ASTM D6329-98(2008)
(Guide)Standard Guide for Developing Methodology for Evaluating the Ability of Indoor Materials to Support Microbial Growth Using Static Environmental Chambers
Standard Guide for Developing Methodology for Evaluating the Ability of Indoor Materials to Support Microbial Growth Using Static Environmental Chambers
SIGNIFICANCE AND USE
The static chambers have several different applications:
4.1.1 The static chambers can be used to compare the susceptibility of different materials to the colonization and amplification of various microorganisms under defined conditions.
4.1.2 Chambers operated at high relative humidities may be used to perform worst case scenario screening tests on materials by providing an atmosphere where environmental conditions may be favorable for microbial growth.
4.1.3 Use of multiple chambers with different environmental parameters, such as a range of relative humidities, permits the evaluation of multiple microenvironments and allows investigation of materials under differing environmental conditions.
4.1.4 Drying requirements for wetted materials may also be investigated. This information may be relevant for determining material resistance to microbial growth after becoming wet. These conditions may simulate those where materials are subjected to water incursion through leaks as well as during remediation of a building after a fire.
4.1.5 Growth rates of microorganisms on the material may also be investigated. Once it has been established that organisms are able to grow on a particular material under defined conditions, investigations into the rate of organism growth may be performed. These evaluations provide base line information and can be used to evaluate methods to limit or contain amplification of microorganisms.
These techniques should be performed by personnel with training in microbiology. The individual must be competent in the use of sterile technique, which is critical to exclude external contamination of materials.
SCOPE
1.1 Many different types of microorganisms (for example, bacteria, fungi, viruses, algae) can occupy indoor spaces. Materials that support microbial growth are potential indoor sources of biocontaminants (for example, spores and toxins) that can become airborne indoor biopollutants. This guide describes a simple, relatively cost effective approach to evaluating the ability of a variety of materials to support microbial growth using a small chamber method.
1.2 This guide is intended to assist groups in the development of specific test methods for a definite material or groups of materials.
1.3 Static chambers have certain limitations. Usually, only small samples of indoor materials can be evaluated. Care must be taken that these samples are representative of the materials being tested so that a true evaluation of the material is performed.
1.4 Static chambers provide controlled laboratory microenvironment conditions. These chambers are not intended to duplicate room conditions, and care must be taken when interpreting the results. Static chambers are not a substitute for dynamic chambers or field studies.
1.5 A variety of microorganisms, specifically bacteria and fungi, can be evaluated using these chambers. This guide is not intended to provide human health effect data. However, organisms of clinical interest, such as those described as potentially allergenic, may be studied using this approach.
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: D6329 − 98(Reapproved 2008)
Standard Guide for
Developing Methodology for Evaluating the Ability of Indoor
Materials to Support Microbial Growth Using Static
Environmental Chambers
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 2. Referenced Documents
2.1 ASTM Standards:
1.1 Many different types of microorganisms (for example,
D1193 Specification for Reagent Water
bacteria, fungi, viruses, algae) can occupy indoor spaces.
D1356 Terminology Relating to Sampling and Analysis of
Materials that support microbial growth are potential indoor
Atmospheres
sources of biocontaminants (for example, spores and toxins)
E104 Practice for Maintaining Constant Relative Humidity
that can become airborne indoor biopollutants. This guide
by Means of Aqueous Solutions
describes a simple, relatively cost effective approach to evalu-
ating the ability of a variety of materials to support microbial 2.2 APHA Standards :
Standard Methods for the Examination of Water and Waste-
growth using a small chamber method.
water
1.2 This guide is intended to assist groups in the develop-
ment of specific test methods for a definite material or groups
3. Terminology
of materials.
3.1 Definitions—For definitions of terms used in this guide,
1.3 Static chambers have certain limitations. Usually, only
refer to Terminology D1356.
small samples of indoor materials can be evaluated. Care must
3.2 Definitions of Terms Specific to This Standard:
be taken that these samples are representative of the materials
3.2.1 amplification—the act or result of increasing the
being tested so that a true evaluation of the material is
quantity of microorganisms.
performed.
3.2.2 CFU—colony forming unit, which may arise from a
1.4 Static chambers provide controlled laboratory microen-
single organism or multiple units, such as spores, in the case of
vironment conditions. These chambers are not intended to
the fungi.
duplicate room conditions, and care must be taken when
3.2.3 colony—macroscopically visible growth.
interpreting the results. Static chambers are not a substitute for
3.2.4 inoculation—the act of introducing a microorganism
dynamic chambers or field studies.
(inoculum) into the test material.
1.5 A variety of microorganisms, specifically bacteria and
3.2.5 inoculum—viable test microorganism introduced onto
fungi, can be evaluated using these chambers.This guide is not
a material by implanting a small amount on the surface or
intended to provide human health effect data. However, organ-
substrate.
isms of clinical interest, such as those described as potentially
3.2.6 plate—petri dish containing microbiological agar me-
allergenic, may be studied using this approach.
dia on which microorganism are grown.
1.6 This standard does not purport to address all of the
3.2.7 static chamber—a small chamber (enclosed space)
safety concerns, if any, associated with its use. It is the
with no internal forced air motion.
responsibility of the user of this standard to establish appro-
3.2.8 susceptibility—the vulnerability of a material or sur-
priate safety and health practices and determine the applica-
face to colonization by microorganisms.
bility of regulatory limitations prior to use.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
This guide is under the jurisdiction of ASTM Committee D22 on Air contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Quality and is the direct responsibility of Subcommittee D22.08 on Indoor Air. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved April 1, 2008. Published July 2008. Originally the ASTM website.
3 th
approved in 1998. Last previous edition approved in 2003 as D6329 - 98(2003). Available from American Public Health Association, 1015 15 St., NW,
DOI: 10.1520/D6329-98R08. Washington, DC 20036.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6329 − 98 (2008)
4. Significance and Use chamber door must provide ready access to the materials but
should be airtight when closed.
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-
4.1.2 Chambers operated at high relative humidities may be
tion that humidities are being maintained, although saturated
used to perform worst case scenario screening tests on mate-
salt solutions are themselves standards. Exercise care that the
rials by providing an atmosphere where environmental condi-
salts selected for use in the chamber are not inhibitory to the
tions may be favorable for microbial growth.
test organisms.
4.1.3 Use of multiple chambers with different environmen-
5.1.2 Temperature—Control the temperature of the cham-
tal parameters, such as a range of relative humidities, permits
bers. The chambers may be externally controlled through the
the evaluation of multiple microenvironments and allows
use of constant temperature environments, such as a room or
investigation of materials under differing environmental con-
incubator. Chart recorders or other data logging devices are
ditions.
recommended to confirm maintenance of temperature. Con-
4.1.4 Drying requirements for wetted materials may also be
trolled temperature is critical for two reasons. First, it can have
investigated.This information may be relevant for determining
a profound effect on the growth of microorganisms. Second,
material resistance to microbial growth after becoming wet.
relative humidity is dependent upon temperature. The control
These conditions may simulate those where materials are
limits may be defined by consulting a psychometric chart and
subjected to water incursion through leaks as well as during
determining the impact of temperature on a specific test RH.
remediation of a building after a fire.
5.1.3 Characterize instrumentation for evaluating other pa-
4.1.5 Growth rates of microorganisms on the material may
rameters if the instruments are to be employed during material
also be investigated. Once it has been established that organ-
testing. Conditions such as light need to be noted and con-
isms are able to grow on a particular material under defined
trolled during the course of an experiment as these conditions
conditions,investigationsintotherateoforganismgrowthmay
may have an effect on the growth of the test organism. Light
be performed. These evaluations provide base line information
may be controlled externally by placing the chambers in a
and can be used to evaluate methods to limit or contain
darkened room to remove light or in a continuously lighted
amplification of microorganisms.
room for a constant light source.
4.2 These techniques should be performed by personnel
5.2 Provide ports, where needed, for the insertion of probes
with training in microbiology. The individual must be compe-
to monitor and record temperature and relative humidity, using
tent in the use of sterile technique, which is critical to exclude
externally located instrumentation as long as it is well sealed
external contamination of materials.
and contamination is avoided.
5. Apparatus 5.3 Decontamination—Decontaminate the chamber before
initiating any analysis. Surface disinfection or vapor phase
5.1 Static Chamber—Chambers should be relatively small
disinfection may be appropriate. Glass may be autoclaved.
and portable, contain three or four shelves, and be easily
Follow the manufacturers’ instructions, especially any safety
decontaminated. In addition, transparent walls are desirable
precautions. If a chemical disinfectant is employed, clear the
becausevisualinspectionofthetestmaterialandmonitoringof
chambers of any residual disinfectant to prevent interference
instruments (that is, hygrometers) without opening the cham-
with the growth of the microorganisms on the material being
ber is preferred. Fig. 1 is a schematic diagram of a possible
evaluated. Thoroughly ventilate the chambers in a clean
static chamber.Acrylic desiccators are readily available, easily
environment. Decontaminate the salt solutions. The method
adaptable, and relatively inexpensive. Other options, such as
used is dependent upon the composition of the salts selected.
glass, are also acceptable. Glass has the advantage of being
Any instrumentation to be used during the evaluations, such as
autoclavable; however, it is frequently much less portable. The
hygrometers, may be removed from the chambers during the
decontamination procedure of the chamber surfaces and de-
contaminated separately; however, it is generally more effec-
tive for them to remain in the chambers. Verify the efficacy of
the decontamination procedure as part of the Quality
Assurance/Quality Control (QA/QC) plan.
5.4 Decontaminate the work area around the chambers
routinely, especially before opening the chamber door. The
chambers should be kept in a clean room, functionally Class
100 000 (M 6.5 or ISO 8) or better.
6. Reagents
6.1 Purity of Reagents—Reagent grade chemicals shall be
FIG. 1 Schematic of Example Static Chamber used in all tests. Unless otherwise indicated, it is intended that
D6329 − 98 (2008)
all reagents conform to the specifications of the Committee on 8.2 Common microbiological practice is to sterilize a sur-
Analytical Reagents of the American Chemical Society where face or material before inoculation to ensure that the test
such specifications are available. Other grades may be used, organism is the only source being evaluated.Autoclaving is an
provided it is first ascertained that the reagent is of sufficiently extremely effective method if such a procedure does not alter
high purity to permit its use without lessening the accuracy of the test material. Other methods, such as ionizing and non-
the determination. ionizing irradiation, ultraviolet, dry heat, and surface or vapor
phase disinfection, are also acceptable if these methods do not
6.2 Purity of Water—Unless otherwise indicated, references
harmthematerialanddonothaveresidueeffectsorifalltraces
to water shall be understood to mean reagent water as certified
of the disinfectant can be removed prior to testing. Consult the
by Type II of Specification D1193. It should conform to the
test material manufacturer or conduct tests with the test
Type A specifications for microbial classification.
material to determine the best method of sterilization. Specific
6.3 Microbiological Media—Choose appropriate media de-
details for decontamination depend upon the method selected
pending upon the test microorganism selected. Commercially
and should be worked out before actual testing begins within
prepared media may be acceptable, but it may be necessary to
the QA/QC framework.
prepare organism specific media. References should be con-
8.3 Equilibrate or bring to near equilibrium samples in the
sulted to determine the proper media for optimal growth of the
chamber before inoculation with the test organism. Equilibra-
test organism.
tion time will depend upon both the material to be tested and
the chamber relative humidity selected for the test. Determine
7. Characterization of Static Chamber
equilibration times for each material prior to testing.
7.1 Characterize static chambers for all environmental pa-
8.3.1 Ascertain equilibration by determining when the bulk
rameters being measured before any material evaluations are
moisture content of the material reaches a constant value. The
performed. Chambers should be characterized for at least
use of a calibrated analytical balance is recommended.
relative humidity and temperature. Take sufficient readings to
8.3.2 Compute the bulk moisture content of the test material
ensure that the conditions will be maintained throughout the
as follows:
course of the experiment and will meet the QA/QC standards
MC 5 M 2 M /M 3100 (1)
@~ ! #
b d d
developed for a specific test.
7.1.1 Equilibration—Equilibrate disinfected chambers con-
where:
taining hygrometers before taking the first relative humidity
MC = bulk moisture content (%),
reading. Place the hygrometers on a shelf for ease of reading
M = mass of the small piece (g), and
b
through the walls of the chamber without opening the door.
M = mass of the small piece after drying (g).
d
Take multiple sequential readings at appropriate intervals that
M maybedeterminedeitherbyovendryingat105to110°C
d
were determined experimentally. The variation of the instru-
or desiccation to constant weight depending upon the test
mentation must be determined and taken into consideration.
material. Time required for drying is determined experimen-
For example, a minimum of four similar readings (65 %) over
tally. A sample can be considered dry when no significant
an 8 h period may be determined to demonstrate equilibrium.
weightchangeisdetectedintwoconsecutiveweighingsatleast
7.1.2 Recovery—Determine the amount of time required for
1 h apart.
the chamber relative humidity to recover to test levels after
opening the door for 1 to 2 min. This determination may be
9. Selection of Test Organism
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
7.2 Check chamber relative humidity daily, and record
organisms, the selection process should include a justification
readings depending on test length.
for the particular organism or organisms chosen. Testing of
materialswithmanydifferentorganismsfromdiversegroupsis
8. Sample Preparation
optimal. At a minimum, representative bacteria and fungi
should both be tested. Initial tests should be performed with
8.1 Specific details on the preparation of the samples will
only one species of microorganism.
depend upon the characteristics of the material to be tested.
9.1.1 Criteria for organism selection are based on a number
Generally, replicate small pieces of the test material should be
offactors.Appropri
...
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