ASTM E3273-21

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3273 − 21
Standard Practice to
Assess Microbial Decontamination of Indoor Air using an
Aerobiology Chamber
This standard is issued under the fixed designation E3273; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Indoor air normally contains a variety of microbes released from animate and inanimate sources
(1). Exposuretoairbornemicrobescanoccureitherdirectlybyinhalationorbycontactwithsurfaces
and objects where airborne particles have settled (2). Such exposure can lead to infections or allergic
reactions, and microbial decontamination of indoor air can reduce the risk of exposure to potentially
harmfulmicrobes.Whilemanydevicesforindoorairdecontaminationareeitheralreadyonthemarket
or under development, the continuing absence of suitable test protocols makes their claims difficult to
assess. This practice gives the design, construction and operation of an economical and versatile
aerobiology chamber to test technologies for a temporary reduction in the number of viable microbes
in indoor air.
1. Scope 1.5 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
1.1 This practice is to assess technologies for microbial
responsibility of the user of this standard to establish appro-
decontamination of indoor air using a sealed, room-sized
3 priate safety, health, and environmental practices and deter-
chamber(~24m )asrecommendedbytheU.S.Environmental
mine the applicability of regulatory limitations prior to use.
Protection Agency (3). The test microbe is aerosolized inside
1.6 This international standard was developed in accor-
the chamber where a fan uniformly mixes the aerosols and
dance with internationally recognized principles on standard-
keeps them airborne. Samples of the air are collected and
ization established in the Decision on Principles for the
assayed,firstlytodeterminetheratesofphysicalandbiological
Development of International Standards, Guides and Recom-
decay of the test microbe, and then to assess the air decon-
mendations issued by the World Trade Organization Technical
taminating activity of the technology under test as log or
Barriers to Trade (TBT) Committee.
percentage reductions in viability per m (1). The air tempera-
ture and relative humidity (RH) in the chamber are measured
2. Referenced Documents
and recorded during each test.
2.1 ASTM Standards:
1.2 Thechambercanbeusedtoassessmicrobialsurvivalin
D1129Terminology Relating to Water
indoorairaswellastotesttheabilityofphysical(forexample,
D1193Specification for Reagent Water
ultraviolet light) and chemical agents (for example, vaporized
E2756Terminology Relating toAntimicrobial andAntiviral
hydrogen peroxide) to inactivate representative pathogens or
Agents
their surrogates in indoor air.
E2197Quantitative Disk Carrier Test Method for Determin-
1.3 This practice does not cover testing of microbial con-
ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,
tamination introduced into the chamber as a dry powder.
and Sporicidal Activities of Chemicals
1.4 This practice does not cover work with human patho-
3. Terminology
genic viruses, which require additional safety and technical
considerations.
3.1 For definitions of general terms used in this practice,
refer to Terminologies D1129 and E2756.
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Oct. 15, 2021. Published November 2021. DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/E3273-21. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
The boldface numbers in parentheses refer to a list of references at the end of Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3273 − 21
3.2 Definitions: the aerosols and keeps them airborne. An air sample is
3.2.1 nebulizer—a device used to create an aerosol from a collected using a slit-to-agar (STA) sampler to determine the
liquid. initial level of microbial contamination.
3.2.2 soil load, n—a solution of one or more organic, or
4.3 The test device is switched on or the test chemical
inorganic substances, or both, added to the suspension of the
introduced into the chamber and allowed to operate for the
test organism to simulate the presence of body secretions,
desired length of time. During this period, additional air
excretions, or other extraneous substances.
samplesarecollectedtodeterminethelevelsofviableairborne
microorganisms over the test period.
3.2.3 test organism, n—an applied inoculum of an organism
that has characteristics which allow it to be readily identified.
4.4 The plates of the recovery medium from the STA are
3.2.3.1 Discussion—The test organism is used to simulate a
incubated for CFU development.
transienttopicalmicrobialcontaminant.Itmayalsobereferred
4.5 The CFU are counted to determine log or percent
to as a marker organism, bacterial simulant, or bacterial
reductions in the viability of the test microorganism/m .
contaminant.
4.6 Between tests, the air in the chamber is flushed with
3.2.4 test substance, n—a formulation that incorporates
freshairtopurgeoutanymicrobialand/orchemicalresiduesin
antimicrobial ingredients.
it.
3.3 Definitions of Terms Specific to This Standard:
5. Significance and Use
3.3.1 test device, n—a piece of equipment meant for micro-
bial decontamination of air using a physical or chemical
5.1 This practice is to help in the development of protocols
process.
to assess the survival, removal and/or inactivation of human
pathogens or their surrogates in indoor air. It accommodates
4. Summary of Practice
thetestingoftechnologiesbasedonphysical(forexample,UV
4.1 The test device is placed in the leak-free aerobiology
light) and chemical agents (for example, vaporized hydrogen
chamber and operated either remotely or via the access gloves peroxide) or simple microbial removal by air filtration or a
built into the chamber (Fig. 1); a port on one side of the
combination thereof.
chamber can be used to attach aerosol cans to release test
5.2 While this practice is designed primarily for work with
chemicals.
aerobic, mesophilic vegetative bacteria, it can be readily
4.2 Aerosols of the test microorganism are released into the adapted to handle other classes of microbial pathogens or their
chamberusinganebulizer;afaninsideituniformlydistributes surrogates.
3 3
(10.5 ft × 11.83 ft × 6.92 ft -860.0 ft or 24.0 m )
FIG. 1 Aerosol Test Chamber with Essential Components
E3273 − 21
5.3 The pieces of equipment given here are as examples 6.23 Vacuum Source,avacuumpump,accesstoanin-house
only. Other similar devices may be used as appropriate. vacuum line or a water faucet vacuum apparatus required to
pull the samples through the membrane filters.
6. General Equipment and Labware
6.24 Vials (Glass), wide-mouth, 20mL, for use as dilution
vials.
6.1 Air Displacement Pipettes, 100 µL to 1000 µL with
disposable tips.
6.25 Vortex Mixer, to vortex mix microbial suspensions.
6.2 Analytical Balance,toweighchemicalsandtostandard-
6.26 Freezer,at-20°C 62°Cforstorageofmedia,reagents
ize inoculum delivery volumes by pipettes.
and additives.
6.3 Centrifuge, for sedimentation and washing of cells of 6.27 Deep freezer,-70°C 62°Corlowertostorestocksof
the test microorganism(s).
test microbes.
6.4 Colony Counter, for example, Quebec Colony Counter. 6.28 Centrifuge, with a minimum speed of 3000 ×g to
sediment bacteria for concentration or washing, or both.
6.5 Filter Sterilization System for Media and Reagents, a
6.29 Glass beads, borosilicate with 5µm in diameter.
membrane or cartridge filtration system (0.22µm pore diam-
eter) is required to sterilize heat-sensitive solutions.
7. General Solutions and Reagents
6.6 Glassware, 1L flasks with a side arm and appropriate
7.1 Purity of Reagents—Reagent grade chemicals shall be
tubing to capture the filtrates from 47mm diameter membrane
used in all tests. Unless otherwise indicated, it is intended that
filters; 250mL Erlenmeyer flasks for culture media.
all reagents conform to the specifications of the Committee on
Analytical Reagents of the American Chemical Society (5).
6.7 Hot Air Oven, an oven at 60°C to dry clean and sterile
glassware.
7.2 Phosphate Buffered Stock Solution—To prepare a stock
solution of phosphate buffer, dissolve 34.0 g of potassium
6.8 Incubators, an aerobic incubator with an adjustable
dihydrogen phosphate (KH PO ) in 500 mL of water. Adjust
temperature range of 30°C-56°C.
2 4
pH to 7.2 6 0.2 with 0.1 N NaOH or 0.1 N HCl and bring to
6.9 Biological safety cabinet, Class II (Type A).
1000 mL with deionized water.
NOTE 1—See reference (4) for details on the proper maintenance and
7.3 Phosphate Buffered Saline (PBS),tobeusedasadiluent
use of such cabinets.
andwashforallmicroorganisms;topreparePBS,add1.25mL
6.10 Magnetic Stir Plate and Stir Bars, large enough for a
of the stock solution and 8.75 g of NaCl to a volumetric flask,
5L beaker or Erlenmeyer flask for preparing culture media or
fill with deionized water to the 1000 mLmark, and mix; adjust
other solutions.
pH to 7.2 6 0.2, if necessary. Sterilize by filtration or
autoclaving.
6.11 Markers, for permanent marking of labware.
7.4 Test formulation,preparedatitsuse-dilutionandbringit
6.12 Disposable or reusable holders for 47mm diameter
to the test temperature.
membrane filters (0.22µm or 0.45µm pore diameter).
7.5 Growth, Recovery Media, and Media Supplements, the
6.13 pH Meter, to measure pH of buffers, reagents and test
required types of materials (see below) can be purchased from
formulations.
a variety of sources specializing in laboratory supplies.
6.14 Positive Displacement Pipette, with pipette tips fitted
7.6 Distilled Deionized Water (DDW), or equivalent high-
with “plungers” that can accurately dispense 10µL.
quality water, for making reagent solutions and media. (See
6.15 Refrigerator, a refrigerator at 5°C 63°C for storage Terminology D1129 and Specification D1193.)
of media, culture plates and reagents.
7.7 Plates of Recovery Media, ready-made plates can be
purchased or prepared in-house according to manufacturer’s
6.16 Serological Pipettes, sterile reusable or single-use
pipettes of 10.0 mL, 5.0 mL, and 1.0 mL capacity. instructions. Sterility and growth-promotion checks must be
performed on all batches of culture media.
6.17 Spectrophotometer, for measuring turbidity of micro-
7.8 Tryptone, Bovine Serum Albumin (BSA), and Bovine
bial suspensions.
Mucin, the three ingredients for the soil load (see Section 9)
6.18 Nitrile Disposable Gloves.
can be purchased from a variety of chemical suppliers.
6.19 Sterile Disposable Plastic Petri Dishes, 100mm by 15
7.9 Antifoam, a concentrate is added to the microbial
mm.
suspension to be nebulized to reduce frothing. It can be
autoclave-sterilized.
6.20 Sterile Polypropylene Centrifuge, Tubes with Caps,
50mL.
ACS Reagent Chemicals, Specifications and Procedures for Reagents and
6.21 Sterilizer, any steam sterilizer suitable for processing
Standard-Grade Reference Materials, American Chemical Society, Washington,
culture media, reagents, and labware is acceptable.
DC. For suggestions on the testing of reagents not listed by theAmerican Chemical
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
6.22 Timer, any stopwatch able to read in minutes and
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
seconds. copeial Convention, Inc. (USPC), Rockville, MD.
E3273 − 21
8. Soil Load (E2197) 11.1.1 An STA programmable sampler: For event-related
collection of bacterial aerosols (for example, Pinpoint Scien-
8.1 In case a soil load is to be added to the test microbial
tific. )
suspension, prepare its stock solutions as follows in PBS (pH
11.1.2 Six-jet Collison nebulizer—To generate microbial
7.2):
aerosols in the respirable range of 0.5 µm-5.0 µm (for example
CH Tech ); cylinder of extra-dry compressed air with pressure
8.2 Add 0.5 g of Tryptone to 10 mL of PBS.
regulator and a back flow preventer.
8.3 Add 0.5 g of BSA to 10 mL of PBS.
11.1.3 Volatile-gas detector with a gas leak probe—To
check for any air leaks from the chamber.
8.4 Add 0.04 g of bovine mucin to 10 mL of PBS.
11.1.4 Air temperature and RH meter—To monitor and
8.5 Prepare the solutions separately and sterilize by passage
record the air temperature and RH in the aerosol chamber via
through a 0.22 µm pore diameter membrane filter, aliquot, and
a wireless data logger.
store at either 5°C 63°C or –20°C 62°C.
11.1.5 Magnehelic—To detect any pressure differential be-
tween the inside and outside of the chamber (Fig. 1).
8.6 To prepare the fluid for each nebulization into the
11.1.6 A muffin fan for an even distribution the aerosols
aerobiology chamber, add to the nebulizer reservoir 0.75 mL
inside the chamber, and keeping them airborne during testing.
BSA, 1.05 mL yeast extract, 3.0 mL mucin, 50 µL of the test
bacterial suspension and 10µL of Antifoam A to 10.14 mL of
12. Microbial Species for Testing
Dulbecco’s PBS.
12.1 The selection of the microbes for the testing should
consider (a) biosafety, (b) ability to grow to high enough titers
9. Preparation of Inocula
for experimentation, (c) ease of handling and detection, (d)
9.1 Appendix X1 lists the microorganisms most often used
relative stability in air and during aerosol generation/capture,
and the media for their cultivation and recovery from indoor
and (e)relevancetothesiteoftechnologyuse.TableX1.1lists
air. The number of CFU/mL of each freshly prepared and
suitable bacterial species representing airborne pathogens.
homogenized microbial test suspension may be estimated
spectrophotometrically, based on a standard curve at a specific 13. Maintenance, Passage and Storage of Test Bacteria
wavelength, but should be confirmed by titration using mem-
13.1 Obtainstandardstrainsofthebacteriatobeusedinthe
brane filtration or alternative methods.
testing from a reputable source such as the American Type
Culture Collection (ATCC).
9.2 The concentration of the viable test microorganisms in
the experimentally contaminated air should be high enough to 13.2 Use the following procedures to initiate and maintain
meet the required tested technology’s performance criterion.
in-house stocks of the cultures.
Thisshouldbeconfirmedbydeterminingthenumbersofviable
13.3 Culture Initiation:
microorganisms in the chamber’s air before its decontamina-
13.3.1 Wipe the outside of the ampule/vial of the freeze-
tion.
dried culture with a towelette prewetted with 70% (v⁄v)
ethanol and open it inside a laminar flow hood.
10. Personal Protective Equipment
13.3.2 Resuspend the freeze-dried material in 1.0 mL of
NOTE2—PPE;allitemslistedareavailablefromsuppliersoflaboratory
sterile TSB.
safety gear.
13.3.3 Usingapipettewithasterilepipettetipplace0.1mL
of the rehydrated suspension into each one of two 10.0 mL
10.1 Facemask,
tubes containing 5.0 mL of sterile TSB. Mix well by shaking.
10.2 Hairnet,
10.3 Booties(externaltoshoeprotectorandscrubbottoms),
The sole source of supply of the apparatus known to the committee at this time
10.4 Lab gown (ties in back),
is Pinpoint Scientific, 1st Floor (SMF), North Road, Bridgend Industrial Estate,
Bridgend, UK, CF31 3TP;
10.5 Safety glasses, and
https://www.pinpointscientific.com/Particle Measuring Systems, Boulder, CO;
http://www.pmeasuring.com/home. If you are aware of alternative suppliers, please
10.6 Laboratory gloves
provide this information toASTM International Headquarters.Your comments will
receive careful consideration at a meeting of the responsible technical committee,
which you may attend.
11. Specialized Pieces of Equipment
The sole source of supply of the apparatus known to the committee at this time
is for example, CH Tech., Westwood, NJ 07675; www.inhalation.org. If you are
11.1 Specialized pieces of equipment. As a wide variety of
aware of alternative suppliers, please provide this information to ASTM Interna-
specialized pieces of equipment is commercially available, the
tional Headquarters.Your comments will receive careful consideration at a meeting
following list gives only examples of those. of the responsible technical committee, which you may attend.
E3273 − 21
13.3.4 Streak a loopful of the suspension onto two 100 mm 14.2.2 It is affixed to a steel-framed structure with polyvi-
diameter TSA plates (predried to remove any accumulated nylchloride(PVC)adhesivetapetorepresentthewalls,ceiling
water on the surface of the agar) to obtain isolated colonies, and floor to maintain an airtight seal.
and incubate the plates at 36°C 61°C for 18h 62h. 14.2.3 Sealable ports, window and door provide access to
the inside of the chamber for maintenance and to place and
13.4 Cryopreservation of cultures—Prepare a broth culture
remove any monitoring devices to be used.
of the desired bacterial species by inoculating with a flamed
14.2.4 The plastic sheeting can be easily and safely
loop a colony from the TSA plate into 9.0 mL of TSB and
removed, decontaminated as biohazardous waste by autoclav-
incubate the tube at 36°C 61°C for 18h 62h (te
...