Reference Guideline for Investigating Indoor Air/Environments
Rajiv R Sahay1, Sandhya Parvateneni1, Francisco Aguirre1, Sandhya T Chandel1, Rony I Iraq1,Alan Wozniak1, and A. B. Singh2
1Environmental Diagnostics Laboratory at Pure Air Control Services, Inc., 4911-C, Clearwater, FL-33760, USA; 2 Institute of Genomics & IntegrativeBiology, DelhiUniversity Campus, DelhiINDIA
Key words: Indoor Air/ Environments, Building Assessment, Allergens, Guideline, EDLab
Abstract
Over 60,000 environmental samples were collected between June 1994 to April 2011 in and around building environments on a proactive or reactive basis by field technicians or individuals. A total of 248,500 environmental specimens were prepared, examined and/or appraised by Environmental Diagnostics Laboratory (EDLab) at Pure Air Control Services (an AIHA accredited environmental microbiology laboratory, EMLAP # 102795) using appropriate techniques.During this study, 439 types of bacteria, 315 types of fungi, and 91 types of pollen grains that were encountered from the indoor environment alongside 36 miscellaneous biogenic or a-biogenic particulates. The mean (after taking the outlier top and bottom 2% or up to 10% in some cases) and the value at the 67th percentile are often very similar to each other in most of the biogenic, as well as a-biogenic, particulates. A numerical guideline for the indoor environment assessment was compiled by pooling these findings with temperature, relative humidity and other animal allergensthat were reported from a likewise environment.
© 2011 EDLab at Pure air Control Services
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1. Introduction
Building related symptoms (BRS) linked to indoor environmental issues are on the rise. Understanding the factors responsible for influencing the environment within closed structures is very complex (Gregory 1973; Burge 1996). These factors may be physical, chemical, biological or environmental in nature (Mandrioli et al. 1998). The lack of information on the above factors is often an obstacle in addressing BRS (Baker 1989).There is no universally accepted indoor environmental reference index/guideline toevaluate indoor air/environments. Increasing evidence suggests that specific risk factors need to be established in order to respond to an indoor environmental concern (Macher et al. 1999). The lack of a universal protocol, uncertainty about what to measure and how to measure the factors responsible for indoor environment related issues makes it very difficult and poses a serious challenge in investigating the indoor air/environments (Flanning et al 2001; NIOSH 2005).
In order to successfully manage the issues associated with indoor air/environmental concerns, it is essential to have a scientifically established reference guideline.Currently there is few criterion for evaluating common indoor contaminants including, but not limited to, bacteria, mold, pollen grains, insect biodetritus, and several other biogenic and a-biogenic entities besides physical factors such as humidity, temperature, water availability, light, etc. (Sahay and Wozniak 2005; Spicer and Gangoff 2005; Sahay et al. 2008). These constituents may influence our day-to-day life in terms of health and hygiene.To minimize risks associated with common indoor pollutants, one should accurately monitor and measure both biogenic and a-biogenic constituents and associated factors to determine healthy living conditions.
Some potential sources of indoor contaminants are dampness, poor housekeeping, ventilation, nature, types of occupants and other building characteristics (Heseltine and Rosen 2009). When a definite source-cause is not established, identification, classification and quantification of constituents are necessary in combating the issues associated with poor indoor air/environment quality.It is in this context that the study was undertaken in order to develop a numerical Reference Guideline as a standard for investigating indoor air/environment quality.
2. Materials and Methods
More than 248,500 environmental specimens were examined and/or appraised by Environmental Diagnostics Laboratory (EDLab) at Pure Air Control Services (an AIHA accredited environmental microbiology laboratory (EMLAP # 102795) by using microscopy and culture based methods. These specimens were made from 60,000 environmental samples, collected between June 1994 to April 2011 in and around building environments on a proactive or reactive basis by field technicians or individuals in and around the United States. Environmental data such as temperature, relative humidity, moisture content and air-borne particulate counts were recorded with temperature and relative humidity data loggers, moisture meters and Laser Diode Particle counters respectively.
Air, surface, bulk, swab and liquid samples were collected from the indoor environment on a proactive or reactive basis by Pure Air Control Services (PACS), Inc. technicians or individuals (Dillon et al 2005). These samples were examined for constituents that are characterized as biological or a-biological in nature. All the collected samples were analyzed or appraised by an American Industrial Hygiene Association’s (LAP, LLC) accredited laboratory (EMLAP# 107295).
Microbiological Assays
(A) Bio-Aerosol Samples
To quantify the extent of airbornemicrobial contamination, an Anderson N6 single-stage cascade impactor is employed. The impactor is cleaned with Isopropyl alcohol at each test site prior to sampling. Collection was performed at a constant flow rate, which was verified in the field utilizing an in-line flow meter. The microbial cultures for bacteria and fungi were performed utilizing the isolated airborne bio-particulates on culture media (Tryptic Soy Agar for bacteria and Malt Agar Extract for fungi). Cultures are properly sealed by Parafilm tape and safely transported to the laboratory in a thermally protected container. Culture plates are incubated for specific times at appropriate temperatures (2 days at 30ºC ± 2°for bacteria and 14 days at 25ºC ± 2° for fungi). The identification and quantification of culture grown microorganisms (bacteria and fungi) was performed based on the EDLab in-house protocol “Analysis of Culturable Bacteria and Fungi from Air and Surface Samples” (Standard Operating Procedure 07-04001). Bacterial characterizations are based on Gram staining techniques along with a series of biochemical tests which include the Analytical Profile Index (API) and Biolog MicrostationIdentification System (GP and GN plates). Fungal specimens are examined mainly by wet/Lactophenol cotton blue preparation with light microscopy (10x40 and/or 10x100 magnification) and using the Biolog MicrostationSystem (FF plates). The results for each growing type of microorganism (bacteria and fungi) are reported in Colony Forming Units per Cubic Meter of air (CFU/m³).
(B) Bulk/Swab/Surface/Liquid Samples
Environmental bulk, swab, surface and liquid samples were collected with appropriate aseptic techniques, placed into a sealable bag and/or container and transported to the laboratory in a thermally protected container. The collected samples were studied for microbial (bacteria, fungi) infestation by using the serial dilution technique. Isolation of microorganisms (bacteria and fungi) was carried out by vortexing a representative portion of pre-weighed environmental sample, uniquely identified in sterilized distilled water (DW) of known volume,and processed by serial dilution technique. EDLab (EMPAT # 102795) in-house protocols “Environmental Bulk Sample Analysis for Bacteria by Culture Method” (07-07001) and “Environmental Bulk Sample Analysis for Fungi by Culture Method” (07-08001) were used for the analysis. In the case of each specimen, the dilution plate with maximum growth and diversity of organisms was selected for qualitative and quantitative study. The same techniques were adapted for identification of culture-grown microorganisms as outlined in the bio-aerosol analysis.
Microscopy
(A) Aerobiology (Spore Trap Assays)
Spore Trap Assays were performed utilizing a cassette containing a gel-coated cover slip. Air was passed over the gel-coated cover slip using a constant flow of air per minute. The gel-coated cover slip was removed from the cassette, stained utilizing Lactophenol cotton blue stain, and placed on a glass microscopy slide (75mmX25mmx1mm). Identification of airborne particles on spore trap slides wasmade by using EDLab (EMPAT31027295) in-house protocol “Analysis of Airborne Particulates Isolated from Ambient Air by Using Light Microscopy” (SOP–07-05001). The total counts for each airborne particle is reported in Counts per Cubic Meter of air (cts/m³).
(B) Surface Microscopy (Bio-Scan400)
Tape Preparation Assays provide insight into the identification and composition of surface pollutants. Clear adhesive tape was applied to a surface and then transferred to a microscopic slide (75 mm X 25 mm x 1mm). The Bio-Scan400 was then examined (under 200-1000x magnification) by following the EDLab in-house protocol “Analysis of Surface Particulates by Light Microscopy (Surface Tape Imprint)” SOP 07-13001. Qualitative results were then recorded in terms of genus and/or speciesand quantitative estimationswere expressed in Counts per squire centimeter (cts/cm2).
All of the above airand surface-borne constituents are categorized into the following groups.
Opaque Particulates: These particles may originate from inorganic or organic sources in nature. They appear opaque when observed under light microscopyand have various shapes and sizes with regular or irregular dimensions. On average,they can be measured as less than 1µm to greater than 50 µm, with some exceptions. These particles commonly include, but are not limited to, dust & debris, paint, combustions, emissions, ash, silica and likewise particulates.
Skin Cell Fragments: Fragments of dead skin cells are dispersed or deposited in and around indoor environments from human or other animal sources. The size range of skin cells varies greatly between less than 5 µm to greater than 50 µm.
Insect Biodetritius: These are either small insects or portions/parts of insects floating or deposited in and around indoor environments. They may vary in size from a few µmto> 1 millimeters.
Total Fibers: This includes fibers such as manmade fibers, fiberglass fibers,etc. Manmade fibers may come from natural raw materials, like cellulose, or from synthetic chemicals like rayon, nylon, etc. The size of these fibers varies from a few microns to a few millimeters; however, an average size range may be 1 µm to over 500 µm.
Plant trichomes:These are the hairy outgrowths from the aerial parts of plants of various shapes (elongated, globose, stellate, pendate, with or without septae, etc.) and sizes (few µm to over 1 mm).Plant trichomes are listed as other types of fiber under the “Total Fibers” category.
Fiberglass Fibers:These are materials made from extremely fine fibers of glass. They appear as a smooth-walled, elongated, tube-like structure under the microscope with varying size ranges (average range 1µm to over 1000 µm).Due to its importance as an irritant, it is accounted for in a separate category.
Pollen Grains: These are microscopic particles of varying shapes (mostly globose, spheroidal to ellipsoidal without pore or with pore, colpi or combination of both) andsizes (5 µm to more than 200 µm).They are colorless or pigmented with various shade of yellow, brown, etc. and originate from angiospermic plants.
Fungal Elements/Spores: These are fungal structures of various shapes (globose, elongated, ellipsoidal and others with or without septation) and sizes (few µm to more than 100 µm). They are hyaline to dark in color.
Others:The following miscellaneous particles that are biogenic or a-biogenic in nature are reported in the “Others” category.
Algae: These are unicellular to multicellular structures of biogenic origin with pigmentation (mostly green, chlorophyll, etc.). They greatly vary in their shape (spherical or longitudinal, etc.) and size (25 µm to > 1 mm).
BlackParticulates: These microscopic particles may originate from an organic source material. They greatly vary in their shape and size depending on their origin. However, an average size ranges between 1µm to 5 µm with some exceptions and it may be regular or irregular in shape. Some important sources/causes of these particles in the indoor environment include, but are not limited to, combustion, burning of oil & candles, chimney soot, automobile exhaust, neoprene (rubber compound that is applied to the inside surface of fiberglass duct liner) and other organic materials emitted by copier machines, printers, abraded paints, etc.
Reddish-brown Particulates: These microscopic particles may originate from inorganic or organic source materials. These particles mainly come originate in indoor environments by rusting, coarse, weathering of materials, etc. These particles greatly vary in shape and size,measuring from 1µm to over 100µm.
Talc-like Particulates: These are thin, disk-like particles of variable size range (10 µm to 50 µm). They may be organic or inorganic in nature. Within an indoor environment, these particles mainly come from cornmeal, other grain flour, talcum powder, etc.
Myxomycetes:This is a group of fungus-like organisms, which exhibit characteristics of bothprotozoans (one-celled microorganisms) and fungi. They greatly vary in their shape and size (10 µm to 500 µm).
(C) Indoor Animal Allergens
House dust samples are collected with a vacuum dust collector by utilizing appropriate sterile techniques. House dust samples were analyzed for allergen identification by using the Enzyme-Linked Immunosorbent Assay (ELISA) or Multiplex Array (MARIATM). In the ELISA method, the house dust extract is run against the calibrated allergen standard; however, the dust sample extract is combined and incubated with captured antibodies. Bound allergens are detected using biotinylated monoclonal (mAb) and a streptavidin-conjugated fluorophore. The identified allergens inhouse dust samples are reported in micrograms per gram (µg/g) of the original sample, except in cockroach where it is reported in Units. The following common allergens are targeted from the house dust:
Dust mites: Mite allergens (common in mite fecal particles), Der p 1 (Dermatophagoidespteronyssinus) and Der f1 (Dermatophagoidesfarina).
Cat:Fed 1 cat allergen, which is common in cat fur, saliva, epithelial cells, sebaceous gland excretions and urine.
Cockroach: Common allergens evaluated are Bla g 1 and Bla g 2 (Blattella), which may originate from cockroach feces and saliva.
Dog: Can f 1 allergen is evaluated in dust samples. It comes from dog saliva or, in some cases, urine or feces.
(D) Respirable-Size Particle Counts
Respirable-size particle counts are determined using a Laser Diode Particle Counter manufactured by Particle Scan TM Model Pro. The sampling flow rate in factory set-up is 0.025cfm. Calibration was checked in the field per manufacturer’s recommendation prior to use in field.
(E) Temperature and Relative Humidity
To assess the thermal indoor environmental conditions, Temperature and Relative Humidity data loggers were used to record the values for temperature and relative humidity. Integrated thermal measurements were obtained by employing Onset Computer Corporation, HOBO H08-003-02 model Data Loggers. The data loggers were set to monitor every fifteen (15) minutes, 24 hours a day and ran continuously during the recording period. Data points and graphs were retrieved via personal computer and Box Car Pro software.
(F) Carbon Dioxide
Real time measurement of Carbon Dioxide is obtained using a Metrosonic Data Logger Model #AQ5000. Prior to using the instrument, value is compared against a Sensidyne Gastec Colometric Tubes.
© 2011 EDLab at Pure air Control Services
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3. Observations
Table 1.Bacteria reported from indoor environments by culture method
1 / Achromobacter xylosoxidans / 33 / Agrobacterium radiobacter / 65 / Bacillus licheniformis / 97 / Buttiauxella species2 / Acidovorax species / 34 / Agrobacterium rhizogenes / 66 / Bacillus megaterium / 98 / Buttiauxella gaviniae
3 / Acidovorax avenae / 35 / Agrobacterium rhizogenes A / 67 / Bacillus mycoides / 99 / Carnobacterium species
4 / Acidovorax delafieldii / 36 / Agrobacterium tumefaciens / 68 / Bacillus pasteurii / 100 / Cardiobacterium hominis
5 / Acinetobacter species / 37 / Agrobacterium tumefaciens A / 69 / Bacillus pumilus / 101 / CDC Group A - 5
6 / Acinetobacter anitratus / 38 / Agrobacterium vitis / 70 / Bacillus sphaericus / 102 / CDC Group B - 1/B - 3
7 / Acinetobacter anitratus gs 4 / 39 / Alcaligenes species / 71 / Bacillus subtilis / 103 / CDC Group DF - 3
8 / Acinetobacter anitratus gs 13 / 40 / Alcaligenes denitrificans / 72 / Bacillus subtilis var globigii / 104 / CDC Group E
9 / Acinetobacter calcoaceticus / 41 / Alcaligenes faecalis / 73 / Bacillus thuringiensis / 105 / CDC Group E, Subgroup A
10 / Acinetobacter calcoaceticus gs 2 / 42 / Alcaligenes faecalis ss homari / 74 / Bergeyella zoohelcum / 106 / CDC Group EF - 4
11 / Acinetobacter calcoaceticus gs 3 / 43 / Alcaligenes latus / 75 / Bordetella bronchiseptica / 107 / CDC Group EO-2 (Eugonic Oxidizer-2)
12 / Acinetobacter genospecies 10 / 44 / Alcaligenes xylosoxidans / 76 / Bordetella trematum / 108 / CDC Group II - H
13 / Acinetobacter genospecies 15 / 45 / Ancylobacter aquaticus / 77 / Brevibacillus brevis / 109 / CDC Group II - I
14 / Acinetobacter johnsonii / 46 / Aquaspirillum species / 78 / Brevibacterium species / 110 / CDC group II-E subgroup A
15 / Acinetobacter lwoffii / 47 / Aquaspirillum dispar / 79 / Brevibacterium acetylicum / 111 / CDC Group IVC - 2
16 / Acinetobacter radioresistens / 48 / Arthrobacter species / 80 / Brevibacterium epidermidis / 112 / Cellulomonas species
17 / Acinetobacter radioresistens gs 12 / 49 / Arthrobacter cumminsii / 81 / Brevibacterium linens / 113 / Cellulomonas cartae
18 / Actinobacillus species / 50 / Aureobacterium species / 82 / Brevibacterium liquefaciens / 114 / Cellulomonas flavigena
19 / Actinomycetes species / 51 / Aureobacterium terregens / 83 / Brevibacterium mcbrellneri / 115 / Cellulomonas hominis
20 / Actinomyces canis / 52 / Aureobacterium testaceum / 84 / Brevibacterium otitidis / 116 / Cellulosimicrobium species
21 / Actinomyces hordeovulneris / 53 / Bacillus species (not B. anthracis) / 85 / Brevundimonas diminuta / 117 / Cellulosimicrobium cellulans
22 / Aeromonas species / 54 / Bacillus alcalophilus / 86 / Brevundimonas vesicularis / 118 / Chryseobacterium species
23 / Aerococcus viridans / 55 / Bacillus amyloliquefaciens / 87 / Brochothrix species / 119 / Chryseobacterium indologenes
24 / Aeromonas caviae / 56 / Bacillus azotoformans / 88 / Brochothrix campestris / 120 / Chryseobacterium indoltheticum
25 / Aeromonas caviae DNA group 4 / 57 / Bacillus badius / 89 / Brochothrix thermosphacta / 121 / Chryseobacterium meningosepticum
26 / Aeromonas hydrophilia DNA group 1 / 58 / Bacillus brevis / 90 / Burkholderia species / 122 / Chryseobacterium scophthalmum
27 / Aeromonas media DNA group 5B / 59 / Bacillus cereus / 91 / Burkholderia andropogonis / 123 / Chryseomonas luteola
28 / Aeromonas media-like DNA group 5A / 60 / Bacillus circulans / 92 / Burkholderia cepacia / 124 / Citrobacter species
29 / Aeromonas salmonicida / 61 / Bacillus coagulans / 93 / Burkholderia cocovenenans / 125 / Citrobacter amalonaticus
30 / Aeromonas schubertii DNA group 12 / 62 / Bacillus fastidious / 94 / Burkholderia gladioli / 126 / Citrobacter freundii
31 / Aeromonas veronii/sobria DNA 8 / 63 / Bacillus laevolacticus / 95 / Burkholderia glumae / 127 / Citrobacter koseri (C. diversus)
32 / Agrobacterium species / 64 / Bacillus lentus / 96 / Burkholderia phenazinium / 128 / Clavibacter agropyri
(Continued)
Table 1. (Continued)
129 / Clavibacter michiganense / 163 / Enterobacter species / 197 / Gordonia species / 231 / Microbacterium arborescens130 / Clavibacter sepedonicum / 164 / Enterobacter aerogenes / 198 / Gordonia bronchialis / 232 / Microbacterium flavescens
131 / Comamonas species / 165 / Enterobacter agglomerans / 199 / Gordonia rubropertinctus / 233 / Microbacterium imperiale
132 / Comamonas acidovorans / 166 / Enterobacter agglomerans Biogp 2A / 200 / Gordonia sputi / 234 / Microbacterium laevaniformans
133 / Comamonas terrigena / 167 / Enterobacter agglomerans Biogp 3B / 201 / Helicobacter species / 235 / Microbacterium saperdae
134 / Comamonas testosteroni / 168 / Enterobacter amnigenus / 202 / Hydrogenophaga flava / 236 / Microbacterium species (CDC Gp A 4)
135 / Corynebacterium species / 169 / Enterobacter asburiae / 203 / Intrasporangium calvum / 237 / Microbacterium species (CDC Gp A 5)
136 / Corynebacterium afermentans / 170 / Enterobacter cancerogenus / 204 / Janthinobacterium lividum / 238 / Microbacterium terregens
137 / Corynebacterium ammoniagenes / 171 / Enterobacter cloacae / 205 / Jonesia denitrificans / 239 / Microbacterium testaceum
138 / Corynebacterium amycolatum / 172 / Enterobacter gergoviae / 206 / Kingella kingae / 240 / Micrococcus species
139 / Corynebacterium aquaticum / 173 / Enterobacter intermedium / 207 / Klebsiella species / 241 / Micrococcus agilis
140 / Corynebacterium aquaticum A / 174 / Enterobacter sakazakii / 208 / Klebsiella oxytoca / 242 / Micrococcus diversus
141 / Corynebacterium aquaticum B / 175 / Enterococcus casseliflavus / 209 / Klebsiella ozaenae / 243 / Micrococcus luteus
142 / Corynebacterium auris / 176 / Enterococcus faecalis / 210 / Klebsiella pneumoniae / 244 / Micrococcus lylae
143 / Corynebacterium jeikeium / 177 / Erwinia species / 211 / Kluyvera cryocrescens / 245 / Micrococcus roseus
144 / Corynebacterium jeikeium A / 178 / Escherichia species / 212 / Kluyvera species / 246 / Micromonospora species
145 / Corynebacterium jeikeium B / 179 / Escherichia blattae / 213 / Kocuria kristinae / 247 / Moraxella species
146 / Corynebacterium minutissimum / 180 / Escherichia coli / 214 / Kocuria rosea / 248 / Morganella morganii
147 / Corynebacterium nitrilophilus / 181 / Escherichia vulneris / 215 / Kocuria varians / 249 / Myroides species
148 / Corynebacterium pseudodiphtheriticum / 182 / Ewingella americana / 216 / Kurthia species / 250 / Neisseria sicca
149 / Corynebacterium urealyticum / 183 / Exiguobacterium species / 217 / Kurthia gibsonii / 251 / Nesterenkonia halobia
150 / Curtobacterium species / 184 / Flavimonas species / 218 / Kurthia sibirica / 252 / Nocardia asteroides
151 / Curtobacterium albidum / 185 / Flavimonas oryzihabitans / 219 / Kurthia zopfii / 253 / Nocardia brasiliensis
152 / Curtobacterium citreum / 186 / Flavobacterium species / 220 / Kytococcus sedentarius / 254 / Nocardia otitidiscaviarum
153 / Curtobacterium flaccumfaciens / 187 / Flavobacterium balustinum / 221 / Lactococcus plantarum / 255 / Nocardia species
154 / Curtobacterium luteum / 188 / Flavobacterium breve / 222 / Leclercia adecarboxylata / 256 / Ochrobactrum anthropi
155 / Curtobacterium pusillum / 189 / Flavobacterium gleum / 223 / Leifsonia aquatica / 257 / Oerskovia (Cellulomonas) turbata
156 / Cytophaga fermentans / 190 / Flavobacterium indologenes / 224 / Leuconostoc paramesenteroides / 258 / Oerskovia xanthineolytica
157 / Cytophaga johnsonae / 191 / Flavobacterium indoltheticum / 225 / Listeria grayi / 259 / Paenibacillus species
158 / Deinococcus species / 192 / Flavobacterium johnsoniae / 226 / Macrococcus species / 260 / Paenibacillus azotofixans
159 / Delftia acidovorans / 193 / Flavobacterium meningosepticum / 227 / Macrococcus carouselicus / 261 / Paenibacillus pabuli
160 / Dermabacter hominis / 194 / Flavobacterium mizutaii / 228 / Mannheimia haemolytica / 262 / Paenibacillus polymyxa
161 / Dermacoccus nishinomiyaensis / 195 / Geobacillus stearothermophilus / 229 / Methylobacterium species / 263 / Pandoraea species
162 / Empedobacter brevis / 196 / Gluconobacter oxydans ss suboxydans / 230 / Microbacterium species / 264 / Pantoea species
(Continued)