ASTM E2111-00
(Test Method)Standard Quantitative Carrier Test Method To Evaluate the Bactericidal, Fungicidal, Mycobactericidal and Sporicidal Potencies of Liquid Chemical Germicides
Standard Quantitative Carrier Test Method To Evaluate the Bactericidal, Fungicidal, Mycobactericidal and Sporicidal Potencies of Liquid Chemical Germicides
SCOPE
1.1 This test method is designed for use in product development and for the generation of product potency data. The loading of each carrier with a known volume of the test organism suspension is possible. The incorporation of control carriers also allows for an assessment of the load of viable test organism on the test carriers.
1.2 This test method is designed to have survivors and also to be used with a performance standard. The surviving microorganisms on each test carrier are compared to the mean of no less than three control carriers to determine if the performance standard has been met. To allow proper statistical evaluation of results, the size of the test inoculum should be sufficiently large to take into account both the performance standard and the experimental variation in the results. For example, if an arbitrary performance standard of 6-log10 reduction in the viability titer of the test organism is used, and an inoculum size of 107 CFU, then theoretically a maximum of ten survivors per carrier is permitted; however, because of experimental variability the exact target may need to be higher than 106 CFU/carrier, thus fewer survivors would be permitted.
1.3 This test method should be performed by persons with training in microbiology and in facilities designed and equipped for work with infectious agents at the appropriate biosafety level (3).
1.4 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.
1.5 In this test method, metric units are used for all applications, except for distance in which case inches are used and metric units follow.
1.6 It is the responsibility of the investigator to determine whether Good Laboratory Practice Regulations (GLPs) are required and to follow them where appropriate (40 CFR, Part 160 for EPA submissions and 21 CFR, Part 58 for FDA submissions).
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Designation:E2111–00
Standard Quantitative Carrier Test Method To
Evaluate the Bactericidal, Fungicidal, Mycobactericidal and
Sporicidal Potencies of Liquid Chemical Germicides
This standard is issued under the fixed designation E 2111; 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.
INTRODUCTION
The need for better tests to assess the germicidal activity of chemicals has been recognized (1) and
several investigations conducted in the past decade have been aimed at developing simpler and fully
quantitative tests while attempting to design protocols suitable for working with a wide variety of
microorganisms (2). The method described here uses glass vials as carriers and the same basic set of
materials and procedures can be used to test the potency of liquid chemical germicides against
vegetative bacteria, fungi, mycobacteria and bacterial spores. It is not appropriate, however, for
working with viruses because of the relatively high levels of eluate dilutions required and the need for
membrane filtration. Further evaluation of products under more stringent test conditions may be
necessary for their registration. Performance standards for the categories of products to be tested and
the specific types of organism(s) to be used may also vary depending on the regulatory agency.
1. Scope 1.3 This test method should be performed by persons with
training in microbiology and in facilities designed and
1.1 This test method is designed for use in product devel-
equipped for work with infectious agents at the appropriate
opment and for the generation of product potency data. The
biosafety level (3).
loading of each carrier with a known volume of the test
1.4 This standard does not purport to address all of the
organism suspension is possible. The incorporation of control
safety concerns, if any, associated with its use. It is the
carriers also allows for an assessment of the load of viable test
responsibility of the user of this standard to establish appro-
organism on the test carriers.
priate safety and health practices and determine the applica-
1.2 This test method is designed to have survivors and also
bility of regulatory limitations prior to use.
to be used with a performance standard. The surviving micro-
1.5 In this test method, metric units are used for all
organisms on each test carrier are compared to the mean of no
applications, except for distance in which case inches are used
less than three control carriers to determine if the performance
and metric units follow.
standard has been met.To allow proper statistical evaluation of
1.6 It is the responsibility of the investigator to determine
results,thesizeofthetestinoculumshouldbesufficientlylarge
whether Good Laboratory Practice Regulations (GLPs) are
to take into account both the performance standard and the
required and to follow them where appropriate (40 CFR, Part
experimental variation in the results. For example, if an
160 for EPA submissions and 21 CFR, Part 58 for FDA
arbitrary performance standard of 6-log reduction in the
submissions).
viabilitytiterofthetestorganismisused,andaninoculumsize
of 10 CFU, then theoretically a maximum of ten survivors per
2. Referenced Documents
carrier is permitted; however, because of experimental vari-
2.1 ASTM Standards:
ability the exact target may need to be higher than 10
D 1129 Terminology Relating to Water
CFU/carrier, thus fewer survivors would be permitted.
D 1193 Specification for Reagent Grade Water
This test method is under the jurisdiction of ASTM Committee E35 on
Pesticides and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Oct. 10, 2000. Published March 2001. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
The boldface numbers in parentheses refer to the list of references at the end of Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2111–00
E 1054 Practices for Evaluating Inactivators of Antimicro- 100-mm diameter petri plate. The plates are held for the
bial Agents Used in Disinfectant, Sanitizer, Antiseptic, or required period at the desired incubation temperature, colonies
Preserved Products counted and log reductions in the viability titer of the test
2.2 CFR Standards: organism calculated.
40 CFR, Part 160
4 5. Significance and Use
21 CFR, Part 58
5.1 This test method is fully quantitative and it also avoids
3. Terminology
any loss of viable organisms through wash off. This makes it
possible to produce statistically valid data using many fewer
3.1 Definitions of Terms Specific to This Standard:
test and control carriers than other quantitative methods based
3.1.1 carrier, n—an inanimate surface or object inoculated
on most probable numbers (MPN).
with the test organism.
5.2 The design of the carriers makes it possible to place into
3.1.2 eluate, n—an eluent, which contains the recovered
each a precisely measured volume of the test suspension. The
organism(s).
use of the threaded stir bars allows for efficient recovery of the
3.1.3 eluent, n—any solution that is harmless to the test
inoculum even after its exposure for several hours to strong
organism(s) and that is added to a carrier to recover the
fixatives such as glutaraldehyde.
organism(s) in or on it.
5.3 The membrane filtration step allows processing of the
3.1.4 neutralization, n—a process to quench the antimicro-
entireeluatefromthetestcarriersandthereforethecaptureand
bial activity of a test formulation. This process may be
subsequent detection of even low numbers of viable organisms
achieved by dilution of the organism/test formulation mixture
that may be present.
and/or by adding to it one or more chemical neutralizers.
5.4 Intheabsenceofauniversalneutralizer,thistestmethod
3.1.5 soil load, n—a solution of one or more organic, or
uses a 10-fold dilution of the test product by phosphate buffer
inorganic substances, or both, added to the suspension of the
or saline immediately at the end of the contact time and
test organism to simulate the presence of body secretions,
filtration of the organism-product mixture through a membrane
excretions, or other extraneous substances.
and the subsequent rinsing of the filter with several changes of
3.1.6 test formulation, n—a formulation that incorporates
the buffer or saline. This approach usually reduces germicide
antimicrobial ingredients.
residues to non-inhibitory levels; however, the test protocol
3.1.7 test organism, n—an applied inoculum of an organism
permits the addition of a specific neutralizer to the eluent/
that has characteristics that allows it to be readily identified. It
diluent, if required (See Practices E 1054 ).
also may be referred to as a surrogate or a marker organism.
5.5 This test can be performed with or without a soil load to
4. Summary of Test Method
determinetheeffectofsuchloadingongermicideperformance.
The soil load developed for this test is a mixture of three types
4.1 This is a fully quantitative carrier test method suitable
of proteins (high molecular weight proteins, low molecular
for assessing the potency of liquid chemical germicides against
weight peptides and mucous material) to represent the body
vegetative bacteria, fungi, mycobacteria, as well as bacterial
secretions, excretions or other extraneous substances that
spores. It is designed primarily for testing formulations to be
chemicalgermicidesmayencounterunderfieldconditions.Itis
used on hard environmentalsurfacesandmedicaldevices.This
suitable for working with the various test organisms included
test method uses the flat inside bottom surface of glass vials as
here. The components of the soil load are readily available and
the carrier. Each vial receives 10 µL of the test organism with
subject to much less variability than animal sera.
or without a soil load. The contamination of the inside surface
5.6 Since the quality of tap water varies considerably both
of the carrier with microaerosols is avoided by the use of glass
geographically and temporally, this test method incorporates
inserts. The inoculum is dried and exposed to 1 mL of the test
the use of water with a specified and documented level of
germicide for the desired contact time at the recommended
hardness to prepare use-dilutions of test products. The U.S.
temperature; control carriers receive 1 mL of phosphate buffer
Environmental Protection Agency’s Scientific Advisory Panel
instead.At the end of the contact time, 9 mLof a diluent, with
(SAP) on Germicide Test Methodology has recommended the
or without a neutralizer, is added to the vial to dilute/neutralize
use of water with a standard hardness of 400 ppm as CaCO .
the germicide and any inoculum adhering to the carrier surface 3
is recovered using a magnetic stir bar with a threaded surface.
6. General Equipment and Labware
The eluate is passed through a membrane filter, the carrier vial
6.1 Laminar Flow Cabinet—AClass II (TypeA) biological
is then rinsed several times with eluate/diluent and the rinses
safety cabinet for this work. The procedures for the proper
also are passed through the same filter. The total rinse volume
maintenance and use of such cabinets are given in Ref (3).
is about 100 mL. Control and test eluates requiring dilution to
6.2 Incubator—An ordinary incubator and an anaerobic
get countable colonies, are subjected first to a series of 10-fold
incubator. If only one ordinary incubator is available, its
dilutions and the material from suitable dilutions is passed
temperature will require adjustment depending on the type of
separately through membrane filters. Each filter is placed on
organism under test.
the agar surface of an appropriate recovery medium in a
6.3 Sterilizer—Any steam sterilizer suitable for processing
culture media, reagents and labware is acceptable. The steam
supplied to the sterilizer must be free from additives toxic to
Available from Superintendent of Documents, U.S. Government Printing
Office, Washington D.C. 20402 the test organisms.
E2111–00
6.4 Filter Sterilization System for Media and Reagents—A 6.23 Stir Bars with Threaded TFE-fluorocarbon Coated
membrane or cartridge filtration system (0.22 µm pore diam- Surface, to dislodge inoculum from the carriers surface. Stir
,
7 6
eter) is required for sterilizing heat-sensitive solutions. bars may be manufactured according to Fig. 1b.
6.5 Membrane Filtration System for Capture of the Test
6.24 Magnet, strong enough to hold the threaded stir bar in
Organisms—Sterile 47-mm diameter membrane filters (0.22 place in the glass carrier while the liquid is being poured out of
µm or 0.45 µm pore diameter) and glass or metal holders for
it for membrane filtration.
such filters are required.
6.25 Aluminum Foil, to wrap items to be sterilized.
6.6 Environmental Chamber/Incubator—To hold the carri-
6.26 Vortex Mixer, to vortex the eluate and rinsing fluid in
ers at the desired test temperature.
the carrier to ensure efficient recovery of the test organism(s).
6.7 Freezers—A freezer at –2062°C is required for the
6.27 Glass Inserts, to be placed inside the glass carriers
storage of media and additives. A second freezer at –70°C or
during their inoculation with the test organism. Such inserts
lower is required to store the stocks of test organisms.
have been found to eliminate the deposition of microaerosols
6.8 Refrigerator—A refrigerator at 462°C for storage of
on the inside walls of the carriers. Glass inserts may be
8,6
media, culture plates and reagents.
manufactured according to Fig. 1c.
6.9 Timer—Any stop-watch that can be read in minutes and
6.28 Centrifuge, to allow for the sedimentation of the
seconds.
cells/spores of the test organism(s) for concentration, or
6.10 Hot Air Oven—An oven at 60°C to dry clean and washing, or both.
sterile glassware.
6.29 Markers, permanent labware marking pens.
6.11 Magnetic Stir Plate and Stir Bars—Large enough for a
6.30 Sterile Polypropylene Centrifuge Tubes with Caps,50
5-L beaker or Erlenmeyer flask for preparing culture media or
mL.
other solutions.
6.31 Colony Counter,forexample,QuebecColonyCounter.
6.12 Positive Displacement Pipette—A pipette and pipette
6.32 Sterile Disposable Gloves, for handling the carriers.
tipsthataccuratelycandispense10-µLvolumesforinoculation
6.33 Hemocytometer, for counting fungal conidia.
of carriers.
6.34 Spectrophotometer, for measuring turbidity of micro-
6.13 Air Displacement Pipettes—Eppendorf or equivalent,
bial suspensions.
100–1000 µL with disposable tips.
6.14 Orbital Shaker—For shaking the broth cultures of
7. General Solutions and Reagents
bacteria during their incubation.
7.1 Purity of Reagents—Reagent grade chemicals shall be
6.15 Sterile Dispenser—10 mL, for dispensing diluent/
used in all tests. Unless otherwise indicated, it is intended that
eluent.
all reagents conform to the specifications of the Committee on
6.16 Glassware—1-Lflaskswithaside-armandappropriate
Analytical Reagents of the American Chemical Society (4).
tubing to capture the filtrates from 47-mm diameter membrane
Other grades may be used (5), provided it is first ascertained
filters; 250-mL Erlenmeyer flasks for culture media; reusable
that the reagent is of sufficiently high purity to permit its use
or disposable glass pipettes capable of handling 10-mL, 5-mL,
without lessening the accuracy of the determination.
and 1-mL volumes; 25-mL test tubes with caps.
7.2 Absolute Alcohol—In a 100-mL plastic or glass beaker
6.17 Vacuum Source, a vacuum pump, access to an in-house
for flame-sterilization of metallic forceps used to handle
vacuum line or a water faucet vacuum apparatus required to
membrane filters.
pull the samples through the membrane filters.
7.3 Phosphate Buffer,preparedaccordingtotheformulation
6.18 Sterile Disposable Plastic Petri Dishes, 100 mm 3 15
given in Ref (6). Adjust buffer pH to 7.2
mm.
7.4 Sterile Normal Saline (0.85 % NaCl)—To be used as an
6.19 Forceps, straight or curved, with smooth tips to handle
eluent for the mycobacterium and the fungus.
membrane filters.
7.5 Test Germicide—Prepared at its use-dilution and
6.20 Flat-Bottomed Glass Vials, 20 mL, with regular and
brought to the test temperature.
septate caps (Fig. 1a). Flat-bottomed glass vials may be
7.6 Growth,
...
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