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: E2721 − 16

Standard Practice for

Evaluation of Effectiveness of Decontamination Procedures
for Surfaces When Challenged with Droplets Containing
Human Pathogenic Viruses1
This standard is issued under the fixed designation E2721; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.

INTRODUCTION

Many communicable diseases can often spread through droplets containing infectious agents. Such
“contagious droplets” may expose susceptible individuals directly or contaminate environmental
surfaces in the immediate vicinity and render them as fomites for further spread of the disease. The
characteristics of the droplets (particle size and composition) will influence the viability of the
microorganisms when exposed to environmental stresses but also shield them from physical and
chemical decontaminants. The wide variations in the types and levels of such protective/shielding
ingredients can impact on the effectiveness of surface decontaminants. This practice is designed to
simulate surface deposition of contagious droplets from human respiratory secretions. It is primarily
focused on influenza viruses but other respiratory viruses or surrogates could be used. Protocols for
assessing the microbicidal activity of disinfectants are also described.

1. Scope 1.6 This practice should be performed only by those trained

in bioaerosols, microbiology, or virology, or combinations
1.1 This practice is designed to evaluate decontamination
thereof.
methods (physical, chemical, self-decontaminating materials)
when used on surfaces contaminated with virus-containing 1.7 The values stated in SI units are to be regarded as
droplets. standard. No other units of measurement are included in this
standard.
1.2 This practice defines the conditions for simulating
respiratory droplets produced by humans and depositing the 1.8 This standard does not purport to address all of the
droplets onto surfaces. safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
1.3 The practice is specific to influenza viruses but could be priate safety and health practices and determine the applica-
adapted for work with other types of respiratory viruses or bility of regulatory limitations prior to use.
surrogates.
2. Referenced Documents
1.4 This practice is suitable for working with a wide variety 2.1 ASTM Standards:2
of environmental surfaces. E1052 Test Method to Assess the Activity of Microbicides
1.5 This practice does not address the performance of against Viruses in Suspension
decontaminants against microbes expelled via blood splatter, E2197 Quantitative Disk Carrier Test Method for Determin-
vomit, or fecal contamination. ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,
and Sporicidal Activities of Chemicals
E2720 Practice for Evaluation of Effectiveness of Decon-
1
tamination Procedures for Air-Permeable Materials when
This practice is under the jurisdiction of ASTM Committee E35 on Pesticides,
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
2
Subcommittee E35.15 on Antimicrobial Agents. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved April 1, 2016. Published May 2016. Originally contact ASTM Customer Service at [email protected]. For Annual Book of ASTM
approved in 2010. Last previous edition approved in 2010 as E2721–10. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E2721–16. the ASTM website.

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 E2721 − 16
Challenged with Biological Aerosols Containing Human 4.1.3 Virus particles are eluted from the test and control
Pathogenic Viruses carriers and viability is assessed by 50 % tissue culture
2.2 EPA Standards: infectious dose assay (log10TCID50).
EPA 600 ⁄4-84 ⁄013 (N16) USEPA Manual of Methods for NOTE 2—Non-viable techniques for viral enumeration such as poly-
Virology3 merase chain reaction (PCR) or hemagglutination cannot be used.
2.3 WHO Standards: 4.1.4 The virucidal activity of the decontamination proce-
WHO Manual on Animal Influenza Diagnosis and Surveil- dure is determined from the log difference in viability between
lance4 treated and test carriers.

3. Terminology 5. Significance and Use

3.1 Definitions: 5.1 The efficacy of disinfection technologies can be evalu-
3.1.1 aerosol, n—a suspension of solid or liquid particles in ated on finished products, as well as on developmental items.
a gas medium. 5.2 This practice defines procedures for validation of the
3.1.2 biological aerosol, n—aerosol comprising particles of droplet generator, preparation of the test specimen, application
biological origin or activity which may affect living things of the challenge virus, enumeration of viable viruses, assessing
through infectivity, allergenicity, toxicity, or pharmacological data quality, and calculation of decontamination efficiency.
and other processes. 5.3 This practice provides defined procedures for creating
3.1.3 contact transmission, n—infections caused by direct droplets that approximate those produced by human respiratory
skin-to-skin contact or indirect contact with objects contami- secretions, with particular emphasis on droplet size distribution
nated with pathogens. and aerosolization media.
3.1.4 contagious respiratory droplet, n—respiratory secre- 5.4 Safety concerns associated with aerosolizing microbial
tions containing infectious microorganisms that form large agents are not addressed as part of this practice. Individual
droplets (≥5 µm) and settle out of the air over short distances. users should consult with their local safety authority, and a
3.1.5 droplet transmission, n—direct transfer of pathogen- detailed biological aerosol safety plan and risk assessment
containing droplets to conjuncitval or mucous membranes. should be conducted prior to using this practice. Users are
3.1.6 influenza, n—an infectious disease of birds and mam- encouraged to consult the manual Biosafety in Microbiological
mals caused by RNA viruses of the family Orthomyxoviridae. and Biomedical Laboratories5 published by the U.S. Centers
for Disease Control and Prevention (CDC).
3.1.7 protective factor, n—soluble or insoluble material
co-deposited with microorganisms that directly protects the 5.5 This practice differs from Test Methods E1052 and
microorganism from environmental stresses or decontami- E2197 in the presentation of virus to the surface. The afore-
nants. mentioned test methods use a liquid inoculum to contaminate
carrier surfaces, whereas this practice presents the virus in
3.1.8 self-sanitizing material, n—a substrate containing an
droplets that are representative of human respiratory secretions
antimicrobial agent that collectively acts as a germicide.
5.6 This practice differs from Practice E2720, because (1)
4. Summary of Practice larger droplets are being formed, (2) the droplets will not be
4.1 The practice describes the steps required to deposit completely dried prior to application to surfaces, (3) the
droplets onto surfaces and quantitatively assess decontamina- droplets can be applied to any surfaces, not just those that are
tion efficiency. air permeable, and (4) unique equipment is required to create
4.1.1 Using an aerosol device capable of meeting the data droplets.
quality objectives set forth in this practice, influenza virus or
6. Apparatus
surrogates are aerosolized to form droplets that are subse-
quently applied to surfaces. 6.1 Droplet Apparatus—The apparatus used to load micro-
4.1.2 The virus-contaminated carriers are subjected to dis- organisms onto a substrate is composed of several commer-
infection protocols and incubated for the specified time and cially available components and can be readily constructed.6,7,8
conditions. Control samples are incubated under identical
conditions, but are not exposed to the disinfection protocols. 5
CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories, 5th
NOTE 1—Carriers with incorporated microbicides do not receive any Edition, U.S. Department of Health and Human Services, Washington, D.C., 2009.
6
additional disinfection treatment. An untreated control is needed to assess Vo, E., Rengasamy, S., Shaffer, R., “Development of a Test System to Evaluate
antimicrobial efficacy. Decontamination Procedures for Viral Droplets on Respirators.” Applied and
Environmental Microbiology, Vol 75, No. 23, 2009, pp. 7303–7309.
7
Woo, M. H., Hsu, Y. M., Wu, C. Y., Heimbuch, B. K., Wander, J. D., “A Device
for a Consistent and Controlled Delivery of Aerosolized Droplets Containing Viral
3
Available from United States Environmental Protection Agency (EPA), Ariel Agents Onto Surfaces.” Journal of Aerosol Science, Vol 41, 2010, pp. 941-952.
8
Rios Bldg., 1200 Pennsylvania Ave., NW, Washington, DC 20460, http:// Heimbuch B. K., Wallace, W. H., Kinney, K., Lumley, A. E., Wu, C-Y, Woo,
www.epa.gov. M-H, Wander, J. D., “A Pandemic Influenza Preparedness Study: Use of Energetic
4
Webster, R., Cox, N., Stohr, K. WHO Manual on Animal Influenza Diagnosis Methods to Decontaminate Filtering Facepiece Respirators Contaminated with
and Surveillance. World Health Organization, Department of Communicable Dis- H1N1 Aerosols and Droplets,” American Journal of Infection Control, 2010, DOI
ease Surveillance and Response. WHO/CDS/CDR/2002.5 Rev. 1. 10.1016/j.ajic.2010.07.004.

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 E2721 − 16
The overall design of the apparatus can take various forms and 7.1.1.1 The WHO Manual on Animal Influenza Diagnosis
can be fashioned in different dimensions while meeting the and Surveillance contains specific procedures for preparing
validation requirements and data quality objectives listed influenza virus and titering samples. Other viruses may be
below. Appendix X1 contains the description of a prototypical used, but conditions for propagation and enumeration are not
device that can be used to load droplets onto surfaces. provided in this practice.
However, it is the responsibility of the user of this standard to 7.1.2 Madin–Darby Canine Kidney (MDCK) Cell Line,
validate the performance of the device prior to use. ATCC CCL-34.
6.1.1 Validation requirements and baseline testing. 7.1.3 Artificial Saliva, see Table 1 in section 6.1.1.4.
6.1.1.1 Environmental Conditions—Generator must be ca- 7.1.4 Eagle’s Minimal Essential Medium With Earle’s Bal-
pable of delivering air with a relative humidity of 50 6 10 %. anced Salts (EMEM).
6.1.1.2 Loading uniformity across the diameter of the test 7.1.5 Heat-Inactivated Fetal Bovine Serum (45 min at
specimen is required to ensure the even distribution of the 56°C).
droplets over the surface of the carrier. A standard deviation of 7.1.6 Penicillin/Streptomycin, 10 000 units penicillin and 10
60.5 log10TCID50 is desired. mg streptomycin per mL.
6.1.1.3 Sample-to-Sample Variation Objective—The vari- 7.1.7 L-Glutamine, 200 mM in 0.85 % NaCl.
ability of virus loading for multiple samples loaded for a single 7.1.8 Crystal Violet.
test must have a standard deviation of 60.5 log10 TCID50. 7.1.9 Glutaraldehyde.
6.1.1.4 Droplet Characteristics—The droplets generated for 7.1.10 TPCK–Trypsin.
this practice will have a number median diameter (CMD) of 7.1.11 Phosphate Buffered Saline (PBS).
~15 6 5 µm. The virus will be aerosolized in a saliva substitute 7.1.12 Bovine Serum Albumin.
(Table 1) that will add the appropriate “protective factors.” 7.1.13 Trypsin–EDTA Solution—0.05 % trypsin, 0.53 mM
This practice would be suitable for simulating other fluids of EDTA in Hank’s balanced salts solution without sodium
interest; however, if a different fluid is used, the formulation bicarbonate, calcium, and magnesium.
and recipe listing the protective factors and droplet size must 7.1.14 Distilled Water and Purified Water.
be reported. 7.1.15 Ethanol, laboratory grade.
6.2 Other Equipment—The list is specific for influenza 7.1.16 Household Bleach.
virus. Other equipment may be needed if a different virus is 7.2 Materials—The list is specific for influenza use. Other
used. reagents may be needed if a different virus is used.
6.2.1 Autoclave, capable of maintaining 121 to 123°C and 7.2.1 Tissue Culture Treated Flasks—T-75, T-175, 12-well,
[15 to 17 lbs per in.2-gauge (psig)]. and 96-well plates.
6.2.2 CO2 Incubator, capable of maintaining 35 to 37°C and 7.2.2 Pipettes, 1, 5, 10, and 25 mL.
5 6 0.5 % CO2. 7.2.3 Test Tube Rack.
6.2.3 Vortex Mixer. 7.2.4 Micropipettes, capable of delivering 0.001 mL accu-
6.2.4 Analytical Balance, capable of weighing 0.001 g. rately and consistently.
6.2.5 Refrigerator, capable of maintaining 2 to 8°C. 7.2.5 1.7-mL Sterile Microcentrifuge Tubes.
6.2.6 Stopwatch or Electronic Timer. 7.2.6 15-mL Sterile Centrifuge Tubes.
6.2.7 Pipettor, with a precision of 0.001 mL. 7.2.7 50-mL Sterile Centrifuge Tubes.
7.2.8 Test Materials.
7. Reagents and Materials
8. Sampling, Test Specimens, and Test Units
7.1 Reagents—The list is specific for influenza use. Other
reagents may be needed if a different virus is used. 8.1 Cut test specimens from finished products or from
7.1.1 Influenza virus (H1N1; A/PR/8/34)—cell culture specimens that can be documented as representative of finished
adapted, ATCC VR-1469. products. The configuration of the particular aerosol device
dictates the size and type of each specimen. Place specimens
into the droplet loader in the proper orientation. In some cases,
the complete finished product may be used, which obviates the
TABLE 1 Artificial Saliva8 need for cutting “coupons.”
Reagent Amount
MgCl2 ·7 H2O 0.04 g 9. Experimental Design
CaCl2 · H2O 0.13 g
NaHCO3 0.42 g 9.1 A minimum of three independent test and control
0.2 M KH2PO4 7.70 mL
samples must be evaluated so that fundamental statistical
0.2 M K2HPO4 12.3 mL
NH4Cl 0.11 g analysis of the data can be performed.
KSCN 0.19 g
(NH2)2CO 0.12 g 10. Test Procedure
NaCl 0.88 g
KCl 1.04 g 10.1 Apparatus Operation—Appendix X1 describes a drop-
Mucin 3.00 g let loading device and details the standard protocols for
Distilled water 1000 mL
pH 7 operation of the device. General information that is indepen-
dent of the droplet devices is listed below.

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 E2721 − 16
10.2 Perform Neutralizer Effectiveness Test—The objective 11. Calculation or Interpretation of Results
of this test is to determine whether toxic effects from the 11.1 Virus Quantification—The Spearman–Kärber formula9
chemical or physical decontamination method have been is used to determine the virus titer of each sample.
neutralized by the extraction buffer prior to virus enumeration.
Treat a test specimen not exposed to virus with the decontami-
nation procedure following the experimental protocol. Follow-
L 5 log10TCID50 titer 5 X 0 2 SDd
2
1d 3 (n
ri
i
(1)
where:
ing the completion of the decontamination procedure, place the X0 = log10 of the reciprocal of the lowest
test specimen in 10 mL of the extraction buffer and perform the dilution at which all test inocula are
extraction procedure following the experimental protocol. positive,
d = log10 of the dilution factor (that is, the
Remove and discard the test specimen, then split the sample difference between the log dilution
into two equal volumes. Set aside sample A as it will be used intervals),
ni = number of test inocula used at each
to determine toxicity to the MDCK host cells. Add 10 µL of a individual dilution,
virus suspension of known titer (for example, 105 TCID50 per ri = number of positive test inocula (out of
ni), and
mL) to sample B and incubate at room temperature (18 to
24°C) for a minimum of 1 h. Serially dilute sample B (1/10) o S D
ri
ni
= sum of the proportion of positive tests
beginning at the lowest dilution showing
into serum-free EMEM and determine titer using the TCID50 100 % positive result.

assay. Compare the number of viable viruses recovered from 11.2 Average Loading (TCID 50 per cm2)—Determine the
the test specimen extraction buffer to the number recovered average amount of viable viruses recovered from each test
from the fresh buffer solution to determine toxicity. Inoculate article to ensure the loading specification meets the require-
sample A onto MDCK cells and incubate for 96 6 4 h at 35 to ments.
37°C in 5 % CO2. The cells must remain healthy and viable to
For determining surface loading (La) in TCID50/cm2
pass the test.
L a 5 L̄ u 1log~ V÷A ! (2)
10.3 Load Samples With the Droplets—The desired loading where:
should be high enough that no less than 3 log10TCID50/cm2 is
recovered from the test samples. This value is achieved by
altering concentration of the virus in the nebulizer and by V = volume of extraction medium,
adjusting loading times. Appendix X1 reports these values for A = surface area of samples, and
the specific test rig. If a different test rig is used, the values will L̄ = mean loading ((ΣL i ⁄n ) of the untreated sample
have to be determined empirically. In general, loading is 11.3 Data Quality Objectives—Calculate standard deviation
carried out by diluting the stock of viruses in artificial saliva for the control and test populations.
buffer, which is subsequently added to the nebulizer. After
priming, the test articles are exposed to the droplets for the For determining standard deviation:
required amount of time.
10.4 Decontamination—Remove samples from the droplet
Standard deviation ~ σ ! 5 S Œ( N
i51

N21
~Li 2 L!2
D (3)

loader and expose a subset (at least three) to the decontamina- where:
tion method: either a physical or chemical method. Incubate
the samples (treated and control replicates) for the specified L̄= mean of (L1...N), for the treated and untreated sample set,
amount of time at the required environmental conditions and
(temperature and humidity). A control set (at least three) is not n = number of samples.
treated with the decontamination method, but is incubated at
11.4 Decontamination Effıciency—Efficacy of decontamina-
the identical conditions (time, humidity, and temperature) as
tion is determined by comparing the number of viable viruses
the decontaminated samples. recovered from treated test specimens and untreated test
10.5 Virus Extraction: specimens. The results are reported as log reduction using the
10.5.1 Coupon—place the coupon in a 50-mL sterile centri- equation below.
fuge tube containing 10 mL of serum-free EMEM (Sample size For determining log reduction:
may vary depending on the test article being used. An
extraction buffer-to-sample ratio of 1.0 mL per cm2 should be ∆L̄ U2T 5 L̄ U 2 L̄ T (4)
used). Extract the samples for 20 min using a vortex mixer. where:
10.5.1.1 “Large Items”—Cut representative samples (for
example, 38-mm diameter circles) from the device and extract L̄ U = mean of the titers (L, log10TCID50) recovered from the
as described in 10.5.1. A minimum of 25 % of the test article untreated test specimens, and
should be sampled.
10.6 Determine the presence of viable virus by performing 9
Finney, D. J., Statistical Methods in Biological Assays. 2nd ed. New York:
a TCID50 assay on each sample. Hafner Publishing; 1964.

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 E2721 − 16

L̄ T = mean of the titers (L, log10TCID50) recovered from the 12.9 The duration of the exposure to the aerosol.
decontaminated specimens. 12.10 The temperature and relative humidity in the chamber
11.5 Statistical Analysis—An unpaired two-tailed t-test at during the exposure.
the 95 % confidence interval is performed to determine if the 12.11 The Conditions of Decontamination—
means of the test and control population are significantly decontaminating agent and concentration, plus any activating
different. p-values ≤ 0.05 indicate that there is a 95 % prob- factors (for example, intensity, frequency and duration of
ability that the differences in the means were not simply due to illumination, voltage applied and time of application, and other
chance. applicable parameters).
12. Report 12.12 Results of neutralization tests.
12.1 Statement that the test was conducted as directed in 12.13 Coefficient of variation for the control and test
Practice E2721. samples.
12.2 Sample Identification—Description of the material 12.14 The mean viable recoveries in log10TCID50/cm2 for
tested. the control and test samples.
12.3 The microorganism used for conducting the testing. 12.15 Log reduction.
12.4 Description of test device including the device used to 12.16 p-value comparing the control and test populations
generate the droplets.
NOTE 3—There are no specific pass/fail criteria for this practice. This
12.5 Aerosolization buffer used to aerosolize the microor- practice as written is intended to quantify the effectiveness of biological
ganism. decontamination methods, including antimicrobial technologies that have
been incorporated directly into the materials.
12.6 The exposed surface area for each test specimen.
12.7 The liquid flow rate in the droplet loader. 13. Keywords
12.8 Composition of the neutralization buffer used to extract 13.1 bioaerosol challenge; contagious droplet; decontami-
the virus. nation; influenza; virus

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 E2721 − 16

APPENDIX

(Nonmandatory Information)

X1. EXAMPLE DEVICE: OPERATION OF THE DROPLET CHAMBER TESTING SYSTEM

X1.1 Diagram of the Droplet Loader (see below) 12.5 cm from the bottom of the chamber. A fractional-
horsepower DC gear motor is mounted on the bottom of the
X1.2 Parameters of the Droplet Loader chamber that is attached to a 55-cm diameter circular turntable
X1.2.1 The system was designed to mimic respiratory and perforated with 0.31-cm holes. The turntable is positioned
droplet transmission of viruses onto any surface. Droplets are 15 cm above the bottom of the chamber. The motor is wired to
created by using an air atomizing nozzle that produces a a DC speed controller that is used to set the speed of the
droplet at the source that has a number median diameter of turntable. An air atomizing nozzle (Passche, Chicago, IL; part
~15 6 5µm. Water evaporates from the droplets as they ap- number SA 2000), is mounted into a 0.93-cm diameter fitting
proach the test samples but they remain as liquid droplets when using epoxy. The nozzle is fitted into the forwardmost port in
they impact the test samples. Adequate distribution of the the top of the chamber. The other ports on the top of the
droplets onto the test specimens is achieved by rotating the chamber are fitted with set screws. All six ports on the bottom
samples on a turntable at 3 r/min.
of the chamber are fitted with high-efficiency particulate air
X1.2.2 The chamber is composed of a stainless steel shell (HEPA) filters. An external compressor and vacuum pump
that is has a dimension of 60 by 60 by 70 cm (L by W by H). capable of moving 2 CFM of air are needed to operate the
The chamber has six ports on the bottom and top of the droplet loader. A bubbler or other humidifying device is
chamber to allow for introduction and exit of the droplets and required for operating at high humidity.
dilution air. The ports are 0.93-cm NPT threaded openings
spaced 15 cm from the center of the chamber. The ports are X1.3 Test Procedure
spaced 15 cm apart in a circular pattern. The rear panel of the
chamber also contains two 0.93-cm NPT threaded ports, which X1.3.1 Plumb HEPA-filtered air line to the top of the droplet
are used to install humidity and temperature probes. The loader and set flow to 2 CFM. Flush the chamber for at least 1 h
chamber contains an access door (55 by 32.5 cm) located prior to beginning test.

FIG. X1.1 Diagram of the Droplet Loader

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 E2721 − 16
X1.3.2 Place test articles into settling chamber so they are X1.3.7 Turn off the pressure to the air atomizing nozzle.
equally spaced relative to one another and so they are 1 in.
X1.3.8 Evacuate vagrant aerosols remaining in the chamber
from the outer edge of the turntable.
by drawing vacuum at the bottom chamber at a rate of ~1.5 ft3
X1.3.3 Set the turntable to rotate at 3 r/min. per min for a minimum of 15 min.
X1.3.4 Add 25 mL of the virus diluted to log 8 TCID50 per
mL in mucin buffer to the reservoir. X1.3.9 Remove the samples from the droplet loader and
perform decontamination tests. Control and test sample should
X1.3.5 Connect the virus reservoir to the air atomizing
be spaced alternately.
nozzle and apply 2.5 to 3.0 psig of pressure.
X1.3.6 Adjust the liquid flow rate into the air atomizing X1.3.10 Flow dilution air into the chamber at >2 CFM for at
nozzle to ~2 mL per min. Expose the samples until the entire least 4 h to dry the chamber.
volume in the reservoir is consumed.

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