ASTM D6329-98(2003)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:D6329–98 (Reapproved 2003)
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 D 6329; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
1.1 Many different types of microorganisms (for example, 2.1 ASTM Standards:
bacteria, fungi, viruses, algae) can occupy indoor spaces. D 1193 Specification for Reagent Water
Materials that support microbial growth are potential indoor D 1356 Terminology Relating to Sampling and Analysis of
sources of biocontaminants (for example, spores and toxins) Atmospheres
that can become airborne indoor biopollutants. This guide E 104 Practice for Maintaining Constant Relative Humidity
describes a simple, relatively cost effective approach to evalu- by Means of Aqueous Solutions
ating the ability of a variety of materials to support microbial 2.2 APHA Standards :
growth using a small chamber method. Standard Methods for the Examination of Water and Waste-
1.2 This guide is intended to assist groups in the develop- water
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
small samples of indoor materials can be evaluated. Care must refer to Terminology D 1356.
3.2 Definitions of Terms Specific to This Standard:
be taken that these samples are representative of the materials
being tested so that a true evaluation of the material is 3.2.1 amplification—the act or result of increasing the
quantity of microorganisms.
performed.
1.4 Static chambers provide controlled laboratory microen- 3.2.2 CFU—colony forming unit, which may arise from a
vironment conditions. These chambers are not intended to single organism or multiple units, such as spores, in the case of
the fungi.
duplicate room conditions, and care must be taken when
interpreting the results. Static chambers are not a substitute for 3.2.3 colony—macroscopically visible growth.
3.2.4 inoculation—the act of introducing a microorganism
dynamic chambers or field studies.
1.5 A variety of microorganisms, specifically bacteria and (inoculum) into the test material.
3.2.5 inoculum—viable test microorganism introduced onto
fungi, can be evaluated using these chambers.This guide is not
intended to provide human health effect data. However, organ- a material by implanting a small amount on the surface or
substrate.
isms of clinical interest, such as those described as potentially
allergenic, may be studied using this approach. 3.2.6 plate—petri dish containing microbiological agar me-
dia on which microorganism are grown.
1.6 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the 3.2.7 static chamber—a small chamber (enclosed space)
with no internal forced air motion.
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica- 3.2.8 susceptibility—the vulnerability of a material or sur-
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 ofASTM Committee D22 on Sampling and contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Analysis of Atmospheres and is the direct responsibility of Subcommittee D22.05 Standards volume information, refer to the standard’s Document Summary page on
on Indoor Air. the ASTM website.
3 th
CurrenteditionapprovedOctober1,2003.PublishedNovember2003.Originally Available from American Public Health Association, 1015 15 St., NW,
approved in 1998. Last previous edition approved in 1998 as D 6329 - 98. Washington, DC 20036.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D6329–98 (2003)
4. Significance and Use 5.1.1 Relative Humidity—Maintain humidities through the
use of saturated salt solutions contained in trays on the bottom
4.1 The static chambers have several different applications:
of the chambers (see Practice E 104). It is essential that the
4.1.1 The static chambers can be used to compare the
chambers be tightly sealed so that the desired humidity will be
susceptibility of different materials to the colonization and
maintained. Place hygrometers in the chambers for confirma-
amplification of various microorganisms under defined condi-
tion that humidities are being maintained, although saturated
tions.
salt solutions are themselves standards. Exercise care that the
4.1.2 Chambers operated at high relative humidities may be
salts selected for use in the chamber are not inhibitory to the
used to perform worst case scenario screening tests on mate-
test organisms.
rials by providing an atmosphere where environmental condi-
5.1.2 Temperature—Control the temperature of the cham-
tions may be favorable for microbial growth.
bers. The chambers may be externally controlled through the
4.1.3 Use of multiple chambers with different environmen-
use of constant temperature environments, such as a room or
tal parameters, such as a range of relative humidities, permits
incubator. Chart recorders or other data logging devices are
the evaluation of multiple microenvironments and allows
recommended to confirm maintenance of temperature. Con-
investigation of materials under differing environmental con-
trolled temperature is critical for two reasons. First, it can have
ditions.
a profound effect on the growth of microorganisms. Second,
4.1.4 Drying requirements for wetted materials may also be
relative humidity is dependent upon temperature. The control
investigated.This information may be relevant for determining
limits may be defined by consulting a psychometric chart and
material resistance to microbial growth after becoming wet.
determining the impact of temperature on a specific test RH.
These conditions may simulate those where materials are
5.1.3 Characterize instrumentation for evaluating other pa-
subjected to water incursion through leaks as well as during
rameters if the instruments are to be employed during material
remediation of a building after a fire.
testing. Conditions such as light need to be noted and con-
4.1.5 Growth rates of microorganisms on the material may
trolled during the course of an experiment as these conditions
also be investigated. Once it has been established that organ-
may have an effect on the growth of the test organism. Light
isms are able to grow on a particular material under defined
may be controlled externally by placing the chambers in a
conditions,investigationsintotherateoforganismgrowthmay
darkened room to remove light or in a continuously lighted
be performed. These evaluations provide base line information
room for a constant light source.
and can be used to evaluate methods to limit or contain
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
chambers of any residual disinfectant to prevent interference
decontaminated. In addition, transparent walls are desirable
becausevisualinspectionofthetestmaterialandmonitoringof with the growth of the microorganisms on the material being
evaluated. Thoroughly ventilate the chambers in a clean
instruments (that is, hygrometers) without opening the cham-
ber is preferred. Fig. 1 is a schematic diagram of a possible environment. Decontaminate the salt solutions. The method
used is dependent upon the composition of the salts selected.
static chamber.Acrylic desiccators are readily available, easily
adaptable, and relatively inexpensive. Other options, such as Any instrumentation to be used during the evaluations, such as
hygrometers, may be removed from the chambers during the
glass, are also acceptable. Glass has the advantage of being
autoclavable; however, it is frequently much less portable. The decontamination procedure of the chamber surfaces and de-
contaminated separately; however, it is generally more effec-
chamber door must provide ready access to the materials but
should be airtight when closed. 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
used in all tests. Unless otherwise indicated, it is intended that
all reagents conform to the specifications of the Committee on
FIG. 1 Schematic of Example Static Chamber Analytical Reagents of the American Chemical Society where
D6329–98 (2003)
such specifications are available. Other grades may be used, 8.2 Common microbiological practice is to sterilize a sur-
provided it is first ascertained that the reagent is of sufficiently face or material before inoculation to ensure that the test
high purity to permit its use without lessening the accuracy of organism is the only source being evaluated.Autoclaving is an
the determination. extremely effective method if such a procedure does not alter
6.2 Purity of Water—Unless otherwise indicated, references the test material. Other methods, such as ionizing and non-
to water shall be understood to mean reagent water as certified ionizing irradiation, ultraviolet, dry heat, and surface or vapor
by Type II of Specification D 1193. It should conform to the phase disinfection, are also acceptable if these methods do not
Type A specifications for microbial classification. harmthematerialanddonothaveresidueeffectsorifalltraces
6.3 Microbiological Media—Choose appropriate media de- of the disinfectant can be removed prior to testing. Consult the
pending upon the test microorganism selected. Commercially test material manufacturer or conduct tests with the test
prepared media may be acceptable, but it may be necessary to material to determine the best method of sterilization. Specific
prepare organism specific media. References should be con- details for decontamination depend upon the method selected
sulted to determine the proper media for optimal growth of the and should be worked out before actual testing begins within
test organism. the QA/QC framework.
8.3 Equilibrate or bring to near equilibrium samples in the
7. Characterization of Static Chamber
chamber before inoculation with the test organism. Equilibra-
tion time will depend upon both the material to be tested and
7.1 Characterize static chambers for all environmental pa-
the chamber relative humidity selected for the test. Determine
rameters being measured before any material evaluations are
equilibration times for each material prior to testing.
performed. Chambers should be characterized for at least
8.3.1 Ascertain equilibration by determining when the bulk
relative humidity and temperature. Take sufficient readings to
ensure that the conditions will be maintained throughout the moisture content of the material reaches a constant value. The
course of the experiment and will meet the QA/QC standards use of a calibrated analytical balance is recommended.
developed for a specific test.
8.3.2 Computethebulkmoisturecontentofthetestmaterial
7.1.1 Equilibration—Equilibrate disinfected chambers con-
as follows:
taining hygrometers before taking the first relative humidity
MC 5 [ M – M / M 3 100 (1)
~ ! #
b d d
reading. Place the hygrometers on a shelf for ease of reading
through the walls of the chamber without opening the door. where:
Take multiple sequential readings at appropriate intervals that MC = bulk moisture content (%),
M = mass of the small piece (g), and
were determined experimentally. The variation of the instru-
b
M = mass of the small piece after drying (g).
mentation must be determined and taken into consideration.
d
For example, a minimum of four similar readings (65 %) over M maybedeterminedeitherbyovendryingat105to110°C
d
an 8 h period may be determined to demonstrate equilibrium. or desiccation to constant weight depending upon the test
7.1.2 Recovery—Determine the amount of time required for material. Time required for drying is determined experimen-
the chamber relative humidity to recover to test levels after tally. A sample can be considered dry when no significant
opening the door for 1 to 2 min. This determination may be weightchangeisdetectedintwoconsecutiveweighingsatleast
crucial, especially at the higher relative humidities. Exercise 1 h apart.
care to utilize hygrometers that have a rapid response time.
7.2 Check chamber relative humidity daily, and record
9. Selection of Test Organism
readings depending on test length.
9.1 Selection of the appropriate test organisms is extremely
important. Since growth requirements vary for different organ-
8. Sample Preparation
isms, the selection process should include a justification for the
8.1 Specific details on the preparation of the samples will
particular organism or organisms chosen. Testing of materials
depend upon the characteristics of the material to be tested.
with many different organisms from diverse groups is optimal.
Generally, replicate small pieces of the test material should be
At a minimum, representative bacteria and fungi should both
used. Depending upon the material, pieces as small as 4 by 4
be tested. Initial tests should be performed with only one
cm may be used. Pieces should be placed on ste
...