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: D6329 − 98 (Reapproved 2023)

Standard Guide for

Developing Methodology for Evaluating the Ability of Indoor
Materials to Support Microbial Growth Using Static
Environmental Chambers1
This standard is issued under the fixed designation D6329; 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.

1. Scope responsibility of the user of this standard to establish appro-

1.1 Many different types of microorganisms (for example, priate safety, health, and environmental practices and deter-
bacteria, fungi, viruses, algae) can occupy indoor spaces. mine the applicability of regulatory limitations prior to use.
Materials that support microbial growth are potential indoor 1.8 This international standard was developed in accor-
sources of biocontaminants (for example, spores and toxins) dance with internationally recognized principles on standard-
that can become airborne indoor biopollutants. This guide ization established in the Decision on Principles for the
describes a simple, relatively cost effective approach to evalu- Development of International Standards, Guides and Recom-
ating the ability of a variety of materials to support microbial mendations issued by the World Trade Organization Technical
growth using a small chamber method. Barriers to Trade (TBT) Committee.
1.2 This guide is intended to assist groups in the develop- 2. Referenced Documents

of materials. iTeh Standards

ment of specific test methods for a definite material or groups
2.1 ASTM Standards:
D1193 Specification for Reagent Water
2

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1.3 Static chambers have certain limitations. Usually, only
small samples of indoor materials can be evaluated. Care must
D1356 Terminology Relating to Sampling and Analysis of
Atmospheres

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of the material is Preview
be taken that these samples are representative of the materials E104 Practice for Maintaining Constant Relative Humidity
being tested so that a true evaluation by Means of Aqueous Solutions
performed. 2.2 APHA Standards:3
1.4 Static chambers provide controlled laboratory microen- Standard Methods for the Examination of Water and Waste-
vironment conditions. These chambers are not ASTM D6329-98(2023)
intended to water
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duplicate room conditions, and care must be taken when
interpreting the results. Static chambers are not a substitute for 3. Terminology
dynamic chambers or field studies. 3.1 Definitions—For definitions of terms used in this guide,
1.5 A variety of microorganisms, specifically bacteria and refer to Terminology D1356.
fungi, can be evaluated using these chambers. This guide is not 3.2 Definitions of Terms Specific to This Standard:
intended to provide human health effect data. However, organ- 3.2.1 amplification—the act or result of increasing the
isms of clinical interest, such as those described as potentially quantity of microorganisms.
allergenic, may be studied using this approach.
3.2.2 CFU—colony forming unit, which may arise from a
1.6 The values stated in SI units are to be regarded as single organism or multiple units, such as spores, in the case of
standard. No other units of measurement are included in this the fungi.
standard. 3.2.3 colony—macroscopically visible growth.
1.7 This standard does not purport to address all of the 3.2.4 inoculation—the act of introducing a microorganism
safety concerns, if any, associated with its use. It is the (inoculum) into the test material.

1 2
This guide is under the jurisdiction of ASTM Committee D22 on Air For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Quality and is the direct responsibility of Subcommittee D22.08 on Assessment, contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Sampling, and Analysis of Microorganisms. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved Jan. 1, 2023. Published February 2023. Originally the ASTM website.
3
approved in 1998. Last previous edition approved in 2015 as D6329 – 98 (2015). Available from American Public Health Association (APHA), 800 I St., NW,
DOI: 10.1520/D6329-98R23. Washington, DC 20001, http://www.apha.org.

Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States

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 D6329 − 98 (2023)
3.2.5 inoculum—viable test microorganism introduced onto
a material by implanting a small amount on the surface or
substrate.
3.2.6 plate—petri dish containing microbiological agar me-
dia on which microorganism are grown.
3.2.7 static chamber—a small chamber (enclosed space)
with no internal forced air motion.
3.2.8 susceptibility—the vulnerability of a material or sur-
face to colonization by microorganisms.
FIG. 1 Schematic of Example Static Chamber
4. Significance and Use
4.1 The static chambers have several different applications: 5.1.1 Relative Humidity—Maintain humidities through the
4.1.1 The static chambers can be used to compare the use of saturated salt solutions contained in trays on the bottom
susceptibility of different materials to the colonization and of the chambers (see Practice E104). It is essential that the
amplification of various microorganisms under defined condi- chambers be tightly sealed so that the desired humidity will be
tions. maintained. Place hygrometers in the chambers for confirma-
tion that humidities are being maintained, although saturated
4.1.2 Chambers operated at high relative humidities may be
salt solutions are themselves standards. Exercise care that the
used to perform worst case scenario screening tests on mate-
salts selected for use in the chamber are not inhibitory to the
rials by providing an atmosphere where environmental condi-
test organisms.
tions may be favorable for microbial growth.
5.1.2 Temperature—Control the temperature of the cham-
4.1.3 Use of multiple chambers with different environmen- bers. The chambers may be externally controlled through the
tal parameters, such as a range of relative humidities, permits use of constant temperature environments, such as a room or
the evaluation of multiple microenvironments and allows incubator. Chart recorders or other data logging devices are
ditions. iTeh Standards
investigation of materials under differing environmental con- recommended to confirm maintenance of temperature. Con-
trolled temperature is critical for two reasons. First, it can have

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4.1.4 Drying requirements for wetted materials may also be a profound effect on the growth of microorganisms. Second,
investigated. This information may be relevant for determining relative humidity is dependent upon temperature. The control
material resistance to microbial growth after becoming wet. limits may be defined by consulting a psychometric chart and
Document Preview
These conditions may simulate those where materials are
subjected to water incursion through leaks as well as during
determining the impact of temperature on a specific test RH.
5.1.3 Characterize instrumentation for evaluating other pa-
remediation of a building after a fire. rameters if the instruments are to be employed during material
4.1.5 Growth rates of microorganisms on the material
ASTM may
D6329-98(2023)
testing. Conditions such as light need to be noted and con-
also be investigated. Once it has been established that organ- trolled during the course of an experiment as these conditions
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isms are able to grow on a particular material under defined may have an effect on the growth of the test organism. Light
conditions, investigations into the rate of organism growth may may be controlled externally by placing the chambers in a
be performed. These evaluations provide base line information darkened room to remove light or in a continuously lighted
and can be used to evaluate methods to limit or contain room for a constant light source.
amplification of microorganisms.
5.2 Provide ports, where needed, for the insertion of probes
4.2 These techniques should be performed by personnel to monitor and record temperature and relative humidity, using
with training in microbiology. The individual must be compe- externally located instrumentation as long as it is well sealed
tent in the use of sterile technique, which is critical to exclude and contamination is avoided.
external contamination of materials.
5.3 Decontamination—Decontaminate the chamber before
initiating any analysis. Surface disinfection or vapor phase
5. Apparatus
disinfection may be appropriate. Glass may be autoclaved.
5.1 Static Chamber—Chambers should be relatively small Follow the manufacturers’ instructions, especially any safety
and portable, contain three or four shelves, and be easily precautions. If a chemical disinfectant is employed, clear the
decontaminated. In addition, transparent walls are desirable chambers of any residual disinfectant to prevent interference
because visual inspection of the test material and monitoring of with the growth of the microorganisms on the material being
instruments (that is, hygrometers) without opening the cham- evaluated. Thoroughly ventilate the chambers in a clean
ber is preferred. Fig. 1 is a schematic diagram of a possible environment. Decontaminate the salt solutions. The method
static chamber. Acrylic desiccators are readily available, easily used is dependent upon the composition of the salts selected.
adaptable, and relatively inexpensive. Other options, such as Any instrumentation to be used during the evaluations, such as
glass, are also acceptable. Glass has the advantage of being hygrometers, may be removed from the chambers during the
autoclavable; however, it is frequently much less portable. The decontamination procedure of the chamber surfaces and de-
chamber door must provide ready access to the materials but contaminated separately; however, it is generally more effec-
should be airtight when closed. tive for them to remain in the chambers. Verify the efficacy of

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 D6329 − 98 (2023)
the decontamination procedure as part of the Quality 8. Sample Preparation
Assurance/Quality Control (QA/QC) plan. 8.1 Specific details on the preparation of the samples will
5.4 Decontaminate the work area around the chambers depend upon the characteristics of the material to be tested.
routinely, especially before opening the chamber door. The Generally, replicate small pieces of the test material should be
chambers should be kept in a clean room, functionally Class used. Depending upon the material, pieces as small as 4 by 4
100 000 (M 6.5 or ISO 8) or better. cm may be used. Pieces should be placed on sterile petri dishes
or other appropriate holders on the shelves in the chamber.
6. Reagents Include controls and blanks within the QA/QC framework.
6.1 Purity of Reagents—Reagent grade chemicals shall be 8.2 Common microbiological practice is to sterilize a sur-
used in all tests. Unless otherwise indicated, it is intended that face or material before inoculation to ensure that the test
all reagents conform to the specifications of the Committee on organism is the only source being evaluated. Autoclaving is an
Analytical Reagents of the American Chemical Society where extremely effective method if such a procedure does not alter
such specifications are available.4 Other grades may be used, the test material. Other methods, such as ionizing and non-
provided it is first ascertained that the reagent is of sufficiently ionizing irradiation, ultraviolet, dry heat, and surface or vapor
high purity to permit its use without lessening the accuracy of phase disinfection, are also acceptable if these methods do not
the determination. harm the material and do not have residue effects or if all traces
6.2 Purity of Water—Unless otherwise indicated, references of the disinfectant can be removed prior to testing. Consult the
to water shall be understood to mean reagent water as certified test material manufacturer or conduct tests with the test
by Type II of Specification D1193. It should conform to the material to determine the best method of sterilization. Specific
Type A specifications for microbial classification. details for decontamination depend upon the method selected
6.3 Microbiological Media—Choose appropriate media de- and should be worked out before actual testing begins within
pending upon the test microorganism selected. Commercially the QA/QC framework.
prepared media may be acceptable, but it may be necessary to 8.3 Equilibrate or bring to near equilibrium samples in the
prepare organism specific media. References should be con- chamber before inoculation with the test organism. Equilibra-

test organism.
iTeh Standards
sulted to determine the proper media for optimal growth of the tion time will depend upon both the material to be tested and
the chamber relative humidity selected for the test. Determine

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7. Characterization of Static Chamber
equilibration times for each material prior to testing.
8.3.1 Ascertain equilibration by determining when the bulk
7.1 Characterize static chambers for all environmental pa- moisture content of the material reaches a constant value. The
rameters being measured before any Document
material evaluations are Preview use of a calibrated analytical balance is recommended.
performed. Chambers should be characterized for at least 8.3.2 Compute the bulk moisture content of the test material
relative humidity and temperature. Take sufficient readings to as follows:
ensure that the conditions will be maintained ASTM D6329-98(2023)
throughout the MC 5 @ ~ M b 2 M d ! /M d # × 100 (1)
course of the experiment and will meet the QA/QC standards
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developed for a specific test. where:
7.1.1 Equilibration—Equilibrate disinfected chambers con- MC = bulk moisture content (%),
taining hygrometers before taking the first relative humidity Mb = mass of the small piece (g), and
reading. Place the hygrometers on a shelf for ease of reading Md = mass of the small piece after drying (g).
through the walls of the chamber without opening the door. Md may be determined either by oven drying at 105 to
Take multiple sequential readings at appropriate intervals that 110 °C or desiccation to constant weight depending upon the
were determined experimentally. The variation of the instru- test material. Time required for drying is determined experi-
mentation must be determined and taken into consideration. mentally. A sample can be considered dry when no significant
For example, a minimum of four similar readings (65 %) over weight change is detected in two consecutive weighings at least
an 8 h period may be determined to demonstrate equilibrium. 1 h apart.
7.1.2 Recovery—Determine the amount of time required for
the chamber relative humidity to recover to test levels after 9. Selection of Test Organism
opening the door for 1 to 2 min. This determination may be
crucial, especially at the higher relative humidities. Exercise 9.1 Selection of the appropriate test organisms is extremely
care to utilize hygrometers that have a rapid response time. important. Since growth requirements vary for different
organisms, the selection process should include a justification
7.2 Check chamber relative humidity daily, and record for the particular organism or organisms chosen. Testing of
readings depending on test length. materials with many different organisms from diverse groups is
optimal. At a minimum, representative bacteria and fungi
4
ACS Reagent Chemicals, Specifications and Procedures for Reagents and should both be tested. Initial tests should be performed with
Standard-Grade Reference Materials, American Chemical Society, Washington, only one species of microorganism.
DC. For suggestions on the testing of reagents not listed by the American Chemical
9.1.1 Criteria for organism selection are based on a number
Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma- of factors. Appropriateness of the organism for the test material
copeial Convention, Inc. (USPC), Rockville, MD. and the environment where the material is used are key factors.

3
 D6329 − 98 (2023)
9.1.2 Literature reviews or reference articles reporting on moisture should be allowed to evaporate from the test material
indoor field investigations are good sources of suggestions for after inoculation. If large inocula volumes are desired, first
potential test organisms. calculate the moisture content of the material determined in 8.3
9.2 Documentation of the test organism is critical. Standard for equilibrated samples. Exercise care that the materials are
strains from established culture collections may be selected or kept in a sterile or clean environment, such as biological safety
environmental organism, isolated from contaminated materials, cabinet or High Efficiency Particulate Arrestor (HEPA) filtered
may be used. Identification and verification of the identification area, while the moisture is evaporating.
and source of the organism must be included as part of the 10.3.2.1 Select an appropriate liquid carrier for the test
QA/QC plan. organism with care. Attention should be paid to any potential
nutrients in the diluent that could artificially enhance the ability
10. Inoculation of Material with Test Organism of a material to support microbial growth throughout the course
of the experiment. Use of sterile distilled and deionized water
10.1 Small (30 to 40 mm square) pieces of material are minimizes the amount of possible residual nutrient sources.
usually sufficient for testing. Multiple replicates of the material Some microbial forms, such as vegetative organisms, may not
should be run to minimize possible error and to demonstrate survive in unbuffered water. Others, such as spores, are much
reproducibility. hardier forms and are resistant to the osmotic pressure of
10.2 Determination of Amount of Inoculum—The number of unbuffered water.
CFU of an organism used to inoculate each piece of material 10.4 To determine the recovery efficiency of the test organ-
should be sufficiently high to provide an adequate challenge ism from the test specimen, immediately after inoculation,
but at a level that is realistic to quantitate. Challenges ranging assay triplicate test material samples.
from 5000 to 7000 CFU per cm2 have been reported.5,6
Experimentally appropriate levels for each material are calcu- 11. Procedure
lated based upon recovery during processing of the test sample, 11.1 Inoculate sufficient replicates (at least three) of the test
including dilution factors, amount plated, and minimum detec- material with the test organism to ensure reproducibility. The
tion limit.
iTeh Standards
number of replicates should be defined within the QA/QC plan.
10.2.1 Calculate minimum detection limits (MDL) as fol- For tests with several test dates, multiple replicates of the
lows: material should be run for each test date. Quantitative evalua-

where:
~
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!
MDL 5 MCFUp ÷VP × DF × TV (2) tion of microbial growth usually involves the destruction of the
material. Usually only one type of test material inoculated with

Document
that can be counted Preview
one type of test organism may be placed in a chamber at one
MDL = minimum detection limit, time, but multiple pieces of the same material inoculated with
MCFUp = minimum number of colonies
the same organism should be evaluated simultaneously.
on one plate (1 CFU/plate),
VP = aliquot (volume) of suspension spread on plate 11.2 After inoculation, carefully transport and place the
(ml),
ASTM D6329-98(2023)
pieces of material in the chamber. Avoidance of contamination
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DF = dilution factor of original sample, and by ambient air or physical contact is essential. This may be
TV = total volume of diluent containing sample mate- accomplished by covering the samples and carrying them on a
rial (ml). clean, sterilized tray.
10.3 Inoculate organisms onto the test material as either a 11.3 To ensure that the chamber or other samples do not
suspension or as a dry inoculum. become contaminated, do not replace them into the chamber
10.3.1 Dry inocula are at best qualitative or semi- after they have been removed. This will assist in ensuring that
quantitative and may be sufficient for visible growth/no growth contamination does not occur. Only as many pieces as required
evaluations. for evaluation at each time should be removed.
10.3.2 Inoculation of materials with a known number of
CFU suspended in a known volume of liquid carrier results in 12. Determination of Microbial Growth
more reproducible data and quantification of growth. The 12.1 Microbial growth on materials may be evaluated quali-
suspension with an appropriate concentration of CFUs may be tatively or quantitatively.
introduced into the samples as either an aerosol or pipetted 12.2 Qualitative evaluation requires inspection of the test
directly onto the samples. Use a small inoculum, sufficient to material for evidence of microbial growth. Inspection of the
deliver an adequate amount of CFU, while minimizing the material may be microscopic or macroscopic. Scales for visual
amount of moisture added to the system. The small amount of examination are available.3,7 Most qualitative evaluations in-
volve either measuring the diameter of the growth on the
material or applying a subjective scale.
5
Foarde, K., VanOsdell, D., and Chang, J., “Static Chamber Method for
Evaluating the Ability of Indoor Materials to Support Microbial Growth,” Charac- 12.3 Quantitative evaluation yields an indication of the
terizing Sources of Indoor Air Pollution and Related Sink Effects, ASTM STP 1287, amount of growth within a specific time frame on a test
B. Tichenor, ed., American Society for Testing and Materials, 1996, pp. 87–97.
6
Foarde, K., VanOsdell, D., and Chang, J., “Evaluation of Fungal Growth of
7
Fiberglass Duct Materials for Various Moisture, Soil, Use, and Temperature Block, S.S., “Humidity Requirements for Mold Growth,” Applied
Conditions,” Indoor Air, Vol 61996, pp. 83–92. Microbiology, Vol 1, 1953, pp. 287–293.