Designation: E 2111 05

Standard Quantitative Carrier Test Method to

Evaluate the Bactericidal, Fungicidal, Mycobactericidal, and
Sporicidal Potencies of Liquid Chemical Microbicides1
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 microbicidal activity of chemicals was recognized (1)2 and
several simpler and quantitative test methods have been developed for working with a wide variety of
microorganisms (2). The test method described here uses glass vials as carriers; the same basic set of
materials and procedures can be used to test the potency of liquid microbicides against vegetative
bacteria, fungi, mycobacteria, and bacterial spores. However, the test method is not appropriate for use
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

1.1 This test method is designed for use in product devel- training in microbiology and in facilities designed and
opment and for the generation of product potency data. This equipped for work with infectious agents at the appropriate
test method permits the loading of each carrier with a known biosafety level (3).
volume of the test organism. The incorporation of controls can 1.4 In this test method, SI units are used for all applications,
also determine the initial load of colony forming units (CFU) except for distance, in which case inches are used and SI units
of organisms on the test carriers and any loss in CFU after the follow.
mandatory drying of the inoculum. 1.5 It is the responsibility of the investigator to determine
1.2 This test method is designed to have survivors and also whether Good Laboratory Practice Regulations (GLPs) are
to be used with a performance standard. The surviving micro- required and to follow them where appropriate (40 CFR, Part
organisms on each test carrier are compared to the mean of no 160 for EPA submissions and 21 CFR, Part 58 for FDA
less than three control carriers to determine if the performance submissions).
standard has been met. To allow proper statistical evaluation of 1.6 This standard does not purport to address all of the
results, the size of the test inoculum should be sufficiently large safety concerns, if any, associated with its use. It is the
to take into account both the performance standard and the responsibility of the user of this standard to establish appro-
experimental variation in the results. For example, if an priate safety and health practices and determine the applica-
arbitrary performance standard of 6-log10 reduction in the bility of regulatory limitations prior to use.
viability titer of the test organism is used, and an inoculum size
2. Referenced Documents
of 107 CFU, then theoretically a maximum of ten survivors per
carrier is permitted; however, because of experimental vari- 2.1 ASTM Standards: 3
ability, the exact target may need to be higher than 106 D 1129 Terminology Relating to Water
CFU/carrier, thus fewer survivors would be permitted. D 1193 Specification for Reagent Grade Water
E 1054 Practices for Evaluating Inactivators of Antimicro-
bial Agents Used in Disinfectant, Sanitizer, Antiseptic, or
1
This test method is under the jurisdiction of ASTM Committee E35 on
Preserved Products
Pesticides and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
3
Current edition approved Nov. 1, 2005. Published December 2005. Originally For referenced ASTM standards, visit the ASTM website, www.astm.org, or
approved in 2000. Last previous edition approved in 2000 as E 2111 00. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
2
The boldface numbers in parentheses refer to the list of references at the end of Standards volume information, refer to the standards 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.

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 E 2111 05
2.2 CFR Standards: filters. Each filter is placed on the agar surface of an appropri-
40 CFR, Part 1604 ate recovery medium in a 100-mm diameter petri plate. The
21 CFR, Part 584 plates are held for the required period at the desired incubation
temperature, colonies counted, and log10 reductions in the
3. Terminology viability titer of the test organism calculated.
3.1 Definitions of Terms Specific to This Standard:
NOTE 1Do not soak the magnetic stir bars in ethanol or other solvents
3.1.1 carrier, ninanimate surface or object inoculated
for decontamination as this may damage the sealant on them.
with the test organism.
3.1.2 eluate, neluent, which contains the recovered organ- 5. Significance and Use
ism(s).
3.1.3 eluent, nany solution that is harmless to the test 5.1 This test method is fully quantitative and it also avoids
organism(s) and that is added to a carrier to recover the any loss of viable organisms through wash off. This makes it
organism(s) in or on it. possible to produce statistically valid data using many fewer
3.1.4 neutralization, nprocess to quench the antimicrobial test and control carriers than other quantitative methods based
activity of a test formulation. This process may be achieved by on most probable numbers (MPN).
dilution of the organism/test formulation mixture and/or by 5.2 The design of the carriers makes it possible to place into
adding to it one or more chemical neutralizers. (Refer to each a precisely measured volume of the test suspension. The
Practices E 1054 in 2.1 for further details use of the threaded stir bars allows for efficient recovery of the
3.1.4.1 DiscussionThis process may be achieved by dilu- inoculum even after its exposure for several hours to strong
tion of the organism/test formulation mixture or by adding to it fixatives such as glutaraldehyde.
one or more chemical neutralizers, or both. 5.3 The membrane filtration step allows processing of the
3.1.5 soil load, nsolution of one or more organic, or entire eluate from the test carriers and therefore the capture and
inorganic substances, or both, added to the suspension of the subsequent detection of even low numbers of viable organisms
test organism to simulate the presence of body secretions, that may be present.
excretions, or other extraneous substances. 5.4 This test can be performed with or without a soil load to
3.1.6 test formulation, nformulation that incorporates an- determine the effect of such loading on microbicide perfor-
timicrobial ingredients. mance. The soil load developed for this test is a mixture of
3.1.7 test organism, napplied inoculum of an organism three types of proteins (high molecular weight proteins, low
that has characteristics that allows it to be readily identified. It
molecular weight peptides, and mucous material) to represent
also may be referred to as a surrogate or a marker organism.
the body secretions, excretions, or other extraneous substances
4. Summary of Test Method that chemical microbicides may encounter under field condi-
tions. It is suitable for working with the various test organisms
4.1 This is a fully quantitative carrier test method suitable included here. The components of the soil load are readily
for assessing the potency of chemicals against vegetative
available and subject to much less variability than animal sera.
bacteria, fungi, mycobacteria, as well as bacterial spores. It is
designed primarily for testing formulations to be used on hard 5.5 Since the quality of tap water varies considerably both
environmental surfaces and medical devices. This test method geographically and temporally, this test method incorporates
uses the flat inside bottom surface of glass vials as the carrier. the use of water with a specified and documented level of
Each vial receives 10 L of the test organism with or without hardness to prepare use-dilutions of test products. The U.S.
a soil load. The contamination of the inside surface of the Environmental Protection Agencys Scientific Advisory Panel
carrier with microaerosols is avoided by the use of glass (SAP) on Germicide Test Methodology has recommended the
inserts. The inoculum is dried and exposed to 1 mL of the test use of water with a standard hardness of 400 ppm as CaCO3.
microbicide for the desired contact time at the recommended
temperature; control carriers receive 1 mL of normal saline 6. General Equipment and Labware
instead. At the end of the contact time, 9 mL of an eluent 6.1 Laminar Flow CabinetA Class II (Type A) biological
without or with a neutralizer, is added to the vial to dilute/ safety cabinet for this work. The procedures for the proper
neutralize the microbicide and any inoculum adhering to the maintenance and use of such cabinets are given in Ref 3.
carrier surface is recovered using a magnetic stir bar with a 6.2 IncubatorAn ordinary incubator and an anaerobic
threaded surface. The eluate is passed through a membrane incubator. If only one ordinary incubator is available, its
filter, the carrier vial is then rinsed several times with eluent/ temperature will require adjustment depending on the type of
diluent and the rinses are also passed through the same filter. organism under test.
The total rinse volume is no less than 100 mL. Control and test
eluates requiring dilution to get countable colonies are first 6.3 SterilizerAny steam sterilizer suitable for processing
subjected to a series of tenfold dilutions and the material from culture media, reagents and labware is acceptable. The steam
suitable dilutions is passed separately through membrane supplied to the sterilizer must be free from additives toxic to
the test organisms.
6.4 Filter Sterilization System for Media and ReagentsA
4
Available from U.S. Government Printing Office Superintendent of Documents, membrane or cartridge filtration system (0.22-m pore diam-
732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401. eter) is required for sterilizing heat-sensitive solutions.

2
 E 2111 05

FIG. 1 Components of a Carrier for the Quantitative Carrier Test

6.5 Membrane Filtration System for Capture of the Test 6.11 Magnetic Stir Plate and Stir BarsLarge enough for a
OrganismsSterile 47-mm diameter membrane filters (0.22- 5-L beaker or Erlenmeyer flask for preparing culture media or
or 0.45-m pore diameter) and glass, metal, or plastic holders other solutions.
for such filters are required. 6.12 Positive Displacement PipetteA pipette and pipette
6.6 Environmental Chamber/IncubatorTo hold the carri- tips that accurately can dispense 10-L volumes for inoculation
ers at the desired test temperature. of carriers.
6.7 FreezersA freezer at 20 6 2C is required for the 6.13 Air Displacement PipettesEppendorf or equivalent,
storage of media and additives. A second freezer at 70C or 100 to 1000 L with disposable tips.
lower is required to store the stocks of test organisms. 6.14 Orbital ShakerFor shaking the broth cultures of
6.8 RefrigeratorA refrigerator at 4 6 2C for storage of bacteria during their incubation.
media, plates, and reagents. 6.15 Sterile Dispenser10 mL, for dispensing diluent/
6.9 TimerAny stopwatch that can be read in minutes and eluent.
seconds. 6.16 GlasswareOne-liter flasks with a side-arm and ap-
6.10 Hot Air OvenAn oven at 60C to dry and sterile propriate tubing to capture the filtrates from 47-mm diameter
clean glassware. membrane filters; 250-mL Erlenmeyer flasks for culture media;

3
 E 2111 05
100 mL and 5 L beakers, reusable or disposable glass pipettes Analytical Reagents of the American Chemical Society (4).
capable of handling 10-, 5-, and 1-mL volumes; and 25-mL test Other grades may be used (5), provided it is first ascertained
tubes with caps. that the reagent is of sufficiently high purity to permit its use
6.17 Vacuum SourceA vacuum pump, access to an in- without lessening the accuracy of the determination.
house vacuum line or a water faucet vacuum apparatus 7.2 Absolute AlcoholIn a 100-mL plastic or glass beaker
required to pull the samples through the membrane filters. for flame-sterilization of metallic forceps used to handle
6.18 Sterile Disposable Plastic Petri Dishes, 100 by 15 mm. membrane filters.
6.19 Forceps, straight or curved, with smooth tips to handle 7.3 Normal Saline (0.85 % NaCl; pH 7.2)To be used as
membrane filters. an eluent and control fluid.
6.20 Flat-Bottomed Glass Vials, 20 mL, with regular and 7.4 Test MicrobicidePrepared at its use-dilution and
septate caps (Fig. 1A). Flat-bottomed glass vials may be brought to the test temperature.
manufactured such that the bottom of the vials is completely 7.5 Growth, Recovery Media and Media SupplementsThe
flat with no ridges.5,6 required types of materials (see below) can be purchased from
6.21 Vials, wide-mouth, glass, 25 mL, for use as dilution a variety of sources specializing in laboratory supplies.
vials. 7.6 MnSO4H2O, added to Columbia broth to promote the B.
6.22 Desiccator, recommended size is 25 cm wide by 20 cm subtilis sporulation.
deep, with an active desiccant for drying the inocula on the 7.7 Test Product Diluent, water with a standard hardness of
carriers. 400 ppm as CaCO3 may be used as the diluent, for test products
6.23 Stir Bars with Threaded TFE-Fluorocarbon-Coated requiring dilution in water to obtain a use-dilution.
Surface, to dislodge inoculum from the carriers surface. Stir 7.8 Deionized Distilled Water (DDW), for making reagent
bars may be manufactured according to Fig. 1B.7 solutions and media. For terminology and specifications for
6.24 Magnet, strong enough to hold the threaded stir bar in water to be used refer to Terminology D 1129 and Specification
place in the glass carrier while the liquid is being poured out of D 1193 under 2.1.
it for membrane filtration. 7.9 Plates of Recovery MediaMedia must be prepared and
6.25 Aluminum Foil, to wrap items to be sterilized. sterilized according to manufacturers instructions and then
6.26 Vortex Mixer, to vortex the eluate and rinsing fluid in aseptically dispensed into culture plates.
the carrier to ensure efficient recovery of the test organism(s).
6.27 Glass Inserts, to be placed inside the glass carriers 8. Carriers
during inoculation with the test organism. Such inserts have 8.1 Preparation of the CarriersPlace a clean glass insert
been found to eliminate the deposition of microaerosols on the inside each flat-bottomed vial and position the insert in place
inside walls of the carriers. Glass inserts may be manufactured with the help of a septate cap loosely screwed on to the vial
according to Fig. 1C.6,8 (see Fig. 1D). Sterilize the required number of carriers, along
6.28 Centrifuge, for concentration, or washing, or both of with an equivalent number of regular caps for the carrier vials,
the cells/spores of the test organism(s). in a container such that they can be stored without any
6.29 Markers, permanent labware marking pens. contamination.
6.30 Sterile Polypropylene Centrifuge Tubes with Caps, 50
mL. 9. Soil Load
6.31 Colony Counter, for example, Quebec Colony Counter. 9.1 When a soil load is to be incorporated in the suspension
6.32 Sterile Disposable Gloves, for handling the carriers. of the test organism, it will consist of a mixture of the
6.33 Hemocytometer, for counting fungal conidia. following stock solutions in saline (pH 7.2):
6.34 Spectrophotometer, for measuring turbidity of micro- 9.1.1 Add 0.5 g of tryptone to 10 mL of saline.
bial suspensions. 9.1.2 Add 0.5 g of bovine serum albumin (BSA) to 10 mL
6.35 Bunsen Burner, for aseptic technique of saline.
9.1.3 Add 0.04 g of bovine mucin to 10 mL of saline.
7. General Solutions and Reagents 9.1.4 Prepare the solutions separately and sterilize by pas-
7.1 Purity of ReagentsReagent grade chemicals shall be sage through a 0.22 m pore diameter membrane filter, aliquot
used in all tests. Unless otherwise indicated, it is intended that and store at either 4 6 2C or 20 6 2C.
all reagents conform to the specifications of the Committee on 9.2 To obtain a 500 L inoculum of the test organism, add
to 340 L of the microbial suspension 25, 100, and 35 L of
BSA, mucin, and tryptone stock solutions, respectively.
5
The sole source of supply of flat-bottomed vials (catalog #5260G) known to the
committee at this time is Galaxy Environ. Products, P.O. Box 238, 7 Greenwood
NOTE 2Animal sera, often used as a soil load, vary widely in their
Ave., Newfield, NJ 08344. composition and may also contain microbial inhibitors. The soil load
6
If you are aware of alternative suppliers, please provide this information to mixture given above contains a level of protein roughly equal to that in
ASTM Headquarters. Your comments will receive careful consideration at a meeting 5 % serum. Preliminary screening of albumin and mucin is recommended
of the responsible technical committee, which you may attend. to ensure compatibility with test organism(s).
7
The sole source of supply of stir bars known to the committee at this time is
Engineering Department, Rehabilitation Centre, 505 Smyth Rd., Ottawa, ON, 10. Preparing Inocula of Specific Types of Organisms
Canada K1H 8M2; phone: 613-737-7350, ext. 75320.
8
The sole source of supply of glass inserts known to the committee at this time 10.1 This test method can be used with most species of
is Galaxy Environ. Products (Newfield, NJ) . vegetative and spore-forming bacteria as well as mycobacteria

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 E 2111 05
and fungi; however, Table 1 summarizes the species and strains 10.4.4.4 Refer to Section 9 for the soil load.
of the test organisms most often used. The number of CFU/mL 10.5 Trichophyton mentagrophytes:
of each freshly prepared and properly homogenized microbial 10.5.1 MaterialsStock culture of T. mentagrophytes
test suspension may be estimated spectrophotometrically, (ATCC #9533).
based on a standard curve at a specific wavelength, but should 10.5.1.1 Plates of Sabourauds Dextrose Agar (SDA) as
be confirmed by membrane filtration. growth and recovery media.
10.2 The concentration of the test organism in the dried 10.5.1.2 Sterile stainless steel spatula.
inoculum on each carrier must be equal to or higher than the 10.5.1.3 Sterile normal saline.
product performance criterion to be met. This is confirmed in 10.5.1.4 250-mL flask with glass beads (sterile).
each test by titrating the eluates from the control carriers. 10.5.1.5 Sterile absorbent cotton.
NOTE 3TSA and TSB, which are based on soybean-casein digests, 10.5.1.6 Sterile 150-mL glass beaker.
were used in the development of the test method described here. Other 10.5.1.7 Bunsen burner.
media with similar formulations may be used instead. 10.5.1.8 Incubator set at 29 6 2C.
10.3 Staphylococcus aureus: 10.5.1.9 Hemocytometer to count fungal conidia.
10.3.1 MaterialsFrozen stock of S. aureus (ATCC 6538). 10.5.2 Method:
10.3.2 Tryptose soy broth (TSB). 10.5.2.1 Streak a loopful (10 L) of thawed stock culture of
10.3.3 Trypticase soy agar (TSA). T. mentagrophytes at the center of each of four SDA plates.
10.3.4 MethodPrepare 100 mL of TSB according to the 10.5.2.2 Incubate plates at 29 6 2C for not less than 10
manufacturers instructions and distribute aliquots of approxi- days and not more than 15 days.
mately 10 mL into the appropriate number of test tubes. 10.5.2.3 Remove mycelial mats from the surface of agar
Sterilize as per manufacturers instructions. plates using a sterile spatula.
10.3.5 Inoculate a test tube of broth with 100 L of thawed 10.5.2.4 Transfer to 250-mL flask containing 25- to 50-mL
stock culture. sterile saline (0.85 % NaCl) with glass beads; shake flask
10.3.6 Incubate for 18 h at 35 6 2C (should yield > 109 vigorously enough to break off the conidia from the hyphae.
CFU/mL). 10.5.2.5 Filter suspension through sterile absorbent cotton
10.3.7 Refer to Section 9 for the soil load. into a beaker (conidia are collected in the filtrate in the beaker).
10.4 Pseudomonas aeruginosa: 10.5.2.6 Estimate density of conidial suspension by count-
10.4.1 MaterialsFrozen stock of P. aeruginosa (ATCC ing in hemocytometer.
15442). 10.5.2.7 Standardize suspension as needed by diluting it
10.4.2 TSB. with sterile saline so that it contains about 1 3 107 conidia/mL.
10.4.3 TSA. 10.5.2.8 Store at 2 to 10C for up to four weeks in preparing
10.4.4 MethodPrepare diluted TSB by adding 1 mL of test suspension of conidia for disinfection experiments.
regular TSB to 999 mL of DDW, distribute it in 10-mL aliquots 10.5.2.9 Maintain stock culture of fungus on SDA plate at 4
in test tubes, and sterilize by autoclaving at 121C for 20 min. 6 2C. At three-month intervals, inoculate a fresh agar plate
10.4.4.1 Inoculate each tube of broth with 100 L of thawed and incubate plate for ten days at 29 6 2C.
stock culture. 10.5.2.10 Refer to Section 9 for the soil load.
10.4.4.2 Incubate for three days at 35 6 2C (should yield 10.6 Mycobacterium terrae:
about 108 CFU/mL). 10.6.1 MaterialsFrozen stockM. terrae (ATCC 15755).
10.4.4.3 Concentrate suspension by centrifugation and by 10.6.1.1 Sterile deionized distilled water (DDW).
resuspending the pellet in 110 the initial volume of TSB. 10.6.1.2 Sterile normal saline.

TABLE 1 Cultivation and Recovery of the Various Test Organisms to be Used in the Carrier Test
Organism (ATCC #) Culture Medium Recovery Medium
Staphylococcus aureus (6538) Trypticase soy broth; incubation at 35 6 2C for 18 hours Trypticase soy agar; plates observed daily and final reading
recorded after 5 days at 35 6 2C

Pseudomonas aeruginosa(15442) Trypticase soy broth diluted 1:1000 with deionized distilled Trypticase soy agar; plates observed daily and final reading
water; incubation at 35 6 2C for 3 days recorded after 5 days at 35 6 2C

Conidia of Trichophyton mentagrophytes Sabouraud Dextrose Agar; incubation for 12 days at Sabouraud Dextrose Agar; plates observed first after 72
(9533) 29 6 2C hours and final reading recorded after 10 days at 29 6 2C

Mycobacterium terrae (15755) Middlebrook 7H9 broth with glycerol and ADC enrichment; Middlebrook 7H11 agar with OADC; plates observed
incubation at 35 6 2C for 21 days weekly and final reading after 30 days at 35 6 2C

Spores of Bacillus subtilis (19659) Columbia broth diluted 1:10 with deionized distilled water; Trypticase soy agar; plates observed daily and final reading
incubation for 72 hours at 35 6 2C recorded after 5 days at 35 6 2C

Spores Clostridium sporogenes (7955) Columbia broth; incubation at 29 6 2C under anaerobic Fastidious anaerobic agar; plates observed first after 48
conditions for 5 days hours and final reading recorded after 5 days at 29 6 2C

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 E 2111 05
10.6.1.3 Sterile bijoux bottles with ten glass beads (5 mm in 10.8.1.3 Anaerobic incubator set at 29 6 2C or incubator
diameter) in each. using anaerobic jars.
10.6.1.4 Sterile Middlebrook 7H9 broth containing glycerol 10.8.2 Method:
and albumin-dextrose-catalase (ADC) Enrichment. 10.8.2.1 Add 100 L of thawed bacterial culture to each 100
10.6.1.5 Middlebrook 7H11 Agar supplemented with oleic mL of the broth.
acid-albumin-dextrose-catalase (OADC) Enrichment. 10.8.2.2 Incubate at 29 6 2C for five days (should produce
10.6.1.6 Plastic cell culture flasks (75 cm2) with a canted approximately 108 viable spores/mL).
neck and a cap with a 0.2-m filter in it. 10.8.2.3 Wash spore suspension three times by centrifuging
10.6.1.7 Incubator set at 35 6 2C. it at 1500 xg and resuspending the pellet in DDW. After the last
10.6.1.8 Black, gridded membrane filters 47 mm in diam- centrifugation, resuspend the pellet in DDW using 110 the
eter (0.45 m pore diameter). volume of the original culture medium.
10.6.2 Method: 10.8.2.4 Heat the spore suspension at 70C for 10 min in a
10.6.2.1 Place 100 mL of sterile 7H9 broth in each of four waterbath to inactivate vegetative cells.
culture flasks.
10.8.2.5 Refer to Section 9 for the soil load.
10.6.2.2 Add 500 L of thawed stock culture to each flask.
10.6.2.3 Incubate at 35 6 2C for 21 days.
11. Carrier Test
10.6.2.4 Put 21-day-old culture of M. terrae grown in 7H9
broth into sterile centrifuge tubes. 11.1 Inoculation of the CarriersWearing sterile gloves,
10.6.2.5 Centrifuge at 1500 xg for 15 min. gently tighten the septate caps on the carriers such that insert is
10.6.2.6 Decant supernatant. positioned at the center of the bottom of the vial.
10.6.2.7 Wash cells by resuspending in sterile distilled NOTE 4The septate cap must not be screwed on too tightly to avoid
water. the touching and grinding of the narrow end of the insert on the inside
10.6.2.8 Repeat centrifugation and washing steps a total of bottom surface of the carrier vial.
three times.
11.1.1 Vortex the test suspension to distribute evenly cells/
10.6.2.9 Place the suspension into a bijoux bottle with ten spores. Withdraw 10 L of the suspension with a positive
glass beads (5-mm in diameter) and vortex it to break up displacement pipette and place it onto the inside bottom surface
clumps of the cells (the suspension should contain no less than of each carrier (Fig. 1D). For consistency, the same pipette tip
109 CFU/mL. can be used throughout the inoculation of a batch of carriers.
10.6.2.10 Refer to Section 9 for the soil load. Make sure that the inoculum does not touch the walls of the
10.7 Bacillus subtilis: insert.
10.7.1 MaterialsFrozen stock of B. subtilis (ATCC
11.1.2 Allow the inoculum to dry at room temperature by
19659).
first holding the carriers in a laminar flow hood for 1 h follwed
10.7.1.1 Sterile Columbia broth diluted 1:10 with sterile by drying under vacuum in a desiccator for one more hour.
DDW.
10.7.1.2 TSA. NOTE 5The ability of microorganisms to survive drying varies
10.7.1.3 Sterile 10 mM MnSO4.4 H2O. depending on air temperature and relative humidity. The drying times
10.7.1.4 Incubator set at 35 6 2C. indicated here are a general guide only and care must be taken to ensure
that the inoculum in the carriers becomes visibly dry while retaining
10.7.1.5 Orbital platform shaker. sufficient viable organisms to assure a valid test.
10.7.2 Method:
10.7.2.1 Add 1 mL of 10 mM MnSO4.4 H2O solution to 99 11.1.3 Observe the dried inoculum on each carrier and
mL of 110 Columbia broth. discard any carrier in which the inoculum has touched the
10.7.2.2 Add 100 L of thawed bacterial culture to each 100 insert.
mL of the broth. 11.1.4 Aseptically remove the septate caps and inserts and
10.7.2.3 Incubate at 35 6 2C for 72 h on an orbital shaker place them in a bucket for subsequent decontamination and
and shake at 150 rpm (should produce approximately 108 cleaning. Replace the septate caps with sterile regular caps and
viable spores/mL). tighten.
10.7.2.4 Wash spore suspension three times by centrifuging 11.1.5 These carriers are now ready for the test procedure.
it at 1000 xg and resuspending the pellet in sterile DDW. After 11.2 Exposure of the Organism(s) to the Formulation Under
the last centrifugation, resuspend the pellet in DDW using 110 TestThe number of test carriers to be used in each run is ten;
the volume of the original culture medium. however, in preliminary tests during product development
10.7.2.5 Heat the spore suspension at 70C for 10 min to three to five carriers may be sufficient to assess the potency of
inactivate vegetative cells. experimental formulation(s) against the test organism(s).
10.7.2.6 Refer to Section 9 for the soil load. 11.2.1 Place 1 mL of test microbicide into each carrier over
10.8 Clostridium sporogenes: the dried inoculum and hold the carriers at the desired
10.8.1 MaterialsFrozen stock of C. sporogenes (ATCC temperature for the desired contact period. At the end of the
7955). exposure time, aseptically place a sterile threaded stir bar into
10.8.1.1 Sterile full-strength Columbia broth. each carrier and immediately add 9 mL of an eluent/neutralizer
10.8.1.2 Fastidious anaerobic agar (FAA). to neutralize/dilute the microbicide and arrest its activity.

6
 E 2111 05
11.2.2 Place the carrier onto a magnetic stir plate and stir the separate membrane filters. Rinse the vial by adding 10 mL of
contents of the carrier for 30 s to ensure the removal and the eluent/neutralizer, vortex, and filter the rinse through the
resuspension of the inoculum from the bottom of the carrier. same filter. Repeat the procedure two more times. Rinse the
Vortex for 30 s. sides of the funnel unit with approximately 40 mL of saline.
11.2.3 Using a magnet to hold the stir bar in place, pour the Aseptically transfer the filter to the recovery medium.
contents of the vial into the membrane filter holder. Rinse the NOTE 7Separate membrane filters, but the same filtration unit, can be
carrier vial with 20 mL of saline, vortex, and filter the rinse. used for processing all dilutions for a given carrier starting with the most
Repeat rinse two more times. Rinse the sides of the funnel unit dilute sample first.
with at least 40 mL of saline. Aseptically transfer the mem-
11.4.3 Incubate the plates of the recovery medium at the
brane filter to the plate of a suitable recovery medium. Incubate
required temperature for the desired length of time (see Table
the plates at the desired temperature for the required length of
1). Count the colonies and calculate the log10 reductions
time.
obtained.
NOTE 6Presence of large numbers of colonies on the membrane
filters from the test vials indicates no or weak activity of the test 12. Precision and Bias
formulation against the challenge organism(s) under the specific condi- 12.1 PrecisionThe test method has been subjected to
tions used in the test. To obtain a more precise indication of the log10 extensive intra-laboratory testing using a variety of test organ-
reduction in viability by the test product, ten-fold dilutions of the eluate isms to determine the extent of variability in the test data from
may be necessary (see Fig. 2).
operator to operator. A collaborative study of 15 laboratories
11.3 Control CarriersThe minimum number of control also has been carried out to determine the reproducibility of the
carriers to be used in each test is three regardless of the number data for the sporicidal activity of several blinded test samples.
of test carriers. The carriers as well as the spore suspensions of Bacillus
11.3.1 Instead of the test formulation, add 1 mL of sterile subtilis were provided to the participating laboratories, which
saline to each control carrier. The contact time and temperature tested the microbicide samples against the spores without any
for the control carriers must be the same as that for the test soil load in the dried inocula. The test itself contributed only
carriers. 5 % to the variability observed.
11.3.2 At the end of the contact time, add 9 mL of an 12.2 Other researchers have used the method for studies of
eluent/neutralizer and a sterile threaded stir bar to each control germicidal activity (7, 8).
carrier. Place the carrier on a magnetic stir plate for 30 s to 12.3 Target performance standards may vary depending on
remove the inoculum from the bottom of the carrier. Vortex the the regulatory agency.
carrier for approximately 30 s or until any visible clumps have NOTE 8The development of this test method was made possible with
been broken. financial assistance from the Antimicrobials Division of the U.S. Envi-
11.4 Dilution of the Eluates: ronmental Protection Agency.
11.4.1 The extent to which the eluates from the control
carriers are to be diluted will depend on the number of viable 13. Keywords
cells in the inoculum and it will be necessary to determine the 13.1 Bacillus subtilis spores; bactericides; chemical micro-
dilution range before hand to generate countable numbers of bicides; Clostridium sporogenes spores; eluate; eluent; envi-
CFU for an accurate measurement of the challenge titer. ronmental surfaces; fungicides; germicides; log10 reductions;
Similarly, eluates from test carriers may require dilution to medical devices; membrane filtration; mycobactericides; My-
permit the calculation of log10 in the viability titer after cobacterium terrae; Pseudomonas aeruginosa; quantitative
exposure of the target organism(s) to the test formulation. carrier test; soil load; sporicides; Staphylococcus aureus;
11.4.2 Make ten-fold dilutions of the eluates from control standard hard water; surrogate; Trichophyton mentagrophytes
and test carriers. Pass the material from each dilution through conidia

7
 E 2111 05

FIG. 2 Flow Chart

8
 E 2111 05

REFERENCES

(1) U.S. General Accounting Office, Disinfectants: EPA Lacks Assurance (USPC), Rockville, MD, 1979.
They Work, Document #GAO/RCED-90-139, Washington, DC, 1990. (6) Am. Public Health Assoc. Standard Methods for the Examination of
(2) Springthorpe, V. S. and Sattar, S. A., Carrier Tests to Assess Water and Wastewater, Washington, DC, 1998.
Microbicidal Activities of Chemical Disinfectants for Use on Medical
(7) Tilt, N. and Hamilton, M.A., Repeatability and Reproducibility of
Devices and Environmental Surfaces, J. AOAC International, Vol 88,
Germicide Tests: A Literature Review, J. AOAC International, Vol 82,
2005, pp. 182-201.
1999, pp. 384-389.
(3) CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories,
4th ed., U.S. Department of Health and Human Services, Washington, (8) Walsh, S.E., Maillard, J.Y. and Russell, A.D. Orthophthaldehyde: a
DC, 1999. Possible Alternative to Glutaraldehyde for High Level Disinfection, J.
(4) American Chemical Society, Reagent Chemicals, American Chemical Appl. Microbiol., Vol 86, 1999, pp. 10391046.
Society Specifications, Washington, DC, 1993. (9) Sattar, S.A., Springthorpe, V.S. and Rochon, M., A Product-Based on
(5) United States Pharmacopoeia and National Formulary, Analar Stan- Accelerated and Stabilized Hydrogen Peroxide: Evidence for Broad-
dards for Laboratory Chemicals, U.S. Pharmacopeial Convention, Inc. Spectrum Activity, Can. J. Infect. Control, Vol 13, 1998, pp. 123130.

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