MICROBIOLOGICAL METHODS
Modification to the AOAC Sporicidal Activity of Disinfectants
Test (Method 966.04): Collaborative Study
STEPHEN F. TOMASINO
U.S. Environmental Protection Agency, Office of Pesticide Programs, Microbiology Laboratory, Environmental Science
Center, Ft. Meade, MD 20755-5350
MARTINA. HAMILTON
Montana State University, Center for Biofilm Engineering, Bozeman, MT 59717-3980
Collaborators: M. Buen; H. Chan Myers; A. Garza; E. Gonzales; K. Kallander; A. Rodriguez; P. Stahnke; T. To
In an effort to improve AOAC Method 966.04, the
Sporicidal Activity of Disinfectants Test, selected
modifications to the procedure were evaluated in a
collaborative study. Method 966.04 is used to
generate efficacy data to support the product
registration of sporicides and sterilants. The
method is a carrier-based test that provides a
qualitative measure of product efficacy against
spores of Bacillus subtilis and Clostridium
sporogenes. The use of garden soil extract and the
lack of standard procedures for the enumeration of
spores and neutralization of the test chemicals
have been considered problematic for many years.
The proposed modifications were limited to the
B. subtilis and hard surface carrier (porcelain
penicylinder) components of the method. The
study included the evaluation of a replacement for
soil extract nutrient broth and an establishment of
a minimum spore titer per carrier, both considered
crucial for the improvement and utilization of the
method. Additionally, an alternative hard surface
material and a neutralization confirmation procedure
were evaluated. To determine the equivalence of the
proposed alternatives to the standard method,
3 medium/carrier combinations, (1) soil extract
nutrient broth/porcelain carrier (current method),
(2) nutrient agar amended with 5 g/mL manganese
sulfate/porcelain carrier, and (3) nutrient agar
amended with 5 g/mL manganese sulfate/stainless
steel carrier were analyzed for carrier counts, HCl
resistance, efficacy, quantitative efficacy, and spore
wash-off. The test chemicals used in the study
represent 3 chemical classes and are commercially
available antimicrobial liquid products: sodium
hypochlorite (bleach), glutaraldehyde, and a
combination of peracetic acid and hydrogen
peroxide. Four laboratories participated in the
study. The results of the spore titer per carrier, HCl
resistance, efficacy, and wash-off studies demonstrate
that amended nutrient agar in conjunction with the
porcelain is comparable to the current method, soil
extract nutrient broth/porcelain. The nutrient agar
method is simple, inexpensive, reproducible, and
provides an ample supply of high quality spores.
Due to the current use of porcelain carriers for
testing C. sporogenes, it is advisable to retain the
use of porcelain carriers until stainless steel can
be evaluated as a replacement carrier material for
Clostridium. The evaluation of stainless steel for
Clostridium has been initiated by the Study
Director. Study Director recommendations for First
Action revisions are provided in a modified method.
T
he U.S. Environmental Protection Agency (EPA)
Office of Pesticide Programs (OPP) has responsibility
for regulating antimicrobial products, including
sporicides and sterilants, which are used to control pathogenic
bacteria, viruses, and other microorganisms on inanimate
surfaces. The U.S. Food and Drug Administration (FDA)
Center for Devices and Radiological Health regulates
antimicrobial chemicals (e.g., sterilants) used to process
critical and semicritical medical devices. For the purpose of
this paper, the terms sporicide and sterilant are considered as
synonymous. In response to the intentional release of Bacillus
anthracis spores in 2001 and the associated need for verifying
the performance of sporicidal chemicals for building
decontamination, OPP initiated a research program to
evaluate and improve efficacy test methods for sporicides.
One component of this effort involves the assessment of the
AOAC Sporicidal Activity of Disinfectants, AOAC® Official
MethodSM 966.04 (1), the method currently accepted by EPA
and FDA for generating efficacy data to support the
registration of sporicides (sterilants). AOAC Method 966.04
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1373
Submitted for publication August 1, 2006.
The recommendation was approved by the Methods Committee on
Microbiology and Extraneous Materials as First Action. See “Official
Methods Program Actions,” (2006) Inside Laboratory Management,
September/October issue.
Corresponding author’s e-mail: tomasino.stephen@epa.gov
is a carrier-based test that provides a qualitative measure of
product efficacy against spores of Bacillus subtilis
(ATCC 19659) and Clostridium sporogenes (ATCC 3584).
For regulatory purposes, 60 carriers representing each of
2 types of surfaces (hard surface—porcelain penicylinders;
porous surface—silk suture loops) are required to be tested
against spores of both organisms on 3 product samples
representing 3 different batches. Killing on all of the
720 carriers is an EPA regulatory requirement for sporicidal
products.
The quality of the efficacy data generated with
Method 966.04 has been considered problematic for many
years. Potential sources of variability have been previously
published, and several authors have proposed corrective
solutions, including designing quantitative approaches to
efficacy testing (2–6). The most significant concerns are
associated with the qualitative nature of the method, the use of
raw garden soil extract as a source of minerals for spore
production, the carrier materials (unglazed porcelain and silk
suture loops), the lack of a standardized procedure for
enumeration of spores, spore wash-off, and the long
incubation time (21 days). Recently, a modification to use
polyester (Dacron®) loops instead of silk suture loops for
testing peracetic acid-based sterilants was approved by the
AOAC Methods Committee on Microbiology and Extraneous
Materials—a First Action modification (7).
In this paper, we report on the conduct of a collaborative
study to modify the method, present results, and provide
recommendations. In addition, the modified Method 966.04,
including numerous editorial revisions, is presented. The
order of discussion is based on the order of studies provided in
the collaborative study protocol. Test methodology for each
study component is largely reproduced from the actual
collaborative study protocol with only minor changes in
content and format.
Proposed Modifications—An Overview
Replacement for soil extract nutrient broth (SENB), a
procedure modification.—In the current method, SENB is
used for the cultivation (i.e., spore production) of B. subtilis;
the procedure uses a nonstandardized extract of raw garden
soil, is very complicated to prepare, and results in a highly
variable preparation of spores (i.e., spore titer and quality).
The use of a synthetic, standardized sporulation medium is
highly recommended. Nutrient agar amended with 5 g/mL
manganese sulfate was evaluated as a replacement.
Alternative hard surface material (a procedure
modification).—In Method 966.04, unglazed porcelain
(penicylinders) is used to represent a hard surface. Porcelain
penicylinders are somewhat porous, easily damaged, and may
become variable upon reuse. Stainless steel (penicylinders)
was proposed as an alternate hard surface material. Stainless
steel carriers, unlike porcelain, are easily cleaned and highly
reusable, making them a viable option for replacement. In
addition, the vendor who supplies the porcelain carriers,
CeramTec (Laurens, SC), requires a 5000 piece minimum
order. Fisher Scientific (Pittsburgh, PA), a vendor for stainless
steel carriers, has a minimum order of 12 pieces. With
stainless steel, the basic concept and carrier dimensions are
retained, and the same carrier is used in the AOAC
use-dilution methods (Methods 955.14, 955.15, and 964.02).
Spore enumeration procedure and a target spore
titer/carrier (a new procedure and standard).—Method
966.04 does not provide a procedure for measuring the
number of spores per carrier (carrier counts). A carrier count
procedure was provided and used to generate data to support
the establishment of target carrier counts—a minimum of
1.0  105 (log 10 density = 5.0) and a maximum of
approximately 1.0  106 spores/carrier.
Neutralization confirmation (a new procedure).—Method
966.04 does not provide a procedure to confirm neutralizer
effectiveness. The neutralization of active ingredients is one
of the most important steps in efficacy testing of antimicrobial
products. The selected neutralizer is used to stop the activity
of the antimicrobial agent, a process essential to measuring
effectiveness at a desired contact time, and allows for the
recovery of viable inoculum. The neutralizer itself or in
combination with the recovery medium must not exhibit
bacteriostatic activity against the test microbe. A
neutralization confirmation procedure that simulates Method
966.04 was proposed and conducted by 2 laboratories in this
study.
Original concepts for the proposed modifications for the
use of nutrient agar, stainless steel, and the target spore counts
were previously identified and evaluated by other scientists
(2–6; Rodriquez, 2002, unpublished data. The spore
enumeration and neutralization procedures are based on
existing protocols developed and used by the OPP
Microbiology Laboratory. Prior to initiation of this
collaborative effort, AOAC INTERNATIONAL assembled a
review panel, the AOAC Sporicidal Method Expert Review
Panel (ERP), to evaluate the collaborative study protocol. The
ERP, along with members of the AOAC Methods Committee
on Microbiology and Extraneous Materials, were engaged
early in the development of the study design to ensure that the
format would be acceptable to the AOAC Official MethodsSM
process. Based on the nature of the proposed modifications, a
4 laboratory study was agreed upon by the Study Director, the
ERP, and the AOAC Methods Committee.
Collaborative Study
Applicability
The modifications are applicable to liquid formulations
when tested against B. subtilis on a hard surface (porcelain
carrier). The test chemicals used in the efficacy component of
the study represent 3 different chemical classes: sodium
hypochlorite (bleach), glutaraldehyde, and a combination of
peracetic acid and hydrogen peroxide. The suitability of the
modifications for porous materials (i.e., silk and polyester
carriers) and gaseous formulations will require additional
collaborative studies. Also, modifications to the
1374 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
C. sporogenes component of the method (e.g., finding a
suitable replacement for egg meat medium) will be evaluated
under a separate study protocol.
Collaborators and Quality Management
Collaborators were chosen from government and private
laboratories. Four laboratories participated in the study. A
vendor supplied inoculated carriers to 2 of the 4 laboratories.
The vendor verified the spore titer before shipment. The
preparedness of each laboratory was assessed by the Study
Director and Quality Assurance personnel prior to initiation of
the study to ensure compliance with a project-specific EPA
Quality Assurance Project Plan.
The Study Director provided method protocols, data
sheets, media preparation sheets, test chemicals, selected
media and reagents, garden soil, and porcelain penicylinders.
The level of quality assurance was consistent with EPA Good
Laboratory Practices (FIFRA 40 CFR Part 60). The garden
soil used in the study was analyzed for pH as well as, nutrient
and micronutrient content, and each production lot of test
chemical was subjected to chemical analysis to verify that the
formulations were within the certified specifications. Each
laboratory designated a technician team to conduct the study.
Based on the Study Director’s experience with qualitative,
technique-sensitive, carrier-based tests such as
Method 966.04, the data can be variable and the variance
associated with technicians is usually negligible compared to
the other sources of test variability. Nevertheless, if the same
technician team always conducted the testing, the technician
effect would be minimized.
Study Design
The current and modified methods are presented in Table 1
and the 3 medium/carrier combinations evaluated in this study
are identified. Finding a suitable replacement for SENB was
prioritized. Testing of a SENB/stainless steel combination
received low priority and was not considered in this study.
Each laboratory was instructed to determine carrier counts
and HCl resistance for each medium/carrier combination, and
if the established requirements were met, the carriers were
considered suitable for efficacy testing.
In addition, 2 laboratories (1 and 2) conducted a new
procedure to evaluate the effectiveness of the prescribed
neutralizers. Two laboratories (2 and 3) enumerated viable
spores from individual carriers following exposure to the
3 low chemical treatments when tested with Method 966.04
(see Quantitative Efficacy). Two laboratories (2 and 3)
performed a wash-off study to determine the impact of the
carrier type on adherence properties of spores to the carriers
using solutions of saline and saline + polysorbate 80. The
various activities and distribution of the laboratory
responsibilities are listed in Table 2. The procedures for each
study are provided in this report.
Due to the instability of the test chemicals (sporicidal
products), it was necessary for each laboratory to prepare test
formulations on-site; thus, a blind assessment of each
chemical treatment was not possible. To minimize any
potential bias by the analysts, the order of testing chemicals
against the medium/carrier combinations for each laboratory
was randomized. The Study Director provided a randomized
test plan to each laboratory. In order to have a balanced design
that was conducive to a straight-forward statistical analysis,
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1375
Table 1. Current method and proposed replacements
for Method 966.04 evaluated in the collaborative study
Carrier (penicylinder) type
Sporulation medium
Current method:
Porcelain (PC)
Modified method:
Stainless steel
(SS)
Current method
Soil extract nutrient
broth (SENB)
SENB/PC SENB/SS
(not studied)
Modified method
Amended nutrient
agar (NA)
NA/PC NA/SS
Table 2. Collaborative activities and responsibilities of the laboratories
Activity Lab No. 1 Lab No. 2 Lab No. 3 Lab No. 4
Produce spores with SENB and NA Conducted by vendora Conducted by vendor Yes Yes
Inoculate carriers (PC and SS) Conducted by vendor Conducted by vendor Yes Yes
Conduct spore enumeration Yes Yes Yes Yes
Conduct HCl resistance tests Yes Yes Yes Yes
Conduct neutralization tests Yes Yes No No
Conduct efficacy tests Yes Yes Yes Yes
Conduct quantitative efficacy No Yes Yes No
Conduct wash-off in saline No Yes Yes No
a Conducted per instructions provided by the Study Director. Vendor was Presque Isle Cultures (Erie, PA).
each laboratory performed the same number of efficacy tests
per day.
Due to the cost and the labor-intensive nature of
conducting Method 966.04, 30 carrier efficacy tests rather
than 60 carrier tests were conducted. Each medium/carrier
combination was tested against one chemical type (both high
and low treatments) on the same day (a total of six 30 carrier
tests per day). Two of the 4 laboratories used inoculated
carriers prepared by a vendor (Presque Isle Cultures,
Erie, PA); 2 laboratories prepared and inoculated their own
carriers. The vendor, per instructions provided by the Study
Director, followed the same protocol for the generation of the
spore suspensions and inoculation of carriers as was provided
to the collaborators.
Test Chemicals
Test chemicals are 3 commercially available antimicrobial
products; the active ingredient(s) for each are (1) a
combination of 0.08% peracetic acid/1.0% hydrogen
peroxide; (2) 2.6% glutaraldehyde; and (3) 6.0% sodium
hypochlorite (household bleach) without whiteners. For the
purpose of this study, the test chemicals were used as
experimental components only and were not tested to support
or verify product label claims. Each chemical was tested as
“high” (efficacious) and “low” (nonefficacious) to provide a
range of efficacy, i.e., the high treatments were designed to
generate passing results (0/30 positive carriers) and low
treatments were designed to generate failing results
(1/30 positive carriers). The test conditions used to generate
the range are shown in Table 3. Thus, each medium/carrier
combination was tested against a total of 6 chemical
treatments.
Handling and Preparation of Test Chemicals
Test chemicals and the Material Safety Data Sheets for
each were provided to each collaborator by the Study
Director. Multiple lots of the chemicals were allowed into the
study. Formulation chemistry analysis was performed on each
lot to confirm the percent active ingredient(s). Four lots of the
bleach product were available for testing; the lots ranged from
7.0 to 6.1% sodium hypochlorite. Three lots of the peracetic
acid/hydrogen peroxide product were available for testing; the
lots ranged from 0.08 to 0.75% and 1.0 to 0.97% for peracetic
acid and hydrogen peroxide, respectively. Three lots of the
glutaraldehyde product were used in the study; the lots ranged
from 2.61 to 2.55% glutaraldehyde. Test parameters for each
test chemical, describing the conditions for testing (e.g.,
dilution, neutralizer, contact time, temperature, etc.), were
provided to each laboratory.
Media and Reagents
(a) Culture media.—(1) Nutrient broth.—For use in
preparing nutrient agar. Add 5 g beef extract (paste or
powder), 5 g NaCl, and 10 g peptone (anatone, peptic
hydrolysate of pork tissues, manufactured by American
Laboratories, Inc., Omaha, NE) to ca 1 L water. Boil mixture
for 20 min with constant stirring. Readjust volume to 1 L with
water and allow cooling to around 50C. Adjust pH to
6.8  0.2 with 1 N HCl or 1 N NaOH, if necessary. Filter
through paper (e.g., Whatman No. 4). Dispense 10 mL
portions into 20  150 mm culture tubes or 20 mL portions
into 25  150 mm culture tubes. Dehydrated nutrient broth
may be substituted; prepare according to manufacturer’s
instructions. (2) Nutrient agar.—For stock cultures slants.
Add 1.5% (w/v) Bacto-agar to unsterilized nutrient broth. Boil
mixture until agar is dissolved. Adjust pH to 7.2  0.2 if
necessary. Dispense 5 mL portions into 16  100 mm
screw-cap tubes. Larger tubes may be used as well. Autoclave
for 20 min at 121C. Remove from autoclave and slant tubes
to form agar slopes. (3) Nutrient agar with 5 g/mL
MnSO4–H2O (amended nutrient agar).—For spore
production. Suspend 11.5 g nutrient agar in 495 mL water, and
add 5 mL 500 ppm MnSO4–H2O. Dissolve by boiling. Adjust
pH to 6.8  0.2 if necessary. Autoclave for 15 min at 121C.
Pour agar into plates. (4) SENB.—Extract 1 lb (454 g) garden
soil (from Odenton, MD, collected and supplied by Study
Director) in 1 L H2O, filter several times through Schleicher &
Schuell (Keene, NH) No. 588 paper, and dilute to volume
(pH should be 5.2). Add 5 g beef extract (paste or powder),
5 g NaCl, and 10 g peptone. Boil 20 min, dilute to volume,
adjust with 1 M NaOH to pH 6.9  0.2, and filter through
paper. Dispense in 10 mL portions into 25  150 mm tubes,
and autoclave 20 min at 121C. (5) Trypticase soy agar
(TSA).—Suspend 40 g dehydrated TSA in 1 L water and heat
gently while stirring. Boil 1 min or until completely dissolved.
Adjust pH to 7.3  0.2. Autoclave 15 min at 121C. Pour agar
into plates. (6) Fluid thioglycollate medium
(FTM).—Suspend 29.5 g dehydrated FTM in 1 L water. Heat
to boiling to dissolve completely. Adjust pH to 7.1  0.2 if
necessary. Dispense 10 mL portions into 20  150 mm culture
tubes and autoclave for 15 min at 121C. Store at room
temperature. Protect from light. Note: If after autoclaving, the
aerated portion of media consumes more than one-third of
tube, media must be reboiled by placing tubes in beaker of
boiling water. Media can only be reboiled once. (7) FTM
with 1 M NaOH (modified FTM).—For subculturing spores
exposed to 2.5 M HCl. Suspend 29.5 g FTM in 1 L water.
Heat boiling to dissolve completely. Cool and adjust pH to
7.1  0.2 if necessary. Add 20 mL 1 M NaOH and mix well.
1376 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
Table 3. Test conditions used to establish range of
efficacy
Test conditions
Test chemical High treatment Low treatment
Peracetic acid/hydrogen
peroxide
30 min exposure 5 min exposure
Glutaraldehyde 8 h exposure 1 h exposure
Sodium hypochlorite
(bleach)
pH Adjusted to ca
7.0; 60 min exposure
pH Unadjusted
(ca 10.0);
10 min exposure
Check final pH and record. No set pH for final product.
Dispense 10 mL into 20  150 mm culture tubes and autoclave
for 15 min at 121C. Store at room temperature. Protect from
light. Note: If after autoclaving, the aerated portion of media
consumes more than one-third of tube, media must be reboiled
by placing tubes in beaker of boiling water. Media can only be
reboiled once. (8) Letheen broth.—Suspend 25.7 g letheen
broth in 1 L water. Heat to boiling to dissolve medium
completely. Adjust pH to 7.0  0.2. Dispense 10 mL portions
into 20  150 mm culture tubes. Autoclave 15 min at 121C.
(9) Modified letheen broth with 0.1% (w/v) sodium
thiosulfate.—Suspend 25.7 g letheen broth in 1 L water and
add 1.0 g sodium thiosulfate. Heat to boiling to dissolve
completely. Adjust pH to 7.0  0.2. Dispense into tubes.
Autoclave 15 min at 121C.
(b) Manganese sulfate monohydrate.—500 ppm. Add
0.25 g manganese sulfate to 500 mL water. Filter-sterilize for
use.
(c) Dilute hydrochloric acid.—2.5 M. Use to determine
resistance of dried spores. Standardize and adjust to 2.5 M as
in Method 936.15.
(d) Water.—Use sterile reagent grade water or sterile high
performance liquid chromatography (HPLC) grade water or
tap water where specified. Reagent grade water should be free
of substances that interfere with analytical methods. Any
method of preparation of reagent grade water is acceptable
provided that the requisite quality can be met. Reverse
osmosis, distillation, and deionization in various
combinations all can produce reagent grade water when used
in the proper arrangement. See Standard Methods for the
Examination of Water and Wastewater for details on reagent
grade water.
(e) Sterile 5% acetic acid.—Pour ca 500 mL 5% acetic
acid into sterile disposable 0.22 m filter unit in the biosafety
cabinet (BSC). Filter-sterilize, and cap bottle before removing
from BSC.
(f) 0.85% saline.—Add 8.5 g NaCl to 1 L volumetric flask
partially filled with water. Add water to bring to volume.
Dissolve by stirring. Dispense into media bottles. Autoclave
for 20 min at 121C.
(g) Saline + polysorbate 80.—Measure 999 mL sterile
0.85% saline. Add 1 mL polysorbate 80. Mix thoroughly.
Filter-sterilize, using 0.22 m filter. Dispense into media
bottles.
(h) Test organism.—Bacillus subtilis (ATCC No. 19659)
obtained directly from a reputable supplier (e.g., ATCC).
(i) Test chemicals.—(1) Bleach-unadjusted pH (pH about
10.0), 1:10 overall dilution (about 6000 ppm).—One part
bleach, 9 parts sterile HPLC grade water. Bleach-unadjusted
pH must be tested within ca 90 min after preparation. Used for
the low treatment. (2) Bleach-adjusted pH (pH 7.0  0.5),
1:10 overall dilution (about 6000 ppm).—One part bleach,
0.6 parts 5% acetic acid, 8.4 parts sterile HPLC grade water.
Bleach-adjusted pH must be tested within ca 90 min after
preparation. Used for the high treatment. (3) Peracetic
acid/hydrogen peroxide product.—Ready-to-use product and
no dilution required, must initiate testing within ca 3 h after
dispensing. (4) 2.6% glutaraldehyde product.—Activated
according to directions; product has a 14 day shelf life after
activation. Testing was initiated within 3–5 days after
activation.
(j) Garden soil.—From Odenton, MD, collected and
supplied by Study Director.
Apparatus
(a) Carriers.—Penicylinders, porcelain, 8  1 mm od,
6  1 mm id, 10  1 mm length (available from CeramTec
Ceramic, Laurens, SC, USA, www.ceramtec.com, Cat. No.
LE15819). Penicylinders, polished stainless steel, 8  1 mm
od, 6  1 mm id, 10  1 mm length; type 304 stainless steel, SS
18-8 (Fisher Scientific Cat. No. 07-907-5, Pittsburgh, PA).
(b) Inoculated carriers.—Inoculated porcelain and
stainless steel carriers from Presque Isle Cultures (Erie, PA).
Note: The same production lot of inoculated carriers was
tested by 2 laboratories.
(c) Glassware.—For disinfectant, 25  150 mm or
25  100 mm culture tubes (Bellco Glass Inc., Vineland, NJ);
reusable or disposable 20  150 mm (for cultures/
subcultures); 16  100 mm screw-cap tubes for stock cultures.
Cap with closures before sterilizing. Sterilize all glassware 2 h
in hot air oven at 180C or steam-sterilize for a minimum of
20 min at 121C with drying cycle.
(d) Sterile centrifuge tubes.—Polypropylene, 50 mL
conical tubes with conical bottoms (Fisher Scientific;
Corning, Inc., New York, NY), Cat. No. 05-538-53D, or
equivalent.
(e) Chiller unit.—Constant temperature for test chemical,
capable of maintaining 20  1C temperature.
(f) Water bath.—For heat shock and for tests conducted at
25  1C, constant temperature, capable of maintaining
80  2C temperatures.
(g) Petri dishes.—Plastic (sterile), Fisher Scientific Cat.
No. 08-757-103-C; VWR (West Chester, PA), Cat. No.
25373-143, or equivalent.
(h) Filters.—Schleicher & Schuell (S&S) filters
(No. 588), 32 cm pleated: VWR Cat. No. 14224-414; used for
preparation of SENB.
(i) Filter paper.—Whatman No. 2, Fisher Scientific Cat.
No. 09-8100; VWR Cat. No. 28455-085, or equivalent;
placed in Petri dishes for storing carriers.
(j) Analytical filter unit.—Nalgene 150 mL filter unit,
0.2 m filter.
(k) Test tube racks.—Any convenient style.
(l) Inoculating loop.—Any convenient inoculation/transfer
loop for culture transfer.
(m) Wire hook.—For carrier transfer. Make 3 mm right
angle bend at end of 50–75 mm nichrome wire No. 18 B&S
gage. Have other end in suitable holder.
(n) Tissue homogenizer.—Thomas Scientific
(Swedesboro, NJ) 3431-E20 size B, or equivalent, used for
preparation of SENB.
(o) Centrifuge.—Eppendorf 5804 R, or equivalent.
(p) Sonicator.—Branson (Danbury, CT) Model 1510, or
equivalent.
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1377
(q) Orbital shaker.—VWR, Model DS-500, or
equivalent.
(r) Vacuum desiccator.—For carrier storage. With
adequate gauge for measuring 27 in. (69 cm) Hg and fresh
desiccant.
(s) Certified BSC (Class I or II).—Recommended for use
to maintain aseptic work environment.
(t) Certified timer.—For managing timed activities.
Calibration of the timers, against a National Institute of
Science and Technology (NIST; Gaithersburg, MD) traceable
standard, is recommended.
(u) Glass wool.—Fisher Scientific Cat. No. 11-390, used
for preparation of SENB.
(v) Inoculating loop.—Make 4 mm id single loop at end of
50–75 mm (2–3 in.) Pt or Pt alloy wire No. 23 B&S gage or
4 mm loop fused on 75 mm (3 in.) shaft (available from
Johnson Matthey, West Chester, PA). Fit other end in suitable
holder. Bend loop at 30 angle with stem. Volumetric transfer
devices may be used instead of transfer loops.
Procedure
(a) Culture initiation.—Reconstitute lyophilized
B. subtilis culture with nutrient broth. Using several drops of
the suspension, inoculate a second tube of nutrient broth.
Incubate culture 24  2 h at 37  1C. Use the 24  2 h broth
culture to initiate stock cultures (working slants). Streak
inoculate a set (e.g., 6) nutrient agar slopes and incubate
24  2 h at 37  1C. Concurrently, perform purity and
identification confirmation testing (e.g., colony morphology
on TSA, Gram stain, or other identification systems) on the
initial 24  2 h broth culture. After incubation, store at 2–5C.
Maintain stock culture on nutrient agar slants by monthly
(30  2 days) transfers. Every 30  2 days, inoculate a new set
of stock culture tubes from a current stock culture tube.
Perform monthly quality control of stock cultures just prior to
or concurrently with monthly stock culture transfers. Repeat
cycle for 1 year, and then initiate a new culture.
(b) Production of B. subtilis spore suspension from
SENB.—Laboratories prepared and harvested a test culture of
B. subtilis according to Method 966.04. The spores were used
to inoculate porcelain carriers only. Extract 1 lb (454 g)
garden soil (from Odenton, MD, collected and supplied by
Study Director) in 1 L H2O, filter several times through S&S
No. 588 paper, and dilute to volume (pH should be 5.2). Add
5 g beef extract (paste or powder), 5 g NaCl, and 10 g peptone.
Boil 20 min, dilute to volume, adjust with 1 M NaOH to pH
6.9  0.2, and filter through paper. Dispense in 10 mL portions
into 25  150 mm tubes, and autoclave 20 min at 121C.
Inoculate each tube of SENB with several loopfuls of growth
from the nutrient agar slants. Incubate tubes in slanted
position for 72  2 h at 37  1C.
Note: To enhance the level of spore production, use the
following guidance (not part of the current method): Air-dry
and sift soil to break large clumps before preparing the extract.
Soak soil overnight in 1 L water. Filter entire volume. The
filters clog easily; thus, frequently change filters. Allow
filtered soil extract to settle overnight. Decant supernatant and
discard particulate matter before use. Yield for soil extract will
be approximately 600 mL. Also, after autoclaving the SENB
tubes, allow them to settle overnight. If SENB tubes show
settlement in bottom of tubes, remove media without
disturbing pellet and transfer medium to another set of sterile
tubes (adjust final volume in tubes to 10 mL). Alternatively,
SENB may be autoclaved in flask, allowed to settle overnight,
and then dispensed into test tubes. Avoid disturbing settlement
during transfer.
(c) Production of B. subtilis spore suspension from
amended nutrient agar.—Using growth from a stock culture
tube, inoculate 10 mL tubes (e.g., 2 tubes, depending on the
amount of spore preparation desired) of nutrient broth and
incubate tubes on an orbital shaker at ca 150 rpm for 24  2 h
at 37  1C, repeat for a total of two 24  2 h subcultures. Use
this culture to inoculate amended nutrient agar plates.
Inoculate each plate with 500 L broth culture and spread the
inoculum with a sterile bent glass rod or suitable spreading
device. Wrap each plate with parafilm or place in plastic bags.
Incubate plates inverted for 12–14 days at 37  1C. After
incubation, harvest the spores by adding 10 mL cold sterile
water to each plate. Using a spreader (e.g., bent glass rod),
remove growth from plates and pipet suspensions into 50 mL
sterile conical tubes (10 plates = 8 tubes, about 12 mL each).
Centrifuge tubes at 5000 rpm for ca 10 min at room
temperature. Remove and discard supernatant. Resuspend
pellet in each tube with 20 mL cold sterile water and
centrifuge at 5000 rpm for approximately 10 min. Remove
and discard supernatant. Repeat twice. Resuspend the pellet in
each tube with 10 mL sterile water. Store the spore suspension
at 2–5C. Examine spore suspension with a phase contrast
microscope or by staining to assess quality of the spores.
Examine a minimum of 5 fields and determine ratio of spores
to vegetative cells (or sporangia). Percentage of spores vs
vegetative cells should be at least 95%. Note debris if present.
Spore suspension from multiple plates can be combined and
re-aliquoted into tubes for uniformity. Before inoculation of
carriers, determine spore titer of the concentrated spore
suspension by plating serial dilutions (e.g., 1.0  10–6 through
1.0  10–8) using pour or spread plating on TSA plates. For
pour plating, add molten TSA tempered to 45–55C to each
plate. Swirl pour plates to distribute spores evenly and allow
agar to solidify. Invert plates and incubate for 24–48 h at
37  1C. Count colonies (by hand or with colony counter).
Use dilutions yielding between 30 and 300 colony-forming
units (CFU) per plate (target counts) for enumeration;
however, record all counts less than 30. Report plates with
colony counts over 300 as too numerous to count (TNTC).
Note: When harvested and processed, 10 plates of amended
nutrient agar should provide 80–100 mL concentrated spore
suspension (ca 109 CFU/mL). Diluting the suspension before
carrier inoculation will be necessary; a titer of
1.0  108–5.0  108 CFU/mL should be adequate to achieve
the target carrier count.
(d) Preparation of porcelain carriers.—Before use,
examine porcelain carriers individually and discard those with
scratches, nicks, spurs, or discolorations. Rinse unused
1378 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
carriers gently in water 3 times to remove loose material, and
drain. Place rinsed carriers into Petri dishes matted with
2 layers of filter paper in groups of 15 carriers per Petri dish.
Sterilize 20 min at 121C. Cool and store at room temperature.
Note: Handle porcelain carriers with care when placing in
Petri dishes. Minimize carrier movement and avoid excessive
contact between carriers that might result in chips and cracks.
Wash carriers with Triton X-100, and rinse with water 4 times
for reuse.
(e) Preparation of stainless steel carriers.—Physical
screening: examine steel carriers individually and discard
those with scratches or nicks. Soak overnight (ca 12 h) in 1 N
NaOH and rinse 3–4 times with tap water. Collect portion of
last rinse water and add 2–3 drops 1% phenolphthalein. If any
NaOH remains, phenolphthalein turns pink, indicating need
for additional rinsing. Continue to rinse carriers until addition
of phenolphthalein does not produce a color change. Rinse
twice more with water (reagent grade). Place rinsed carriers
into Petri dishes matted with 2 layers of filter paper in groups
of 15 carriers per Petri dish. Sterilize 20 min at 121C. Store
sterile carriers at room temperature for up to 3 months. After
3 months, reclean and sterilize carriers before use.
(f) Inoculation of porcelain carriers with spore
suspensions prepared using SENB.—Pour contents of 1 tube
of a 72  2 h SENB culture into sterile tissue grinder (Thomas
Tissue Homogenizer) and macerate pellicle, several
(15–20) strokes for each tube. Filter through sterile funnel
containing moist glass wool into sterile container. Repeat for
remaining tubes. Pool spore filtrates into separate holding
container. Pipet 10 mL volumes into sterile 25  150 mm test
tubes agitating holding container between each 10 mL transfer
to maintain spores in suspension. Add 10 sterile penicylinders
to each tube containing 10 mL spore filtrate and let stand
10–15 min. Carriers should remain completely immersed in
inoculum during exposure period. Remove carriers with
sterile hook and place upright in sterile, double matted
(2 layers of filter paper) Petri dishes, no more than 30 carriers
per Petri dish. Allow to air-dry in BSC for ca 30 min. Place
Petri dishes containing contaminated carriers in vacuum
desiccator containing CaCl2 and draw vacuum of 69 cm
(27 in.) Hg. Maintain carriers under vacuum 24  2 h before
use. For long-term storage, spore carriers must be maintained
under vacuum and can be used for up to 3 months. Carriers
older than 3 months should be requalified by both standard
HCl test and spore quantitation before use.
(g) Inoculation of porcelain and stainless steel carriers
with spore suspension prepared using amended nutrient
agar.—Add 10 sterile carriers to each tube containing 10 mL
spore suspension, agitate slightly, and let stand 10–15 min.
Remove each carrier with sterile hook and place upright in
sterile Petri dish lined with 2 sheets of filter paper, no more
than 30 carriers per Petri dish. Air-dry in BSC for
ca 30  2 min. Place Petri dishes containing inoculated carriers
in vacuum desiccator containing CaCl2 and draw vacuum of
69 cm (27 in.) Hg for 20 ± 2 min. Dry carriers under vacuum
of 69 cm (27 in.) Hg for a minimum 24  2 h before use in HCl
resistance, efficacy testing, or carrier counts. Spore carriers
should be maintained under vacuum. Note: Ten plates of
amended nutrient agar should provide up to 100 mL spore
suspension which can be diluted with sterile water to provide
spores in the range of 1.0  108–5.0  108 CFU/mL.
(h) Spore enumeration (carrier counts).—Before use,
determine the carrier counts for each preparation of carriers.
Assay 5 randomly selected carriers per preparation. Place
each inoculated carrier into a 50 mL plastic, polypropylene
conical centrifuge tube containing 10 mL sterile water.
Sonicate carriers for 5 min  30 s. Note: For sonication, place
tubes into an appropriately sized beaker with tap water to the
level of sterile water in the tubes. Place beaker in sonicator so
that water level in the beaker is even with water level fill line
on sonicator tank. Fill tank with tap water to water level fill
line. Suspend beaker in sonicator tank so that it does not touch
bottom of tank and all 3 water levels (inside test tubes, inside
beaker, and sonicator tank) are the same. After sonication, mix
tubes in a Vortex mixer for 2 min  5 s. Dilute spore
suspensions by transferring 1 mL aliquots to tubes containing
9 mL sterile water. Dilute spore suspensions out to 1.0  10–5
and plate dilutions 1.0  10–2 through 1.0  10–5. Plate each
dilution in duplicate using pour or spread plating with TSA
and determine titer. Note: The mean carrier counts for each set
of inoculated carriers, including those supplied by the vendor,
must be at an acceptable level (a minimum of
1.0  105 spores/carrier and a maximum of approximately
1.0  106 spores/carrier) to be used in HCl resistance and
efficacy testing. Laboratories were instructed not to use
carriers if average counts fell below the minimum or above the
approximate maximum.
(i) HCl resistance.—Equilibrate water bath to 20  1C.
Pipet 10 mL of 2.5 M HCl into two 25  100 mm tubes and
place into water bath. Allow at least 30 min to reach
temperature equilibrium. Start timer and rapidly transfer
4 inoculated penicylinders into an acid tube (2.5 M HCl) with
flamed hooks or forceps. Do not allow carriers or transfer
device to contact inside of wall of acid tube. Transfer
individual carriers after 2, 5, 10, and 20 min of HCl exposure
to a separate tube of modified FTM. Rotate each tube
vigorously by hand for ca 20 s and then transfer carrier to a
second tube of modified FTM. For viability control, place
1 unexposed inoculated carrier in a separate tube of modified
FTM. For media sterility, use 1 tube of modified FTM.
Incubate all test and control tubes for 21 days at 37  1C.
Record results as growth (+) or no growth (–) at each time
period. Spores should resist HCl for 2 min to be qualified as
resistant test spores. Discard carriers if not resistant and repeat
preparation of carriers as previously described.
(j) Efficacy test.—One chemical type was tested per test
day. Bleach and peracetic acid/hydrogen peroxide were tested
at 20  1C. Glutaraldehyde was tested at 25  1C. For the
bleach treatments, letheen broth with 0.1% sodium thiosulfate
was used as the neutralizer (primary tube) and FTM was the
subculture medium (secondary tube). For the peracetic
acid/hydrogen peroxide and glutaraldehyde treatments, FTM
was used as the neutralizer and subculture medium. For a
30 carrier test, place 10 mL product at dilution recommended
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1379
for use or under investigation into each of six 25  150 mm or
25  100 mm tubes (medication tubes). Place tubes in
20  1C water bath and let equilibrate to temperature. Using a
sterile hook or forceps, transfer inoculated carriers
sequentially at 2 min intervals in groups of 5 from Petri dish to
medication tubes containing sporicidal agent. Use a certified
timer to monitor time. Flame hook and allow cooling after
each transfer. When lowering carriers into medication tube,
neither carriers nor wire hook may touch sides of tubes. If
interior sides are touched, note tube number. Do not count
carrier set if any carrier from that group of 5 yields a positive
result. Inadvertent contamination of this type may result in
retesting. Carriers must be deposited into medication tubes
within 5 s of the prescribed drop time. Return tubes to water
bath immediately after adding carriers. After contact period
has been achieved, transfer carriers in same sequential timed
fashion into primary subculture tubes containing appropriate
neutralizer (10 mL in 20  150 mm tubes). Remove the
carriers one at a time from the sporicidal agent medication
tube with sterile hook, tap against interior side of tube to
remove excess sporicidal agent, and transfer into neutralizer
tube (primary tube). All 5 carriers must be transferred during
each 2 min interval. Flame hook between each carrier transfer.
Move remaining carriers into their corresponding neutralizer
tubes at appropriate time. Carriers may touch interior sides of
neutralizer tube during transfer, but contact should be
minimized. After each carrier is deposited, recap neutralizer
tube and gently shake to facilitate adequate mixing and
efficient neutralization. Within 1 h from when last carrier was
deposited into primaries, transfer carriers using sterile wire
hook to second subculture tube (secondary tube) containing
10 mL appropriate recovery medium, 1 carrier per tube. Move
carriers in order, but movements do not have to be timed.
Gently shake entire rack of secondary tubes after all carriers
have been transferred. Incubate primary (neutralizer) and
secondary subculture tubes for 21 days at 37  1C. Report
results as growth (+) or no growth (–). A positive result is one
in which medium appears turbid. A negative result is one in
which medium appears clear. Shake each tube before
recording results to determine presence or absence of
growth/turbidity. Primary and secondary subculture tubes for
each carrier represent a carrier set. A positive result in either
primary or secondary subculture tube is considered a positive
result for the carrier set. If no growth occurs after 21 days, heat
shock all test and control tubes for approximately 20 min at
80  2C and re-incubate for 72  2 h at 37  1C.
Media sterility controls and system controls (check for
aseptic technique during carrier transfer process) are
recommended. For media controls, incubate 1–3 unopened
subculture medium tubes with the test sample tubes for
21 days at 37  1C. For system controls, use sterile needle
hook to transfer 3 sterile carriers into a tube of test chemical.
Transfer system control carriers to neutralizer medium as
follows: At start of sample test (prior to first tube), transfer
1 sterile carrier to tube of neutralizer medium. After one-half
of test carriers have been transferred to neutralizer tubes,
transfer a second sterile carrier to tube of neutralizer medium.
After all test carriers (last tube) have been transferred to
neutralizer tubes, transfer third sterile carrier to tube of
neutralizer medium. Transfer system control carriers to
secondary subculture medium as follows: Immediately before
initiating transfer of test carriers into secondary subculture
medium tubes, transfer first system control sterile carrier from
neutralizer medium to tube of subculture medium. After
one-half of test carriers have been transferred to secondary
subculture medium tubes, transfer second system control
sterile carrier to tube of subculture medium. After all test
carriers have been transferred to secondary subculture
medium tubes, transfer third system control sterile carrier to
tube of subculture medium. For each test, include a positive
carrier control by placing 1 inoculated carrier into tube of
secondary subculture medium. Incubate controls and test
sample tubes together for 21 days at 37  1C.
Perform identification confirmation on a minimum of
3 positive carrier sets per test, if available, using Gram stain
and/or plating on TSA. Additional confirmation may be
performed using VITEK, API analysis, or comparable
method. If fewer than 3 positive carrier sets, confirm growth
from each positive carrier set. If both tubes are positive in a
carrier set, select only 1 tube for confirmatory testing. For test
with 20 or more positive carrier sets, confirm at least 20% by
Gram stain.
(k) Neutralization confirmation procedure.—The
neutralization (inactivation) of active ingredients is one of the
most important steps in efficacy testing of antimicrobial
products. Neutralization is used to stop the activity of the
product ingredients, a process essential to measuring
effectiveness at a desired contact time. In addition, the
neutralizer itself or in combination with the recovery medium
must not exhibit bacteriostatic activity against the test
microbe. Neutralization can be accomplished by various
means (e.g., dilution, use of specific neutralizing media,
physical and chemical effects).
The neutralizers for the products tested in the collaborative
study were previously determined to be effective by the Study
Director using the procedure below; however, for the purpose
of the collaborative study, 2 laboratories independently
performed the procedure to ensure its clarity and usefulness.
This procedure simulates the conditions of the efficacy test,
except that sterile carriers were used instead of inoculated
carriers, and provides for a quantitative approach to assessing
the effectiveness of the neutralizer and any bacteriostatic
action resulting from the neutralizer itself or
neutralizer/disinfectant interactions. The test chemicals and
parameters were (1) bleach adjusted pH (about 6000 ppm with
60 min exposure); (2) peracetic acid/hydrogen peroxide
(30 min exposure); and (3) 2.6% glutaraldehyde (60 min
exposure). Only the porcelain carrier type was used in this
study. The selection of test chemicals and associated
parameters were used to simulate more challenging scenarios
for chemical inactivation.
Produce a spore preparation according to procedure for
amended nutrient agar. Harvest growth from plates (e.g.,
5 plates) per the method, except resuspend pellet after final
1380 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
centrifugation step in approximately 100 mL aqueous (40%)
ethanol. Store the spore suspension at 2–5C. Determine spore
count by serial dilution and plating (pour or spread) on TSA.
Desirable target of the initial working suspension is 1.0  108
to 1.0  109 CFU/mL. The suspension may require adjustment
to reach target titer. Prepare serial 10-fold dilutions of the
inoculum in sterile water out to 10–8. Use 10–6, 10–7, and 10–8
dilutions to inoculate the neutralizer and subculture media
tubes. The target number of spores to be delivered per tube in
this assay is 5–100 spores/tube. Determine spore titer by
plating (pour or spread) each of 3 dilutions in duplicate on
TSA.
Use a set of 5 sterile carriers (only 3 to be used in the
assay). Within 5 s, place a set of 5 carriers into a glass tube
(25  150 mm or 25  100 mm) containing test chemical;
transfer carriers according to Method 966.04. Allow carriers
to remain in test chemical per the specified contact time and
temperature. After the contact time is complete, aseptically
transfer 3 of the 5 carriers individually into tubes containing
the neutralizer per Method 966.04. This set of tubes is the
neutralizer/primary subculture treatment. After transfering the
last carrier into neutralizer tube, transfer each carrier, in
sequence, into a tube containing secondary subculture
medium. This portion of assay is not timed, but should be
made as soon as possible. This set is the secondary subculture
treatment. After carrier transfer, inoculate each tube
(neutralizer/primary and secondary subculture treatment
tubes) with 1 mL of each of 3 inoculum dilutions (10–6, 10–7,
and 10–8). For controls, use 3 fresh, unexposed tubes of
neutralizer and 3 tubes of the secondary subculture medium;
also inoculate each control tube with 1 mL of each of
3 inoculum dilutions. Include 1 uninoculated tube of
neutralizer and secondary subculture media to serve as
sterility controls. See Table 4 for tube inoculation scheme.
Incubate all tubes 5–7 days at 37  1C. Record results as
growth (+) or no growth (–). Note: The lack of complete
neutralization of the disinfectant or bacteriostatic activity of
the neutralizer itself may be masked when a high level of
inoculum (spores) is added to the subculture tubes.
Confirm a minimum of one positive tube per treatment and
control (if available) using Gram staining and colony
morphology on TSA. For each treatment and control group,
conduct confirmation testing on growth from tube with fewest
spores delivered. B. subtilis is a Gram-positive rod, and
colonies on TSA are opaque, rough, dull, round, with irregular
margins, and low convex. Growth in the inoculated controls
verifies the presence of the spores, performance of the media,
and provides a basis for comparison of growth in the neutralizer
and subculture treatment tubes. Note: There may be cases when
the neutralizer is significantly different from the secondary
subculture media; in these cases, growth may not be comparable.
The uninoculated control tubes are used to determine sterility
and must show no growth for the test to be valid.
The occurrence of growth in the neutralizer/primary
subculture and secondary subculture treatment tubes is used to
assess the effectiveness of the neutralizer. No growth or
growth only in tubes which received a high level of inoculum
(e.g., the dilution with plate counts which are too numerous to
count) indicates poor neutralization and/or presence of
bacteriostatic properties of the neutralizer or
neutralizer-disinfectant interactions. For a neutralizer to be
deemed effective, growth must occur in the secondary
subculture treatment tubes which received lower levels of
inoculum (e.g., 5–100 CFU/tube).
(l) Quantitative efficacy.—This study was conducted
independently of the efficacy evaluation. For each low
chemical treatment, 2 laboratories tested 5 carriers of each
medium/carrier combination according to the randomization
scheme provided by the Study Director. The low treatments
were used to provide a level of detectable viable spores for
comparative analysis.
Conduct efficacy tests (5 carriers) for each of
3 combinations of carriers according to Method 996.04.
Transfer carriers sequentially at 2 min intervals in groups of
5 from the Petri dish into test tubes containing sporicidal agent
using a sterile hook. Immediately after placing carriers into
medication tube, return to water bath held at 20  1C (or
specified temperature). Carriers must be deposited into tube
within 5 s of prescribed drop time. Flame hook and allow
cooling after each carrier transfer. Note: When lowering
carriers into test tube, neither carriers nor wire hook may
touch sides of tubes. If interior sides are touched, note tube
number. Do not count carrier set if any carrier from that group
of 5 yields a positive result. Inadvertent contamination of this
type may result in retesting.
After prescribed contact period, transfer carriers in same
timed fashion into tubes containing sterile water (10 mL in
50 mL plastic conical bottom centrifuge tube). Transfer all
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1381
Table 4. Neutralization confirmation procedure: inoculating treatment and control tubes with diluted spore
suspension
a
Neutralizer–primary
subculture treatment
Secondary subculture
treatment (with carrier)
Neutralizer–primary
inoculated control
Secondary subculture
inoculated control
1 mL of 10–6  Tube 1 1 mL of 10–6  Tube 1 1 mL of 10–6  Tube 1 1 mL of 10–6  Tube 1
1 mL of 10–7  Tube 2 1 mL of 10–7  Tube 2 1 mL of 10–7  Tube 2 1 mL of 10–7  Tube 2
1 mL of 10–8  Tube 3 1 mL of 10–8  Tube 3 1 mL of 10–8  Tube 3 1 mL of 10–8  Tube 3
a 1.0  10–6 through 1.0  10–8 based on starting suspension of approximately 108 spores/mL.
5 carriers during each 2 min interval. Flame hook between
each carrier transfer. Place tubes with carriers into
appropriately sized beaker and fill beaker with tap water to
level of sterile water in tubes. Place beaker in sonicator so
water level in beaker is even with water level fill line. Water in
tank should never fall below 1 in. from top of tank. Place
beaker in sonicator tank so that it is not touching bottom and
all 3 water levels (inside test tubes, inside beaker, and
sonicator tank) are the same. Sonicate samples 5 min  30 s.
After sonication, mix each tube in a Vortex mixer 2 min  5 s.
Dilute spore suspensions by transferring 1 mL aliquots to
tubes containing 9 mL sterile water. Dilute spore suspensions
out to 1.0  10–4 and plate 1 mL of appropriate dilutions
(1.0  10–1 to 1.0  10–4). Plate each dilution using pour plate
or spread plate techniques with TSA. Visually examine
morphology of colonies to confirm identity of culture.
Calculate mean number of viable spores per medium/carrier
combination and the log reduction (LR) for each chemical.
(m) Wash-off of spores in saline.—Analyze a total of
18 carriers. In separate test tubes (25  100 mm), expose sets
of inoculated carriers (1 per tube) to 2 saline treatments:
(1) 10 mL 0.85% saline and (2) 10 mL 0.85% saline +
polysorbate 80 (final concentration 0.01%). Test 1 carrier type
at a time. Without disturbing tubes, allow contact time for
10 min  30 s at 20  1C. Track elapsed time with certified
timer. Remove carriers in sequential order from control
solutions and place each into 50 mL plastic conical bottom
centrifuge tube containing 10 mL sterile water. Place all tubes
(6 total tubes/carrier  medium combination) with carriers
into appropriately sized beaker and fill beaker with tap water
to level of sterile water in tubes. Place beaker in sonicator tank
so that it is not touching bottom and all 3 water levels (inside
test tubes, inside beaker, and sonicator tank) are the same.
Sonicate samples 5 min  30 s. After sonication, mix each tube
in a Vortex mixer for 2 min  5 s. Dilute spore suspensions by
transferring 1 mL aliquots to tubes containing 9 mL sterile
water. Dilute spore suspensions out to 1.0  10–5 and plate
1 mL of appropriate dilutions (1.0  10–2–1.0  10–5). Plate
each dilution in duplicate using pour plate or spread plate
techniques with TSA. Visually examine morphology of
colonies to confirm identity of culture.
To determine wash-off, plate serial dilutions of 1 mL
aliquot of liquid in initial treatment tubes (saline and saline +
polysorbate). For each test tube (18 total), mix briefly in a
Vortex mixer, remove 1 mL and serially dilute in 9 mL sterile
water to 1.0  10–4. Plate 1 mL of appropriate dilutions
(1.0  10–1–1.0  10–4) in duplicate using pour plate or spread
plate techniques with TSA. Visually examine morphology of
colonies to confirm identity of culture.
Data Analysis and Statistical Methods
Statistical comparisons were made between the current
method (SENB/PC) versus each of the modified methods
(NA/PC and NA/SS) with respect to control carrier counts, the
pass/fail HCl resistance result, the pass/fail efficacy result, the
observed numbers of positives among treated carriers, and the
percentage washed-off during a simulated disinfection step.
The mean and reproducibility standard deviation (SDR) were
presented for the quantitative measurements. The mean and SDR
calculations were performed separately for each of the
3 medium/carrier combinations. By submitting the data to a
random effects analysis of variance (ANOVA), it was possible to
estimate the within-experiment variance, the between-laboratory
variance, and SDR. (Note: The SDR is the typical difference, sign
neglected, between the measurement for a single, randomly
chosen carrier or sample and the mean across many independent
experiments in many laboratories. A small value for the SDR
indicates that the medium/carrier method produces about the
same measurement from laboratory to laboratory).
Quantitative aspects of AOAC Method 966.04 (SENB/PC)
were compared with the same aspects for the modified tests
(NA/PC and NA/SS). Two types of statistical tests were
conducted: the conventional hypothesis test and the equivalence
test. For comparing the difference between the AOAC and
modified methods, the results include a P-value and a 90%
confidence interval, which are the key summary statistics for the
conventional hypothesis test. The 90% confidence interval is also
the key summary statistic for the equivalence test.
Many published articles and books describe the
conventional and equivalence testing approaches (8, 9). The
problem of comparing means will be used to motivate the
ideas, i.e., suppose the analysis is to compare the true,
unknown mean for SENB/PC, denoted by S, to the true,
unknown mean for one of the modifications, denoted by N,
based on the difference, denoted by . Then  = S – N.
Because the ideas discussed here are quite general, it is not
necessary to specify the laboratory measurement to which the
mean applies. The measurement could be the log-transformed
spores per carrier or it could be the LR, to list 2 specific
examples.
The conventional hypothesis testing approach is directed at
using the observed data to show that  	 0. The problem is
formulated in the context of hypotheses, specifically the null
hypothesis: Ho:  = 0 and the alternative hypothesis: Ha:  	 0.
A decision rule is devised by which one decides whether or
not to reject Ho in favor of Ha, depending on the observed
laboratory data. The decision rule is established before any
data are collected. It can be visualized as a partitioning of the
list of all possible laboratory data values into 2 exclusive,
exhaustive subsets called the “acceptance region” and the
“rejection region.” If the data realized in the actual experiment
fall within the rejection region, then one would reject Ho and
conclude that the observed difference between means is
statistically significantly different from zero.
The equivalence testing approach is directed at using the
observed data to show that  is negligibly small. The
equivalence test depends on a quantitative definition of
“statistically equivalent.” To put this idea in a specific context,
consider the carrier count data for which the key summary
statistic is the mean log spores per carrier. Equivalence testing
requires specification of a nonzero numerical value, a, for
 such that any  in the “equivalence zone,” –a <  < a, is
judged to be negligibly small for all practical purposes. If  is
outside that equivalence zone, the 2 methods are judged to be
1382 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
nonequivalent. The choice of a is subjective, dependent on
expert judgment about practicality and importance. For the
purpose of this presentation, the Study Director selected the
value a = 1.0. The equivalence test is conducted by
comparing the 90% confidence interval to the equivalence
zone as follows: If the 90% confidence interval for  is
completely contained within the equivalence zone
(–1.0 <  < 1.0), one can conclude that the methods are
statistically equivalent at a 0.05 level. If the 90% confidence
interval extends outside the equivalence zone, then one cannot
conclude that the methods are statistically equivalent.
Although it may seem paradoxical to use a 90% confidence
interval as the basis for a 0.05 level equivalence test, it is a
mathematically justified, valid method (8, 9).
HCl Resistance Test
The quantitative results for both the HCl resistance test and
the efficacy test were recorded as binary pass/fail data,
following established performance standards. (1) AOAC
Method 966.04 calls for a minimum of 2 min resistance to
2.5 M HCl. This is a pass. The outcomes (pass or fail) will be
compared for each medium/carrier combination. The basis of
the comparison is the current method SENB/PC. The
modified version(s) must exhibit a statistically equivalent
pattern of outcomes. (2) EPA/FDA requires killing on all test
carriers; thus no positive carrier sets present. This is a pass. To
validate the modification(s), the modified versions must
exhibit a pass/fail pattern that is statistically equivalent to the
current method (SENB/PC).
Carrier Counts
For each of the 3 medium/carrier combinations, the viable
spores on each of 5 control carriers were counted in each
laboratory. The spores per carrier approximately followed a
log normal distribution. To create a measurement that
exhibited normality and homogeneous variance, statistical
analyses were performed on the log10-transformed spores per
carrier (10). To compare a new medium/carrier combination
NA (either NA/PC or NA/SS) to the standard medium/carrier
combination SENB/PC, the mean log spores per carrier was
calculated for each combination of the 2 methods and the
4 laboratories. Then the means for the methods were
compared using a paired t-test, where the means were paired
by laboratory.
Efficacy
Pass/fail results.—Let S denote the current standard
method (SENB/PC) and N denote a new modification
(NA/PC or NA/SS). Each medium/carrier combination was
tested once for each chemical treatment in each laboratory.
The N and S test outcomes for each combination of sporicide
treatment and laboratory were treated as paired for purposes
of statistical analysis. For each pair of tests, it is of interest to
determine the extent to which the 2 tests resulted in the same
outcome, pass or fail. Such paired binary data are
conventionally presented in the 2  2 table format of Table 5.
Outcome combinations for counts a and d are concordant
pairs (pass for both methods or fail for both methods) and
outcome combinations for counts b and c are discordant pairs
(fail for one method and pass for the other). The true, unknown
probabilities for the 4 cells in Table 5 are denoted by Pa, Pb, Pc,
and Pd, respectively. The probability of a pass for Method N is
PN = Pa + Pc and the probability of a pass for Method S is PS = Pa
+ Pb. The true difference between pass probabilities, denoted by
D, is D = PS – PN = Pb – Pc, the difference between the pass
probabilities for the 2 discordant pairs. The estimate of D is (b –
c)/n (11). To test the null hypothesis that D = 0 (i.e., the 2
methods produce the same pass probabilities), the Tango score
test version of McNemar’s test was used (11–14). The 90%
confidence interval for D was calculated using the Tango
method for the difference between 2 proportions based on
paired data (12–15).
Percentages of agreement were calculated as follows:
(1) the overall percentage agreement between N and S based
on the estimate [(a + d)/n]·100%; (2) the percentage
agreement of N with a passed S based on the estimate
[a/(a + b)]·100%; and (3) the percentage agreement of N with
a failed S based on the estimate [d/(c + d)]·100%.
LR results using the positive/negative (P/N) formula.—The
LR measure of efficacy is the difference on the log10 scale
between the number of spores per control carrier and the
number of spores per treated carrier. Let the mean of the log
spores per carrier be denoted by C for control carriers and by
T for treated carriers. Let LR denote the log reduction value.
Then LR = C – T. Note that because C and T are the means of
logs, LR is a measurement on the log10 scale. In this
investigation, the numbers of spores on control carriers were
counted and C was easily calculated. Although the spores per
treated carrier were not counted, it was possible to estimate
T based on the number of positive carriers among the 30 treated
carriers and to calculate LR using the P/N formula in Equation 1.
Consider a disinfectant test that uses Mc control carriers
that are subjected to quantitative evaluation and Mt treated
carriers that are subjected to qualitative evaluation. Viable
spore counts per carrier are observed for each of the control
carriers, resulting in counts of c1, 
, cMc. Denote the mean of
the log10 counts by C; that is, C = [log(c1) + 
 + log(cMc)]/Mc.
Each of the treated carriers is observed to be either positive
or negative, where a carrier is positive if and only if there is at
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1383
Table 5. Summary of paired binary (pass/fail) data;
each entry (a, b, c, or d) is the number of pairs having
that combination of N and S outcomes
Test outcome for S
Test outcome for N
Pass Fail Total
Pass a b a + b
Fail c d c + d
Total a + c b + d n = a + b + c + d
least one viable spore on the carrier. Let G denote the observed
number of negative carriers and A denote the adjusted fraction
of treated carriers that are negative, A = (G + ½)/(Mt + 1).
Let ln(·) denote the natural logarithm, which is sometimes
denoted by loge(·). The P/N formula for converting the control
and treatment observations into an LR is
P/N formula: LR = C – log10[-ln(A)] (1)
The rationale for this formula will now be described. Suppose
the carriers were prepared so that there were V viable spores per
carrier. Suppose each of the V spores on a treated carrier has
probability p of being killed by exposure to the disinfectant. If we
assume that the spores live or die independently, the number of
spores that survive the disinfectant will follow a binomial (V, p)
probability distribution.
Let Yi denote the positive/negative outcome for the ith
treated carrier, i = 1, 2, 
, Mt; specifically, Yi = 0 or 1, where
Yi = 0 if the carrier is negative and Yi = 1 if the carrier is
positive. Then the probability that the ith carrier is positive is
Pr{Yi = 1} = 1 – (1 – p)
V. If V is large, as it will be in
disinfectant testing where V is usually greater than 105, the
expression (1 – p)V is closely approximated by e–Vp.
If the treated carriers are statistically independent, then the
number of positive carriers, Y1 + … + YMt, will follow a
binomial(Mt, 1 – e
–Vp) probability distribution. Therefore, the
expected number of negative carriers is Mt· e
–Vp. Setting the
observed number of negative carriers equal to the expected
number and solving for p, we find that G = Mt· e
–Vp if and only if
p = –ln(G/Mt)/V (2)
Let LRtrue denote the true, unknown log reduction value.
The LR is related to the survival probability for individual
spores by the equation: LRtrue = –log10(p). Let LR* denote a
preliminary estimate of LRtrue. Substituting for p in Equation 2,
a plausible estimator is LR* = –log10(–ln(G/Mt)/V) or
LR* = log10(V) – log10(–ln(G/Mt)) (3)
For a real disinfectant test, the viable spores per carrier will
not be identical. The typical number of spores per carrier is
estimated by the geometric mean of control carrier counts,
10C. Therefore, it is reasonable to replace the log10(V) term in
Equation 3 with log10(10
C) = C, the mean of log counts for
control carriers. In Equation 3, G/Mt is the observed fraction
of treated carriers that are negative. If G = 0, Equation 3 is not
calculable because ln(0) is not defined mathematically.
DeVries and Hamilton (16) explored a variety of ways to
adjust the fraction A so that it is never zero. They arrived at the
recommendation to replace G/Mt with A = (G + ½) / (Mt + 1).
Although the DeVries and Hamilton work focused on
confidence interval calculations, their recommended adjusted
fraction A was adopted here for the P/N formula. These
practical substitutions for the 2 components of Equation 3
produce the P/N formula of Equation 1.
To compare a new medium/carrier combination NA (either
NA/PC or NA/SS) to the standard medium/carrier
combination SENB/PC, the LR was calculated for each
combination of the 2 methods and the 4 laboratories. Then the
LR values for the methods were compared using a paired
t-test, where the means are paired by the combination of
laboratory and treatment. The test outcome was summarized
by a 2-tailed P-value. The paired t-method was also used to
find a 90% confidence interval for the mean difference of LR
values, SENB/PC – NA.
Quantitative Efficacy
Two laboratories measured the number of viable spores per
treated carriers following exposure to the low treatments. This
allowed the direct calculation of the log reduction,
LR = (mean of log spores per control carriers) – (mean of log
spores per treated carrier). The LR values for SENB/PC and
NA were paired on laboratory by sporicide combination, and
the paired t-test was used to find the p-values and 90%
confidence intervals for the mean differences, SENB/PC –
NA/PC and SENB/PC – NA/SS. For each medium/carrier,
SDR was the square root of the residual mean squared error for
a 1-way ANOVA, where the factor was treatment (2 levels).
Wash-Off in Saline
Three carriers of each medium/carrier combination were
exposed to 2 saline treatments (0.85% saline and 0.85% saline +
polysorbate 80). The saline + polysorbate 80 (final concentration
0.01%) was used to increase the surfactancy of the saline, thus
simulating an actual product. The percent wash-off for each
medium/carrier combination was calculated as follows:
% Wash-off =
100 × mean number of wash-off spores
in medication tubes  by the total
where the total is the mean number of spores in suspension
following wash-off plus the mean number of spores from
subsequently removed by vortexing the carrier.
The percent wash-off and total spores/carrier were
calculated for each saline treatment for each medium carrier
combination; comparisons were made to the current method
(SENB/PC).
Because the percentage washed-off is small for NA/SS, the
homogeneous variance and normality assumptions required
for the ANOVA were violated and it was necessary to
log-transform the percentages washed-off prior to statistical
analysis. A one-factor random effects ANOVA, where
laboratory was the random effect, was conducted for each
medium/carrier. Paired t-tests were used to compare the
log-transformed wash-off percentages for a new test system N
to the standard test system S. The percentages were paired by
the combination of laboratory and treatment, where treatment
refers to saline or saline plus polysorbate 80. Diagnostic
statistical evaluation during the analyses showed that the
log-transformation produced data that did conform to the
1384 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
usual normality assumptions. The log-transformed total
spores per carrier were analyzed by the same methods.
The antilog of the mean log percentage is the geometric
mean of the percentage washed off. The antilog of the
difference between the means of log-transformed percentages
is the ratio of geometric means. The results for wash-off are
presented in terms of geometric means and ratios of geometric
means, although the statistical analyses were conducted on the
log scale. The delta method was used to calculate the SDR that
is applicable to the percentage scale (11).
Results and Discussion
Carrier Counts
Attaining a target number of spores per carrier is desirable
for standardizing Method 966.04 and increasing the method’s
reproducibility and quality of the data. To assess the feasibility
of the proposed modification to establish a target minimum
titer of 5 logs (1.0  105) of B. subtilis spores per carrier, each
of the 4 laboratories independently determined the spore
counts for each medium/carrier combination according to the
provided spore enumeration protocol. The log spores per
carrier are presented in Figure 1. The mean log spore count
values across laboratories for each medium/carrier
combination were above the target minimum of 5 logs of
spores per carrier (Table 6). Conventional hypothesis testing
showed that the mean log spores per carrier for SENB/PC was
statistically significantly different from the mean for NA/PC
(P = 0.02, SENB/PC higher by 0.61 logs). Mean log spores
per carrier for SENB/PC and NA/SS were not statistically
different (P = 0.22, SENB/PC higher by 0.35 logs). For an
equivalence zone where the difference in means is no greater
than 1 (e.g., a =1), both NA/PC and NA/SS were
statistically equivalent to SENB/PC at a 0.05 level of
significance. Although the data discredit the null hypothesis
of no difference between the means of SENB/PC and NA/PC,
the difference is judged to be of no practical importance when
compared to the equivalence zone –1.0 <  < 1.0.
It should be noted that adjustment of the NA spore
preparations used to inoculate PC and SS carriers to arrive at
carrier counts statistically the same as those for SENB/PC was
not emphasized by the Study Director; rather, the goal of
achieving counts within the target range was prioritized. It
would be possible to adjust the NA spore preparation by
dilution or concentration to increase or decrease the carrier
count; however, the goal of the study was to confirm the
practicality of meeting the recommended target carrier load
without additional manipulation of the NA spore preparation.
Based on the Study Director’s experience with carrier-based
qualitative tests, the statistical difference in carrier counts
noted for SENB/PC and NA/PC, 0.61 logs, is likely not of
practical importance to the pass/fail outcome of AOAC
Method 966.04, i.e., the sensitivity of the method is not high
enough to be affected by this difference in carrier counts.
Note: The Study Director assumed that the outcome of the
comparative studies (e.g., HCl resistance, efficacy, wash-off)
for SENB/PC versus NA/PC was not impacted by the
difference in carrier counts.
Table 6 presents the SDR for each medium/carrier
combination. The reproducibility is good relative to other
standardized antimicrobial tests (17). In this respect, each
medium/carrier combination consistently produced an
acceptable number of spores across laboratories, thus
providing support for the enumeration procedure and the
target spore count as modifications to Method 966.04.
HCl Resistance
The test for HCl resistance was conducted on each
medium/carrier combination by each laboratory. AOAC Method
966.04 calls for a minimum of 2 min resistance of spores to 2.5 M
HCl, i.e., growth of B. subtilis in the recovery medium after 2 min
exposure to HCl is considered a pass and lack of growth is
considered a fail. In this study, each test resulted in an outcome of
pass; each laboratory recorded growth in at least the secondary
recovery medium after 2 min exposure for each medium/carrier
combination (Table 7). The pattern of positive tubes (i.e., positive at
2, 5, and 10 min) was variable across the tests. This is not
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1385
Figure 1. Log10 Spore counts per control carrier, arranged to show the variability within experiments, among
methods, and among laboratories. Each point is one carrier. Points aligned vertically are from the same experiment.
Each horizontal line is the mean of log spores per carrier for the experiment.
unexpected and falls within the range of what would be
considered typical for this assay.
Neutralization Confirmation
Two laboratories conducted the neutralization confirmation
procedure. Results are presented in Tables 8–10. The laboratories
generated comparable results. For each test chemical/neutralizer
combination, growth of B. subtilis occurred in both the primary
and secondary treatments at inoculum dilutions providing low
levels of spore challenge (5–100 spores/mL). Acceptable results
were recorded for the inoculated controls and uninoculated
controls (data not shown). The data support the selection and
effectiveness of letheen broth + 0.1% sodium thiosulfate as a
neutralizer for sodium hypochlorite, and FTM as a neutralizer
medium for the peracetic acid/hydrogen peroxide and
glutaraldehyde. This procedure is relevant to Method 966.04, is
simple to conduct, and provides valuable quantitative
information on neutralizer effectiveness and the occurrence of
bacteriostatic activity. This procedure will be a valuable
addition to Method 966.04.
Efficacy
Pass/fail results.—A comparison of results for the current
and modified methods is shown in Tables 11 and 12. The
results are presented as pass (zero positive carriers/30) or as
fail (1 positive carrier/30). The number of positive carrier
sets with growth of B. subtilis is also provided.
The percentages of overall agreement between pass/fail
results for 24 total tests (2 test conditions for each of
3 sporicides tested in each of 4 laboratories) were 96%
between SENB/PC and NA/PC and 88% between SENB/PC
and NA/SS. Each NA method had 100% agreement with a
passed SENB/PC outcome. The NA/PC and NA/SS methods
had 93 and 80% agreement, respectively, with a failed
SENB/PC outcome. Overall, the high percent agreement of
the current to the proposed modified methods supports the
1386 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
Table 6. Statistical summary of log10 spores per control carrier
Medium/carriera
Mean log spores
per carrier SDRb pc
Mean difference (S–N) and
90% confidence limits
SENB/PC 5.87 0.401 — —
NA/PC 5.26 0.181 0.02 0.61 (0.30 to 0.93)
NA/SS 5.52 0.433 0.22 0.35 (–0.19 to 0.89)
a SENB/PC (soil extract nutrient broth/porcelain); NA/PC (amended nutrient agar/porcelain); NA/SS (amended nutrient agar/stainless steel).
b SDR = Reproducibility standard deviation.
c Comparing mean log10 spore counts per carrier for SENB/PC to NA/PC and NA/SS.
Table 7. Outcome of the HCl resistance test for current and modified AOAC Method 966.04
Exposure period and occurrence of tubes with growth (+) or no growth (–)
Lab
Medium/carrier
combinationa
Outcomeb
(pass/fail) 2 min 5 min 10 min 20 min
1 SENB/PC Pass –/+ –/– –/– –/–
NA/PC Pass –/+ +/– –/– –/–
NA/SS Pass +/+ –/– –/– –/–
2 SENB/PC Pass +/+ –/+ –/+ –/–
NA/PC Pass –/+ –/– –/– –/–
NA/SS Pass +/+ –/– –/– –/–
3 SENB/PC Pass +/+ –/– –/– –/–
NA/PC Pass +/+ –/– –/– –/–
NA/SS Pass +/+ +/+ –/– –/–
4 SENB/PC Pass +/+ +/– +/– –/–
NA/PC Pass +/+ +/+ –/– –/–
NA/SS Pass +/+ +/+ +/– –/–
a SENB/PC (soil extract nutrient broth/porcelain); NA/PC (amended nutrient agar/porcelain); NA/SS (amended nutrient agar/stainless steel).
b Pass (spores must resist HCl for 2 min to be qualified as resistant test spores).
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1387
Table 8. Neutralization confirmation of bleach with letheen broth + 0.1% sodium thiosulfate
Treatments/controls
Lab 1 Lab 2
Dilution
10–6 10–7 10–8 10–6 10–7 10–8
Spores/mL
91 13 2 101 12 1
Neutralizer-primary subculturea + + + + + –
Secondary subcultureb + + + + + +
Neutralizer inoculated control + – – + + –
Subculture inoculated control + + – + + –
a Letheen broth + 0.1% sodium thiosulfate.
b Fluid thioglycollate medium (tubes contain a carrier).
Table 9. Neutralization confirmation of peracetic acid/hydrogen peroxide product with fluid thioglycollate medium
Treatments/controls
Lab 1 Lab 2
Dilution
10–6 10–7 10–8 10–6 10–7 10–8
Spores/mL
91 13 2 113 8 1
Neutralizer-primary subculturea + + + + – +
Secondary subcultureb + + + + + +
Neutralizer inoculated control + + + + + +
Subculture inoculated control + + + + + +
a Fluid thioglycollate medium.
b Fluid thioglycollate medium (tubes contain a carrier).
Table 10. Neutralization confirmation of glutaraldehyde with fluid thioglycollate medium
Treatments/controls
Lab 1 Lab 2
Dilution
10–6 10–7 10–8 10–6 10–7 10–8
Spores/mL
89 12 1 108 8 1
Neutralizer-primary subculturea + + – + + –
Secondary subcultureb + + – + + –
Neutralizer inoculated control + + – + + +
Subculture inoculated control + + – + + +
a Fluid thioglycollate medium.
b Fluid thioglycollate medium (tubes contain a carrier).
adoption of the modifications. Note: No additional positive
tubes were recorded following the heat shock step. This step
will not be included in the revised alternate method.
Low treatments.—Exposing carriers (30 carrier tests) to
the low treatments resulted in a fail for 36 out of 36 tests
(Table 11). The number of positive carriers ranged from 1 to
30 out of 30. Thus, the pass/fail outcome for NA/PC and
NA/SS always agreed with SENB/PC, i.e., no discordant pairs
were detected. Of the total positive carriers (684) across all
low treatments and laboratories, 33, 36, and 31% were from
SENB/PC, NA/PC, and NA/SS, respectively. These data
strongly indicate that the modified methods, when used in
place of the current method, will provide the same outcome
for less effective formulations. There was no indication that
the modifications created a less stringent test (i.e., NA/PC or
NA/SS passed a test chemical when the SENB/PC failed the
test chemical). In fact, based on the total percentage of total
positives, the NA/PC was slightly higher, suggesting a more
conservative method. Thus, this component of the study
supports the use of NA/PC and NA/SS as viable replacements
to SENB/PC.
High treatments.—The medium/carrier combinations
exposed to the high treatments produced a pass result in 31 out
of the 36 tests conducted. The number of positive carriers
from the 5 failed tests was low: 1 to 3 positive carrier sets out
of 30. The failed carrier sets occurred for the peracetic
acid/hydrogen peroxide and bleach treatments.
With few exceptions, SENB/PC and the modified
combinations, NA/PC and NA/SS, provided the same results
1388 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
Table 11. Comparative efficacy results for low chemical treatments
Outcome and number of positive carriers
Chemical treatment
Medium/carrier
combinationa Lab No. 1 Lab No. 2 Lab No. 3 Lab No. 4
Peracetic acid and hydrogen peroxide SENB/PC Fail (16+) Fail (28+) Fail (21+) Fail (28+)
NA/PC Fail (29+) Fail (17+) Fail (28+) Fail (30+)
NA/SS Fail (20+) Fail (30+) Fail (30+) Fail (20+)
Glutaraldehyde SENB/PC Fail (15+) Fail (9+) Fail (5+) Fail (23+)
NA/PC Fail (17+) Fail (26+) Fail (22+) Fail (21+)
NA/SS Fail (3+) Fail (27+) Fail (1+) Fail (29+)
Bleach SENB/PC Fail (13+) Fail (20+) Fail (16+) Fail (29+)
NA/PC Fail (28+) Fail (24+) Fail (6+) Fail (2+)
NA/SS Fail (3+) Fail (22+) Fail (11+) Fail (5+)
a SENB/PC (soil extract nutrient broth/porcelain); NA/PC (amended nutrient agar/porcelain); and NA/SS (amended nutrient agar/stainless
steel).
Table 12. Comparative efficacy results for high chemical treatments
Outcome and number of positive carriers
Chemical treatment
Medium/carrier
combinationa Lab No. 1 Lab No. 2 Lab No. 3 Lab No. 4
Peracetic acid and hydrogen peroxide SENB/PC Fail (1+) Pass (0+) Pass (0+) Pass (0+)
NA/PC Fail (1+) Pass (0+) Pass (0+) Pass (0+)
NA/SS Pass (0+) Pass (0+) Pass (0+) Pass (0+)
Glutaraldehyde SENB/PC Pass (0+) Pass (0+) Pass (0+) Pass (0+)
NA/PC Pass (0+) Pass (0+) Pass (0+) Pass (0+)
NA/SS Pass (0+) Pass (0+) Pass (0+) Pass (0+)
Bleach SENB/PC Fail (2+) Fail (2+) Pass (0+) Pass (0+)
NA/PC Fail (3+) Pass (0+) Pass (0+) Pass (0+)
NA/SS Pass (0+) Pass (0+) Pass (0+) Pass (0+)
a SENB/PC (soil extract nutrient broth/porcelain); NA/PC (amended nutrient agar/porcelain); and NA/SS (amended nutrient agar/stainless
steel).
for the high treatments. The outcome for SENB/PC and
NA/PC against the high treatments was the same for 11 of the
12 pairs (Tables 12 and 13). One discordant pair was recorded
by Laboratory 2 (pass with NA/PC and a fail with SENB/PC
when testing the high bleach). Three discordant pairs were
recorded for SENB/PC and NA/SS against the high
treatments, a pass for NA/SS was recorded 3 times when a fail
was recorded for SENB/PC (Tables 12 and 13). The estimated
differences [P-value (90% confidence interval)] between pass
probabilities were –0.08 [P = 0.32, (–0.30, 0.12)] for
SENB/PC minus NA/PC and –0.25 [P = 0.083, (–0.49,
–0.02)] for SENB/PC minus NA/SS. Based on these findings
for the 3 high chemical treatments combined, the SENB/PC
was slightly more stringent than the 2 NA/carrier
combinations.
Although the high treatments were designed to provide
complete spore kill, the occurrence of the fail outcomes does
not impact the quality of the collaborative study; rather it is
likely due to inherent variability with the method and not
associated with a specific method characteristic. In theory, a
single viable spore can produce a tube with growth; the same
result could also be generated by the occurrence of many
survivors. The fail outcomes do not appear to be associated
with significantly higher carrier counts, technical error, or
improper preparation of the test chemicals. The modified
methods do not appear to be more conservative than the
current method; rather there is a slight tendency for the
modifications to be less stringent, most notably for NA/SS.
Overall, the high treatment data provide evidence strongly
supporting the use NA/PC as a replacement for SENB/PC.
The occurrence of the one discordance for the bleach
treatment is recognized and is not considered to be of practical
importance in this instance (2/30 positive for SENB/PC
versus 0/30 positive for NA/PC). NA/SS has excellent
potential as a replacement for SENB/PC as well, although the
occurrence of the 3 discordant pairs indicates a less stringent
test. NA/SS is the combination of 2 method modifications, NA
and SS; thus, achieving equivalency to SENB/PC is more
challenging. Similar to the use of NA as a replacement for
SENB, the advantages of stainless steel over porcelain as a
carrier material in Method 966.04 must also be considered in
the final recommendation to revise the method.
LR results using the P/N formula.—The number of positive
carriers for the high chemical treatments was usually zero;
thus, the variability among LR is primarily due to the
variability among control carrier counts. For this reason, the
usefulness of presenting the P/N formula LRs to compare the
equivalency of the medium/carrier types is limited and will
not be presented for the high chemical treatments.
Figure 2 shows that there is no consistent relationship for
the low chemical treatments between the number of spores per
carrier and the number of positive carriers observed when
testing a sporicide. It appears that greater number of fails
observed with SENB/PC cannot be attributed to the slightly
higher spores per carrier for SENB/PC. The range of the mean
number of spores per carrier among the 3 methods is not wide
enough to show an effect on the efficacy test outcome. These
plots therefore support the conclusion that the observed
differences in mean spores per carrier (Table 6) are not of
practical importance.
The P/N formula was used to convert the number of
positive carriers into an LR value. A statistical summary of the
LR values for each medium/carrier combination is presented
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1389
Table 13. Pass/fail outcome for SENB/PC compared to the NA/PC and NA/SS outcomes for the same treatment and
laboratory; high chemical treatments
Outcome for NA/PCa Outcome for NA/SSa
Outcome for SENB/PCa Pass Fail Total Pass Fail Total
Pass 9 0 9 9 0 9
Fail 1b 2 3 3c 0 3
Total 10 2 12 10 2 12
a SENB/PC (soil extract nutrient broth/porcelain); NA/PC (amended nutrient agar/porcelain); and NA/SS (amended nutrient agar/stainless
steel).
b Discordance observed by Laboratory 2 when testing bleach.
c Discordance observed by Laboratory 1 when testing peracetic acid–hydrogen peroxide and by Laboratories 1 and 2 when testing bleach.
Figure 2. The 4 points in each panel are observations
at 4 laboratories when testing the low chemical
treatments. The same 4 laboratories tested each
combination of medium/carrier and sporicide. The line
in each panel is the least squares regression line.
in Table 14. The SENB/PC – NA/PC difference between LR
values was 0.69, which was statistically significantly different
from zero (P = 0.01). In this case, the LR estimate for NA/PC
is lower, thus providing a more conservative test than
SENB/PC. The SENB/PC – NA/SS difference between LR
values was only 0.26, which was not statistically significantly
different from zero (P = 0.16). For an equivalence zone where
the difference in mean LRs is no greater than 1 (e.g., a =1),
NA/SS was statistically equivalent to SENB/PC at a 0.05 level
of significance. Although NA/PC was not formally
statistically equivalent to SENB/PC, the 90% confidence
interval extended outside the equivalence zone by only
0.09 (Table 14).
Quantitative Efficacy
In this study, the low chemical treatments were selected to
provide a measurable population of survivors for comparison
purposes. The LR values for each medium/carrier
combination were compared. Carriers used in this experiment
were from the same preparations of carriers used throughout
the collaborative study; thus, the control carrier counts
previously discussed in the carrier count section were used in
the LR calculations.
Following exposure, the carriers were handled according
to Method 966.04 except for the following: (1) Treated
carriers were placed into 10 mL water and not into a specific
neutralizer medium; and (2) carriers were processed
quantitatively according to the carrier count procedure.
One mL aliquots of the 1.0  10–1–1.0  10–4 dilutions (serial
dilution blanks with recovered spores) were plated. Wash-off
of spores in the test chemical tube was not accounted for in
this study.
Spores counts were less than detectable for several
chemical treatment  medium/carrier combinations
(Table 15), especially for bleach. Viable spores may have been
recovered if the 1.0  100 dilution had also been plated. Due to
the high frequency of zero spores recovered from the bleach
treatment, only data from the glutaraldehyde and peracetic
acid/hydrogen peroxide treatments were analyzed for
medium/carrier equivalency.
The statistical analysis was conducted on paired
combinations of SENB/PC versus NA/PC and SENB/PC
versus NA/SS. There were 3 pairs of SENB/PC and NA/PC
LRs and 4 pairs of SENB/PC and NA/SS. Mean LR values of
2.92, 2.51, and 2.47 were generated for glutaraldehyde for
SENB/PC, NA/PC, and NA/SS, respectively. For the
peracetic acid–hydrogen peroxide treatment, LR values of
3.06, 1.57, and 4.01 were observed for SENB/PC, NA/PC,
and NA/SS, respectively. Across both chemicals, the mean LR
differences for SENB/PC – NA/PC and SENB/PC – NA/SS
were 0.60 and –0.25, respectively. The differences in the mean
LR values were not statistically significant different from zero
(Table 16). For an equivalence zone where the difference in
means is no greater than ±1 (e.g., a =1), both NA/PC and
NA/SS were not statistically equivalent to SENB/PC at a
0.05 level of significance. However, there is little power to
1390 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
Table 14. Comparison of log reduction (LR) estimates for the low treatments combined
Medium/carrier combinationa Mean LR SDR pb
Mean difference and 90%
confidence limits
SENB/PC 5.90 0.535 — —
NA/PC 5.20 0.416 0.01 0.69 (0.29 to 1.09)
NA/SS 5.64 0.757 0.16 0.26 (–0.05 to 0.57)
a SENB/PC (soil extract nutrient broth/porcelain); NA/PC (amended nutrient agar/porcelain); and NA/SS (amended nutrient agar/stainless
steel).
b Comparing mean log reductions for SENB/PC to NA/PC and NA/SS.
Table 15. Mean log spores per carrier recovered for each medium/carrier type combination following exposure to
the low chemical treatments
Bleach Glutaraldehyde Peracetic acid–hydrogen peroxide
Medium/carrier combinationa Lab 2 Lab 3 Lab 2 Lab 3 Lab 2 Lab 3
SENB/PC LTDb 3.36 3.07 2.44 2.19 3.03
NA/PC LTD LTD 3.60 1.82 LTD 3.55
NA/SS LTD 2.10 3.82 2.64 0.09 3.29
a SENB/PC (soil extract nutrient broth/porcelain); NA/PC (amended nutrient agar/porcelain); and NA/SS (amended nutrient agar/stainless
steel).
b LTD = Less than detectable. For each of the 5 treated carriers, there were zero CFUs at every dilution.
show equivalence for these data because each 90% confidence
interval was very wide due to the small number of differences
being averaged. Based on the analysis of LR values generated
in this study, the current and modified methods are
comparable, thus providing additional information to support
NA and SS as official modifications to Method 966.04.
Wash-Off in Saline
Spore wash-off was investigated as an indicator of
differences in adhesion of spores to the carrier surface after
exposure to liquid. Three carriers of each medium/carrier
combination were exposed to 2 saline treatments (0.85% saline
and 0.85% saline + polysorbate 80). The saline + polysorbate
80 (final concentration 0.01%) was used to increase the
surfactancy of the saline, thus simulating an actual product.
Unlike the exposure of 5 carriers per 10 mL solution in efficacy
testing, carriers were analyzed separately in this study. Carriers
used in this study were selected from the same pool of carriers
used to conduct other studies in this collaborative.
The geometric mean percentage wash-off for the saline
treatment (4.7%) was larger than for saline + polysorbate
treatment (4.0%), but the ratio was not statistically
significantly different from 1.0 (P = 0.71) The 90%
confidence interval for the ratio of geometric means, S to
S + P, was (0.5, 2.8). Because there was no practical difference
between the treatments, subsequent analyses pooled the data
over S and S + P.
In this experiment, the amount of wash-off was low for the
3 medium/carrier combinations. Table 17 shows the statistical
summary of wash-off percentages for each medium/carrier.
The geometric mean percentage washed-off (averaged over S
and S + P and laboratories) was higher for SENB/PC (30%)
than for NA/PC (8%) and NA/SS (0.3%). The mean wash-off
for NA/SS was statistically significantly different from
SENB/PC (P = 0.007). The small amount of wash-off
observed for the NA/SS method is considered an
advantageous characteristic for a carrier-based test. Because
the mean wash-off percentages for all 3 methods were so low,
a formal equivalence test was not necessary.
The total spores used as the denominator in the percentage
washed-off calculation amounted to a 2-step calculation of the
number of spores per control carrier. It is of interest to
compare the counts of total spores in this special task to the
carrier counts observed for the main study. Table 18 shows the
statistical summary for the log total spores. The geometric
means of the total number of spores per carrier were 4.6  105
for SENB/PC, 1.3  105 for NA/PC, and 2.9  105 for NA/SS.
For an equivalence zone where the difference in means is no
greater than 1 (e.g., a = 1), both NA/PC and NA/SS were
statistically equivalent to SENB/PC at a 0.05 level of
significance. The limited data in this special task agree with
the main results in that the 3 medium/carrier methods attained
the target of 5 logs for the number of spores per control carrier.
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1391
Table 16. Statistical comparison of quantitative LR values for the 3 medium/carrier combinations; the LR were
paired by laboratory and treatment (low treatments of glutaraldehyde and peracetic acid–hydrogen peroxide)
Medium/carrier combinationa
Mean LR across
treatments and labs SDR pb
Mean difference and 90%
confidence limits
SENB/PC 2.99 0.553 — —
NA/PC 2.04 1.105 0.25 0.60 (–0.48 to 1.68)
NA/SS 3.24 1.666 0.71 –0.25 (–1.68 to 1.18)
a SENB/PC (soil extract nutrient broth/porcelain); NA/PC (amended nutrient agar/porcelain); and NA/SS (amended nutrient agar/stainless
steel).
b Comparison of mean LR values between SENB/PC and NA/PC, SENB/PC and NA/SS.
Table 17. Comparison of wash-off percentages for the medium/carrier combinations; all statistical calculations were
conducted using the log10-transformed wash-off percentages
Medium/carrier combination
Geometric mean wash-off percentage,
averaged across treatments and labs, % SDR pa
Ratio of geometric
means and 90%
confidence limits
SENB/PC 30 20.14 — —
NA/PC 8 16.42 0.28 4 (0 to 38)
NA/SS 0.3 0.56 0.007 89 (18 to 450)
a Two-tailed p-value for a paired t-test based on log10 percentages; the null hypothesis is that the ratio of geometric mean wash-off
percentages, SENB/PC to NA, equals 1.
Conclusions
The collaborative study was undertaken to compare the
current and modified methods for 966.04 and determine if the
methods are statistically equivalent. To generate data, a series
of comparative experiments were conducted on the set of core
modifications, i.e., use of NA for spore production and SS
carriers. Each experiment provided specific data relevant to
Method 966.04, and in combination, was used to determine
method equivalency. Also, new procedures were introduced
and evaluated, including a spore enumeration and
neutralization confirmation procedure. Both were necessary
to support the overall study and were under consideration as
additions to Method 966.04. On the basis of the results of this
study, it is recommended that NA, the spore enumeration
procedure, the target carrier count, and the neutralization
confirmation procedure be adopted as Official First Action
procedure modifications to Method 966.04.
The results of the HCl resistance, efficacy, and wash-off
studies demonstrate that NA in conjunction with the PC is
comparable to the current method, SENB/PC. The NAmethod
is simple, inexpensive, reproducible, and provides an ample
supply of high quality spores. Based on the titer of spore
suspensions (data not shown) and the associated control
carrier counts, the NA method is a highly effective sporulation
medium. Although the mean carrier counts for NA/PC were
determined to be significantly lower than SENB/PC
(difference of 0.61 logs), the counts were adequate (above
5 logs) and the difference was determined not to be of
practical importance. In addition, the SDR values were small
(range of 0.43 for NA/SS to 0.18 for NA/PC) for the control
carriers compared to other published antimicrobial tests.
Method 966.04 does not specify a standard number of spores
per carrier, maximum or minimum, or a procedure to
enumerate spores from carriers; both aspects are considered
major deficiencies. In this study, a spore enumeration
procedure was introduced and successfully utilized; the
procedure will augment Method 966.04 and allow the user to
monitor carrier counts. Furthermore, the establishment of a
mean spore titer per carrier was proposed (minimum of 5 logs
and a maximum of approximately 6 logs); the target spore
counts were shown to be obtainable. Currently, Method
966.04 does not provide a procedure to confirm neutralizer
effectiveness. A neutralization confirmation procedure that
simulates Method 966.04 was proposed and successfully
conducted by 2 laboratories in this study. SS, in conjunction
with NA, performed similarly to PC in the HCl resistance and
efficacy tests (although a trend for less stringency was noted
for SS); however, based on the wash-off studies, SS retained a
significantly higher number of spores than PC (wash-off of
0.3% for SS vs 30% for PC). Although the data support the
use of stainless steel for B. subtilis, due to the current use of
porcelain carriers for testing C. sporogenes, it is advisable to
retain the use of porcelain carriers until stainless steel can be
evaluated as a replacement carrier material for Clostridium.
The evaluation of stainless steel for Clostridium has been
initiated by the Study Director.
A First Action Modified Method 966.04, which includes
the recommendations discussed, is presented in this report.
The modified method also contains a series of editorial
revisions proposed by the Study Director. The editorial
changes are classified as minor changes. A minor change in a
procedural parameter is one that, in the judgment of the
General Referee and the Methods Committee, does not affect
the method’s performance and does not require new in-house
data, although literature or historical data may exist. Editorial
revisions include the following: (1) guidance on the safe
handling of pathogenic microorganisms; (2) use of reagent
grade water and commercial dehydrated media;
(3) procedures for the maintenance of B. subtilis stock cultures
and purity check; (4) intervals for time and temperature;
(5) recording results for both primary and secondary
subculture tubes as a carrier set; and (6) use of a certified timer
for managing timed activities. Based on its lack of usefulness,
a heat shock step for negative tubes is not incorporated into
the modified method.
Acknowledgments
This work was funded by the EPA Office of Research and
Development, National Homeland Security Research Center,
Safe Buildings Program. The statistical analysis was
supported by contract No. 68-W-02-050 between the EPA and
Montana State University. Maria Nelson, AOAC
INTERNATIONAL, assisted in the preparation of this
1392 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
Table 18. Comparison of log total spores for the medium/carrier combinations
Medium/carrier combination
Mean of log total
spores per carrier SDR pa
Difference between means
of log total spores and 90%
confidence limits
SENB/PC 5.66 0.11 — —
NA/PC 5.10 0.28 0.039 0.56 ( 0.18 to 0.94)
NA/SS 5.47 0.30 0.38 0.19 (–0.25 to 0.63)
a Two-tailed p-value for a paired t-test based on log10 percentages; the null hypothesis is that the ratio of geometric mean wash-off
percentages, SENB/PC to NA, equals 1.
document. Michele Cottrill, EPA, assisted in the preparation
of the collaborative study protocol.
The participation of the following collaborators is
gratefully acknowledged:
Anthony Garza, EPA, Office of Pesticide Programs
Microbiology Laboratory, Fort Meade, MD
Allison Rodriguez and Theresa To, FDA, Winchester
Engineering and Analytical Center, Winchester, MA
Marie Buen, Elizabeth Gonzales, Kevin Kallander, and
Patricia Stahnke, FDA, Denver District Laboratory, Denver, CO
Harriet Chan Myers, Advanced Sterilization Microbiology
Laboratory, Irvine, CA
References
(1) Official Methods of Analysis (2005) 18th Ed., AOAC
INTERNATIONAL, Gaithersburg, MD, Ch. 6, Method 966.04
(2) Danielson, J.W. (1993) J. AOAC Int. 76, 355–360
(3) Gangi, V.J., Leonard, I.E., & Rodriguez, A.A. (1997)
J. AOAC Int. 80, 1361–1367
(4) Miner, N.A., Armstrong, M., Carr, C.D., Maida, B., &
Schlotfeld, L. (1997) Appl. Environ. Microbiol. 63, 3304–3307
(5) Miner, N.A. (1999) J. AOAC Int. 82, 669–675
(6) Miner, N.A., Harris, V., Stumph, S., Cobb, A., & Ortiz, J.
(2004) J. AOAC Int. 87, 429–434
(7) McDonnell, G. (2003) J. AOAC Int. 86, 407–411
(8) McBride, G.B. (1999) Aust. N.Z. J. Statist. 41, 19–29
(9) Richter, S.J., & Richter, C. (2002) Qual. Eng. 14, 375–380
(10) Steiner, E.H. (1975) in Statistical Manual of the Association
of Official Analytical Chemists, W.J. Youden & E.H. Steiner
(Eds), AOAC INTERNATIONAL, Gaithersburg, MD
(11) Fleiss, J.L., Levin, B., & Paik, M.C. (2003) Statistical
Methods for Rates and Proportions, 3rd Ed., John Wiley &
Sons, Hoboken, NJ
(12) Newcombe, R.G. (1998) Stat. Med. 17, 2635–2650
(13) Newcombe, R.G. (1999) Stat. Med. 18, 3513
(14) Tango, T. (1998) Stat. Med. 17, 891–908
(15) Tango, T. (1999) Stat. Med. 18, 3511–3513
(16) DeVries, T., & Hamilton, M. (1996) Biometrics 52, 139–147
(17) Tilt, N., & Hamilton, M.A. (1999) J. AOAC Int. 82, 384–389
AOAC Official Method 966.04
Sporicidal Activity of Disinfectants
Revised First Action 2006
Method II
(Applicable to testing sporicidal activity of liquid
disinfectants using modified Method 966.04 against Bacillus
subtilis on a hard surface (porcelain carrier). Performance
criteria for product efficacy are not impacted. This method has
been validated for products containing sodium hypochlorite,
peracetic acid/hydrogen peroxide, and glutaraldehyde. See
results of the collaborative study supporting the modifications
to Method 966.04.)
Caution: (1) Perform all manipulations of the test organism
in accordance with biosafety practices stipulated
in the institutional biosafety regulations. Use the
equipment and facilities indicated for the test
organism. For recommendations on safe handling
of microorganisms refer to the CDC/NIH
Biosafety in Microbiological and Biomedical
Laboratories manual. (2) Disinfectants may
contain a number of different active ingredients,
such as heavy metals, aldehydes, peroxides, and
phenol. Use personal protective clothing or
devices during the handling of these items for
purpose of activation, dilution, or efficacy testing.
Use a chemical fume hood or other containment
equipment when appropriate during performing
tasks with concentrated products. Consult the
Material Safety Data Sheet for the specific
product/active ingredient to determine best course
of action. (3) References to water mean reagent
grade water, except where otherwise specified.
(4) Commercial dehydrated media made to
conform to the specified recipes may be
substituted. (5) These microbiological methods are
very technique-sensitive and technique-oriented;
thus, exact adherence to the method, good
laboratory practices, and quality control (QC) are
required for proficiency and validity of the
results. (6) Detergents used in washing glassware
may leave residues which are bacteriostatic. Test
for inhibitory residues on glassware periodically.
For procedure, refer to Standard Methods for the
Examination of Water and Wastewater, Section
9020, Quality Assurance/Quality Control.
A. Media and Reagents
(a) Culture Media.—(1) Nutrient broth.—For use in
preparing nutrient agar. Add 5 g beef extract (paste or
powder), 5 g NaCl, and 10 g peptone (anatone, peptic
hydrolysate of pork tissues, manufactured by American
Laboratories, Inc., 4410 S. 102nd St, Omaha, NE 68127) to
approximately 1 L water. Boil mixture for 20 min with
constant stirring. Readjust volume to 1 L with water and cool
to around 50C. Adjust pH to 6.8  0.2 with 1 N HCl or 1 N
NaOH, if necessary. Filter through paper (e.g., Whatman
No. 4). Dispense 10 mL portions into 20  150 mm culture
tubes or 20 mL portions into 25  150 mm culture tubes.
Dehydrated nutrient broth may be substituted; prepare
according to the manufacturer’s instructions. (2) Nutrient
agar.—For stock cultures slants. Add 1.5% (w/v) Bacto-agar
to unsterilized nutrient broth. Boil mixture until agar is
dissolved. Adjust pH to 7.2  0.2 if necessary. Dispense 5 mL
portions into 16  100 mm screw-cap tubes. Larger tubes may
be used as well. Autoclave for 20 min at 121C. Remove from
autoclave and slant tubes to form agar slopes. (3) Nutrient
agar with 5 g/mL MnSO4H2O (amended nutrient
agar).—For spore production. Suspend 11.5 g nutrient agar in
495 mL water, and add 5 mL 500 ppm MnSO4H2O. Dissolve
by boiling. Adjust pH to 6.8  0.2 if necessary. Autoclave for
15 min at 121C. Pour agar into plates. (4) Trypticase soy agar
(TSA).—Suspend 40 g dehydrated TSA in 1 L water and heat
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1393
gently while stirring. Boil 1 min or until completely dissolved.
Adjust pH to 7.3  0.2. Autoclave 15 min at 121C. Pour agar
into plates. (5) Fluid thioglycollate medium
(FTM).—Suspend 29.5 g dehydrated FTM in 1 L water. Heat
to boiling to dissolve completely. Adjust pH to 7.1  0.2 if
necessary. Dispense 10 mL portions into 20  150 mm culture
tubes and autoclave for 15 min at 121C. Store at room
temperature. Protect from light. Note: If after autoclaving the
aerated portion of media consumes more than one-third of
tube, media must be reboiled by placing tubes in beaker of
boiling water. Media can only be reboiled once. (6) FTM with
1 M NaOH (modified FTM).—For subculturing spores
exposed to 2.5 M HCl. Suspend 29.5 g FTM in 1 L water. Heat
boiling to dissolve completely. Cool and adjust pH to
7.1  0.2 if necessary. Add 20 mL 1 M NaOH, and mix well.
Check final pH and record (pH between 8 and 9 is typical).
Dispense 10 mL into 20  150 mm culture tubes and autoclave
for 15 min at 121C. Store at room temperature. Protect from
light. Note: If after autoclaving the aerated portion of media
consumes more than one-third of tube, media must be reboiled
by placing tubes in beaker of boiling water. Media can only be
reboiled once. Note: Media can be stored for up to 2 months.
(b) Manganese sulfate monohydrate.—500 ppm. Add
0.25 g MnSO4H2O to 500 mL water. Filter-sterilize for use.
(c) Dilute hydrochloric acid.—2.5 M. Use to determine
resistance of dried spores. Standardize and adjust to 2.5 M as
in 936.15 (see A.1.06).
(d) Sterile water.—Use reagent grade water. Reagent
grade water should be free of substances that interfere with
analytical methods. Any method of preparation of reagent
grade water is acceptable provided that the requisite quality
can be met. Reverse osmosis, distillation, and deionization in
various combinations all can produce reagent grade water
when used in the proper arrangement. See Standard Methods
for the Examination of Water and Wastewater for details on
reagent grade water.
(e) Triton X-100.
(f) Ethanol (40%).
(g) Test organism.—Bacillus subtilis (ATCC No. 19659)
obtained directly from a reputable supplier (e.g., ATCC).
B. Apparatus
(a) Carriers.—Penicylinders, porcelain, 8  1 mm od,
6  1 mm id, 10  1 mm length (available from CeramTec
Ceramic, PO Box 89, Laurens, SC 29360-0089,
www.ceramtec.com, Cat. No. LE15819).
(b) Glassware.—For disinfectant, 25 × 150 mm or
25  100 mm culture tubes (Bellco Glass Inc., Vineland, NJ;
reusable or disposable 20  150 mm (for cultures/subcultures);
16  100 mm screw-cap tubes for stock cultures. Cap with
closures before sterilizing. Sterilize all glassware 2 h in hot air
oven at 180C or steam-sterilize for a minimum of 20 min at
121C with drying cycle.
(c) Sterile centrifuge tubes.—Polypropylene, 15 mL
conical tubes with conical bottoms (Fisher Scientific,
Pittsburgh, PA; Corning, Inc., New York, NY), or equivalent.
(d) Water bath/chiller unit.—Constant temperature for test
chemical, capable of maintaining 20  1C or specified
temperature for conducting the test.
(e) Petri dishes.—Plastic (sterile).
(f) Filter paper.—Whatman No. 2; placed in Petri dishes
for storing carriers.
(g) Test tube racks.—Any convenient style.
(h) Inoculating loop.—Any convenient
inoculation/transfer loop for culture transfer.
(i) Wire hook.—For carrier transfer. Make 3 mm right
angle bend at end of 50–75 mm nichrome wire No. 18 B&S
gauge with other end in suitable holder.
(j) Centrifuge.—Nonrefrigerated (e.g., Eppendorf 5804 R).
(k) Sonicator.—Ultrasonic cleaner (e.g., Branson,
Danbury, CT; Model 1510).
(l) Orbital shaker.—Speed range from 25 to 500 rpm (e.g.,
VWR, Westchester, PA; DS-500).
(m) Vacuum desiccator.—For carrier storage. With
adequate gauge for measuring 27 in. (69 cm) of Hg and fresh
desiccant.
(n) Certified biosafety cabinet (BSC; Class I or II).— To
maintain aseptic work environment.
(o) Certified timer.—For managing timed activities; any
certified timer that can display time in seconds.
C. Operating Technique
(a) Culture initiation.—Initiate B. subtilis culture (e.g.,
use nutrient broth to rehydrate a lyophilized culture, and
incubate the broth culture for 24  2 h at 36  1C prior to
streak inoculation). Streak inoculate a set (e.g., 6) nutrient
agar slopes and incubate 24  2 h at 36  1C. Concurrently,
perform purity and identification confirmation testing for QC
(e.g., colony morphology on TSA, Gram stain, or other
identification systems). After incubation, store at 2–5C.
Maintain stock culture on nutrient agar slants by monthly
(30  2 days) transfers.
(b) Production of B. subtilis spore suspension.—Using
growth from a stock culture tube, inoculate 10 mL tubes (e.g.,
2 tubes, depending on the amount of spore preparation
desired) of nutrient broth and incubate tubes on an orbital
shaker for 24  2 h at ca 150 rpm at 36  1C. Use this culture
to inoculate amended nutrient agar plates. Inoculate each plate
with 500 L broth culture and spread the inoculum with a
sterile bent glass rod or suitable spreading device. Wrap each
plate with parafilm or place in plastic bags. Incubate plates
inverted for 12–14 days at 36  1C. After incubation, harvest
the spores by adding 10 mL cold sterile water to each plate.
Using a spreader (e.g., bent glass rod), remove growth from
plates, and pipet suspensions into 15 mL sterile conical tubes
(10 plates = 14 tubes, about 10 mL each). Centrifuge tubes at
5000 rpm for ca 10 min at room temperature. Remove and
discard supernatant. Resuspend pellet in each tube with 10 mL
cold sterile water and centrifuge at 5000 rpm for ca 10 min.
Remove and discard supernatant. Repeat twice. Resuspend
the pellet in each tube with 10 mL sterile water. Store the spore
suspension at 2–5C. Examine spore suspension with a phase
contrast microscope or by staining to assess quality of the
1394 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
spores. Examine a minimum of 5 fields and determine ratio of
spores to vegetative cells (or sporangia). Percentage of spores
vs vegetative cells should be at least 95%. Spore suspension
from multiple plates can be combined and re-aliquoted into
tubes for uniformity. Before inoculation of carriers, determine
spore titer of the concentrated spore suspension by plating
serial dilutions (e.g., 1.0  10–6 through 1.0  10–8) using pour
or spread plating on TSA plates. For pour plating, add molten
TSA tempered to 45–55C to each plate, swirl, and allow agar
to solidify. Incubate plates for 24  2 h at 36  1C and
determine titer. Note: When harvested and processed,
10 plates of amended nutrient agar should provide 80–100 mL
of concentrated spore suspension (ca 109 colony-forming
units (CFU)/mL). Diluting the suspension before carrier
inoculation will be necessary; a titer of
1.0  108–5.0  108 CFU/mL should be adequate to achieve
the target carrier count.
(c) Preparation of porcelain carriers.—Before use, examine
porcelain carriers individually and discard those with scratches,
nicks, spurs, or discolorations. Rinse unused carriers gently in
water 3 times to remove loose material, and drain. Place rinsed
carriers into Petri dishes matted with 2 layers of filter paper in
groups of 15 carriers per Petri dish. Sterilize 20 min at 121C.
Cool and store at room temperature. Note: Handle porcelain
carriers with care when placing in Petri dishes. Minimize carrier
movement and avoid excessive contact between carriers that
might result in chips and cracks. Wash carriers with Triton
X-100, and rinse with water 4 times for reuse.
(d) Inoculation of porcelain carriers.—Dilute the
concentrated spore suspension as necessary with sterile water
to achieve carrier counts between 1.0  105 and
ca 1.0  106 spores/carrier. Dispense 10 mL diluted spore
suspension into an appropriate number of 25  150 mm tubes.
Add 10 sterile carriers to each tube containing 10 mL spore
suspension, slightly agitate, and let stand 10–15 min. Remove
each carrier with sterile hook and place upright in sterile Petri
dish lined with 2 sheets of filter paper, no more than
30 carriers per Petri dish. Air-dry in BSC, B(n), for
ca 30  2 min. Place Petri dishes containing inoculated carriers
in vacuum desiccator containing CaCl2 and draw vacuum of
69 cm (27 in.) Hg. Dry carriers under vacuum for 24  2 h
before use in HCl resistance, efficacy testing, or carrier
counts. Maintain under vacuum for up to 3 months. Carriers
may be used after 3 months if they meet the acceptable HCl
resistance and carrier count criteria. Inoculated carriers should
not be used after 1 year of storage. Sterilize and reuse if
necessary [see C(c)].
(e) Spore enumeration (carrier counts).—Before use,
determine the carrier counts for each preparation of carriers.
Assay 3–5 randomly selected carriers per preparation. Place
each inoculated carrier into a 50 mL plastic, polypropylene
conical centrifuge tube containing 10 mL sterile water.
Sonicate carriers for 5 min  30 s. Note: For sonication, place
tubes into an appropriately sized beaker with tap water to the
level of sterile water in the tubes. Place beaker in sonicator so
that water level in the beaker is even with water level fill line
on sonicator tank. Fill tank with tap water to water level fill
line. Suspend beaker in sonicator tank so that it does not touch
bottom of tank and all 3 water levels (inside test tubes, inside
beaker, and sonicator tank) are the same. After sonification,
mix tubes in a Vortex mixer for 2 min  5 s. Dilute spore
suspensions by transferring 1 mL aliquots to tubes containing
9 mL sterile water. Dilute spore suspensions to 1.0  10–5 and
plate dilutions 1.0  10–2 through 1.0  10–5. Plate each
dilution in duplicate using pour or surface spread plating with
TSA. For pour plating, add molten TSA tempered to 45–55C
to each plate. Swirl pour plates to distribute spores evenly, and
allow agar to solidify. Invert plates and incubate for 24–48 h at
36  1C. Count colonies (by hand or with colony counter).
Use dilutions yielding between 30 and 300 CFU per plate
(target counts) for enumeration; however, record all counts
less than 30. Report plates with colony counts over 300 as too
numerous to count (TNTC ). Average spore counts per carrier
should be between 1.0  105 and approximately
1.0  106 spores/carrier. Do not use carriers with counts
outside this range.
(f) HCl resistance.—Equilibrate water bath to 20  1C.
Pipet 10 mL 2.5 M HCl into two 25  100 mm tubes, place in
water bath, and allow to equilibrate. Start timer and rapidly
transfer 4 inoculated penicylinders into an acid tube (2.5 M
HCl) with flamed hooks or forceps. Do not allow carriers or
transfer device to contact inside of wall of acid tube. Transfer
individual carriers after 2, 5, 10, and 20 min of HCl exposure
to a separate tube of modified FTM. Rotate each tube
vigorously by hand for ca 20 s and then transfer carrier to a
second tube of modified FTM. For viability control, place
1 unexposed inoculated carrier in a separate tube of modified
FTM. For media sterility, use 1 tube of modified FTM.
Incubate all test and control tubes for 21 days at 36  1C.
Record results as growth (+) or no growth (–) at each time
period. Spores should resist HCl for 2 min to be qualified as
resistant test spores. Discard carriers if not resistant and repeat
preparation of carriers as previously described.
(g) Efficacy test.–Aseptically prepare disinfectant test
samples as directed. Prepare all dilutions with sterile
calibrated volumetric glassware. For diluted products, use
1.0 mL or 1.0 g test disinfectant to prepare the use-dilution to
be tested. Use v/v dilutions for liquid products and w/v
dilutions for solids. For a 30 carrier test, place 10 mL product
at dilution recommended for use or under investigation into
each of six 25  150 mm or 25  100 mm test tubes, or use
appropriate number of tubes assuming 5 test carriers per tube
of test chemical. Place tubes in 20  1C water bath and let
equilibrate to temperature. Using a sterile hook (or forceps),
transfer inoculated carriers sequentially at 2 min intervals in
groups of 5 from Petri dish to test tubes containing sporicidal
agent. Use a certified timer to monitor time. Flame hook and
let cool after each transfer. When lowering carriers into test
tube, neither carriers nor wire hook may touch sides of tubes.
If interior sides are touched, note tube number. Do not count
carrier set if any carrier from that group of 5 yields a positive
result; test another set of 5 carriers. Carriers must be deposited
into test tubes within 5 s of the prescribed drop time. Return
tubes to water bath immediately after adding carriers. After
TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006 1395
contact period has been achieved, transfer carriers in same
sequential timed fashion into primary subculture tubes
containing appropriate neutralizer (10 mL in 20  150 mm test
tubes). Remove the carriers one at a time from the test tube
with sterile hook, tap against interior side of tube to remove
excess sporicidal agent, and transfer into neutralizer tube
(primary tube). All 5 carriers must be transferred during each
2 min interval. Flame hook between each carrier transfer.
Move remaining carriers into their corresponding neutralizer
tubes at appropriate time. Carriers may touch interior sides of
neutralizer tube during transfer, but minimize contact. After
each carrier is deposited, recap neutralizer tube and gently
shake to facilitate adequate mixing and efficient
neutralization. Within 1 h from when last carrier was
deposited into primaries, transfer carriers using sterile wire
hook to second subculture tube (secondary tube) containing
10 mL appropriate recovery medium, 1 carrier per tube. Move
carriers in order, but movements do not have to be timed.
Gently shake entire rack of secondary tubes after all carriers
have been transferred. Incubate primary (neutralizer) and
secondary subculture tubes for 21 days at 36  1C. Report
results as growth (+) or no growth (–). A positive result is one
in which medium appears turbid. A negative result is one in
which medium appears clear. Shake each tube prior to
recording results to determine presence or absence of
growth/turbidity. Primary and secondary subculture tubes for
each carrier represent a carrier set. A positive result in either
primary or secondary subculture tube is considered a positive
result for the carrier set.
Media sterility controls and system controls (check for
aseptic technique during carrier transfer process) are
recommended. For media controls, incubate 1–3 unopened
subculture medium tubes with the test sample tubes for
21 days at 36  1C. For system controls, use sterile forceps or
needle hooks to transfer 3 sterile carriers into a tube of test
chemical. Transfer system control carriers to neutralizer
medium as follows: at start of test (prior to first tube), transfer
1 sterile carrier to tube of neutralizer medium. After one-half
of test carriers have been transferred to neutralizer tubes,
transfer a second sterile carrier to tube of neutralizer medium.
After all test carriers (last tube) have been transferred to
neutralizer tubes, transfer third sterile carrier to tube of
neutralizer medium. Transfer system control carriers to
secondary subculture medium as follows: immediately before
initiating transfer of test carriers into secondary subculture
medium tubes, transfer first system control sterile carrier from
neutralizer medium to tube of subculture medium. After
one-half of test carriers have been transferred to secondary
subculture medium tubes, transfer second system control
sterile carrier to tube of subculture medium. After all test
carriers have been transferred to secondary subculture
medium tubes, transfer third system control sterile carrier to
tube of subculture medium. For each test, include a positive
carrier control by placing 1 inoculated carrier into tube of
secondary subculture medium. Incubate controls and test
sample tubes together for 21 days at 36  1C.
Perform identification confirmation on a minimum of three
positive carrier sets per test, if available, using Gram stain
and/or plating on TSA. Additional confirmation may be
performed using VITEK, API analysis, or comparable
method. If fewer than 3 positive carrier sets, confirm growth
from each positive carrier set. If both tubes are positive in a
carrier set, select only 1 tube for confirmatory testing. For test
with 20 or more positive carrier sets, confirm at least 20% by
Gram stain. If Gram stains are performed from growth taken
directly from positive tubes, the staining should be performed
within 5–7 days of conducting the efficacy test.
(h) Neutralization confirmation procedure.—A
neutralization confirmation test must be performed in advance
or in conjunction with efficacy testing. This assay is designed
to simulate the conditions (i.e., neutralizer, subculture
medium, contact time, diluent, concentration of test
substance) of the efficacy test and to demonstrate the recovery
of a low level of spores (e.g., 5–100). Diluted inoculum (e.g.,
spores of B. subtilis) is added directly to the various sets of
subculture media tubes (see Table 4). This assay provides for a
quantitative approach to assessing the effectiveness of the
neutralizer and any bacteriostatic action resulting from the
neutralizer itself or neutralizer–disinfectant interactions.
Produce a spore preparation according to the procedure for
amended nutrient agar. Harvest growth from plates (e.g.,
5 plates) per the method, except re-suspend pellet after final
centrifugation step in approximately 100 mL aqueous (40%)
ethanol. Determine spore count by serial dilution and plating
on TSA. Desirable target of the initial working suspension is
1.0  108 to 1.0  109 CFU/mL. The suspension may require
adjustment to reach target titer. Prepare serial 10-fold dilutions
of the inoculum in sterile water out to 10–8. Use 10–6, 10–7, and
10–8 dilutions to inoculate the neutralizer and subculture
media tubes. The target number of spores to be delivered per
tube in this assay is 5–100 per tube. Determine spore titer by
plating (spread plate or pour plate) each of 3 dilutions in
duplicate on TSA. Incubate plates inverted for 24–48 h at
36  1C. Count colonies (by hand or with colony counter).
Report plates with colony counts over 300 as TNTC. Note: A
standardized spore preparation adjusted to deliver
5–100 spores/mL may be substituted for the 3 dilutions of
spore inoculum. In addition, spores sheared from inoculated
carriers may be used as a working suspension.
Use 5 sterile porcelain carriers (only 3 to be used in the
assay). Within 5 s, place a set of 5 carriers into a test tube
(25  150 mm or 25  100 mm) containing test chemical;
transfer carriers according to (g). Allow carriers to remain in
test chemical per the specified contact time and temperature.
After the contact time is complete, aseptically transfer 3 of the
5 carriers individually into tubes containing the neutralizer per
(g). This set of tubes is the neutralizer/primary subculture
treatment. After transfering the last carrier into neutralizer
tube, transfer each carrier, in sequence, into a tube containing
secondary subculture medium. This portion of assay is not
timed, but should be made as soon as possible. This set is the
secondary subculture treatment. After carrier transfer,
inoculate each tube (neutralizer/primary and secondary
1396 TOMASINO & HAMILTON: JOURNAL OF AOAC INTERNATIONAL VOL. 89, NO. 5, 2006
subculture treatment tubes) with 1 mL of each of 3 inoculum
dilutions (10–6, 10–7, and 10–8). For controls, use 3 fresh,
unexposed tubes of neutralizer and 3 tubes of the secondary
subculture medium; also inoculate each control tube with
1 mL of each of 3 inoculum dilutions. Include 1 uninoculated
tube of neutralizer and secondary subculture media to serve as
sterility controls. See Table 4 for tube inoculation scheme.
Incubate all tubes 5–7 days at 36  1C. Record results as
growth (+) or no growth (–). Note: The lack of complete
neutralization of the disinfectant or bacteriostatic activity of
the neutralizer itself may be masked when a high level of
inoculum (spores) is added to the subculture tubes.
Confirm a minimum of one positive tube per treatment and
control (if available) using Gram staining and colony
morphology on TSA. For each treatment and control group,
conduct confirmation testing on growth from tube with fewest
spores delivered. B. subtilis is a Gram-positive rod, and colonies
on TSA are opaque, rough, dull, round, with irregular margins,
and low convex. Colonial variation may be observed and is
typical for this strain. Growth in the inoculated controls verifies
the presence of the spores, performance of the media, and
provides a basis for comparison of growth in the neutralizer and
subculture treatment tubes. Note: There may be cases when the
neutralizer is significantly different from the secondary
subculture media; in these cases, growth may not be comparable.
The uninoculated control tubes are used to determine sterility,
and must show no growth for the test to be valid.
The occurrence of growth in the neutralizer/primary
subculture and secondary subculture treatment tubes is used to
assess the effectiveness of the neutralizer. No growth or
growth only in tubes which received a high level of inoculum
(e.g., the dilution with plate counts which are too numerous to
count) indicates poor neutralization and/or presence of
bacteriostatic properties of the neutralizer or
neutralizer-disinfectant interactions. For a neutralizer to be
deemed effective, growth must occur in the Secondary
Subculture treatment tubes which received lower levels of
inoculum (e.g., 5–100 CFU/mL). Growth in the secondary
subculture inoculated control verifies the presence of the
spores, performance of the media, and provides a basis for
comparison of growth in the neutralizer and subculture
treatment tubes. No growth or only growth in tubes which
received high levels of inoculum (e.g., a dilution with plate
counts which are too numerous to count) indicates poor media
performance. Growth in the neutralizer-primary inoculated
control should be comparable to the secondary subculture
inoculated control if the neutralizer is the same as the
secondary subculture media. There may be cases when the
neutralizer is significantly different from the secondary
subculture media. In these cases, growth may not be
comparable to the secondary subculture inoculated control.
The neutralizer-primary and secondary subculture
uninoculated control tubes are used to determine sterility, and
must show no growth for the test to be valid.
Note: For product registration, the EPA requires the
following to demonstrate sporicidal/sterilant-level efficacy:
Using AOAC Method 966.04, 60 carriers representing each of
2 types of surfaces (porcelain penicylinders and silk suture
loops) must be tested separately against spores of both
Bacillus subtilis (ATCC 19659) and Clostridium sporogenes
(ATCC 3584) on 3 test samples representing 3 different
batches of product, one of which must be at least 60 days old
(2 carrier types  2 test microorganisms  60 carriers/type =
240 carriers per batch sample; 3 product batches 
240 carriers/batch = total of 720 carriers). The product must
kill all of the test spores on all of the 720 carriers without any
failures.
References: J. AOAC Int. 89, 1373 (2006).
ASTM International Method E 1054–Standard Test Methods
for Evaluation of Inactivators of Antimicrobial Agents (2002)
American Society for Testing and Materials, Conshohocken, PA.
Biosafety in Microbiological and Biomedical Laboratories
(1994) 4th Ed., U.S. Department of Health and Human
Services, Centers for Disease Control and Prevention and
National Institutes of Health, Washington, DC.
Standard Methods for the Examination of Water and
Wastewater (1995) 21st Ed., American Public Health
Association, Washington, DC.
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