aspergillus/Penicillum-like spores was done to evaluate the relative capture efficiency of the two methods within the air conditioner ducts. The collection efficiency of the filter cassette was found to be 3.2x greater than the Air-O-Cell. Use of filter cassettes to collect fungal spores in an operating HVAC system was determined to be a better and more reliable method.