ASTM D6329-98

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Designation: D 6329 – 98
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 D 1356 Terminology Relating to Sampling and Analysis of
Atmospheres
1.1 Many different types of microorganisms (for example,
E 104 Practice for Maintaining Constant Relative Humidity
bacteria, fungi, viruses, algae) can occupy indoor spaces.
by Means of Aqueous Solutions
Materials that support microbial growth are potential indoor
2.2 APHA Standards :
sources of biocontaminants (for example, spores and toxins)
Standard Methods for the Examination of Water and Waste-
that can become airborne indoor biopollutants. This guide
water
describes a simple, relatively cost effective approach to evalu-
ating the ability of a variety of materials to support microbial
3. Terminology
growth using a small chamber method.
3.1 Definitions—For definitions of terms used in this guide,
1.2 This guide is intended to assist groups in the develop-
refer to Terminology D 1356.
ment of specific test methods for a definite material or groups
3.2 Definitions of Terms Specific to This Standard:
of materials.
3.2.1 amplification—the act or result of increasing the
1.3 Static chambers have certain limitations. Usually, only
quantity of microorganisms.
small samples of indoor materials can be evaluated. Care must
3.2.2 CFU—colony forming unit, which may arise from a
be taken that these samples are representative of the materials
single organism or multiple units, such as spores, in the case of
being tested so that a true evaluation of the material is
the fungi.
performed.
3.2.3 colony—macroscopically visible growth.
1.4 Static chambers provide controlled laboratory microen-
3.2.4 inoculation—the act of introducing a microorganism
vironment conditions. These chambers are not intended to
(inoculum) into the test material.
duplicate room conditions, and care must be taken when
3.2.5 inoculum—viable test microorganism introduced onto
interpreting the results. Static chambers are not a substitute for
a material by implanting a small amount on the surface or
dynamic chambers or field studies.
substrate.
1.5 A variety of microorganisms, specifically bacteria and
3.2.6 plate—petri dish containing microbiological agar me-
fungi, can be evaluated using these chambers. This guide is not
dia on which microorganism are grown.
intended to provide human health effect data. However, organ-
3.2.7 static chamber—a small chamber (enclosed space)
isms of clinical interest, such as those described as potentially
with no internal forced air motion.
allergenic, may be studied using this approach.
3.2.8 susceptibility—the vulnerability of a material or sur-
1.6 This standard does not purport to address all of the
face to colonization by microorganisms.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
4. Significance and Use
priate safety and health practices and determine the applica-
4.1 The static chambers have several different applications:
bility of regulatory limitations prior to use.
4.1.1 The static chambers can be used to compare the
2. Referenced Documents susceptibility of different materials to the colonization and
amplification of various microorganisms under defined condi-
2.1 ASTM Standards:
2 tions.
D 1193 Specification for Reagent Water
This guide is under the jurisdiction of ASTM Committee D-22 on Sampling and
Analysis of Atmospheres and is the direct responsibility of Subcommittee D22.05
on Indoor Air. Annual Book of ASTM Standards, Vol 11.03.
4 th
Current edition approved Oct. 10, 1998. Published December 1998. Available from American Public Health Association, 1015 15 St., NW,
Annual Book of ASTM Standards, Vol 11.01. Washington, DC 20036.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D 6329
4.1.2 Chambers operated at high relative humidities may be chambers be tightly sealed so that the desired humidity will be
used to perform worst case scenario screening tests on mate- maintained. Place hygrometers in the chambers for confirma-
rials by providing an atmosphere where environmental condi- tion that humidities are being maintained, although saturated
tions may be favorable for microbial growth. salt solutions are themselves standards. Exercise care that the
4.1.3 Use of multiple chambers with different environmen- salts selected for use in the chamber are not inhibitory to the
tal parameters, such as a range of relative humidities, permits test organisms.
the evaluation of multiple microenvironments and allows 5.1.2 Temperature—Control the temperature of the cham-
investigation of materials under differing environmental con- bers. The chambers may be externally controlled through the
ditions. use of constant temperature environments, such as a room or
4.1.4 Drying requirements for wetted materials may also be incubator. Chart recorders or other data logging devices are
investigated. This information may be relevant for determining recommended to confirm maintenance of temperature. Con-
material resistance to microbial growth after becoming wet. trolled temperature is critical for two reasons. First, it can have
These conditions may simulate those where materials are a profound effect on the growth of microorganisms. Second,
subjected to water incursion through leaks as well as during relative humidity is dependent upon temperature. The control
remediation of a building after a fire. limits may be defined by consulting a psychometric chart and
4.1.5 Growth rates of microorganisms on the material may determining the impact of temperature on a specific test RH.
also be investigated. Once it has been established that organ- 5.1.3 Characterize instrumentation for evaluating other pa-
isms are able to grow on a particular material under defined rameters if the instruments are to be employed during material
conditions, investigations into the rate of organism growth may testing. Conditions such as light need to be noted and con-
be performed. These evaluations provide base line information trolled during the course of an experiment as these conditions
and can be used to evaluate methods to limit or contain may have an effect on the growth of the test organism. Light
amplification of microorganisms. may be controlled externally by placing the chambers in a
4.2 These techniques should be performed by personnel darkened room to remove light or in a continuously lighted
with training in microbiology. The individual must be compe- room for a constant light source.
tent in the use of sterile technique, which is critical to exclude 5.2 Provide ports, where needed, for the insertion of probes
external contamination of materials. to monitor and record temperature and relative humidity, using
externally located instrumentation as long as it is well sealed
5. Apparatus
and contamination is avoided.
5.1 Static Chamber—Chambers should be relatively small
5.3 Decontamination—Decontaminate the chamber before
and portable, contain three or four shelves, and be easily initiating any analysis. Surface disinfection or vapor phase
decontaminated. In addition, transparent walls are desirable
disinfection may be appropriate. Glass may be autoclaved.
because visual inspection of the test material and monitoring of Follow the manufacturers’ instructions, especially any safety
instruments (that is, hygrometers) without opening the cham-
precautions. If a chemical disinfectant is employed, clear the
ber is preferred. Fig. 1 is a schematic diagram of a possible chambers of any residual disinfectant to prevent interference
static chamber. Acrylic desiccators are readily available, easily with the growth of the microorganisms on the material being
adaptable, and relatively inexpensive. Other options, such as evaluated. Thoroughly ventilate the chambers in a clean
glass, are also acceptable. Glass has the advantage of being environment. Decontaminate the salt solutions. The method
autoclavable; however, it is frequently much less portable. The used is dependent upon the composition of the salts selected.
chamber door must provide ready access to the materials but Any instrumentation to be used during the evaluations, such as
should be airtight when closed.
hygrometers, may be removed from the chambers during the
5.1.1 Relative Humidity—Maintain humidities through the decontamination procedure of the chamber surfaces and de-
use of saturated salt solutions contained in trays on the bottom
contaminated separately; however, it is generally more effec-
of the chambers (see Practice E 104). It is essential that the 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
D 6329
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. harm the material and do not have residue effects or if all traces
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 Compute the bulk moisture content of the test material
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 (6 5 %) over M may be determined either by oven drying at 105 to 110°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 weight change is detected in two consecutive weighings at least
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 3 4
be tested. Initial tests should be performed with only one
cm may be used. Pieces should be placed on sterile petri dishes
species of microorganism.
or other appropriate holders on the shelves in the chamber.
9.1.1 Criteria for organism selection are based on a number
Include controls and blanks within the
...