US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
AOAC Use Dilution Method for Testing Disinfectants
SOP Number: MB-05-07
Date Revised: 08-18-09
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SOP No. MB-05-07
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EPA/OPP MICROBIOLOGY LABORATORY
ESC, Ft. Meade, MD
Standard Operating Procedure
for
AOAC Use Dilution Method for Testing Disinfectants
SOP Number: MB-05-07
Date Revised: 08-18-09
Initiated By:
Print Name:
Date: / /
Technical Review:
QA Review:
Approved By:
Print Name:
Technical Staff
Print Name:
QA Officer
Print Name:
Branch Chief
Date:
Date: / /
Date: / /
Date Issued: / /
Controlled Copy No.:
Withdrawn By:
Date:
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TABLE OF CONTENTS
Contents Page Number
1.0 SCOPE AND APPLICATION 3
2.0 DEFINITIONS 3
3.0 HEALTH AND SAFETY 4
4.0 CAUTIONS 4
5.0 INTERFERENCES 5
6.0 PERSONNEL QUALIFICATIONS 6
7.0 SPECIAL APPARATUS AND MATERIALS 6
8.0 INSTRUMENT OR METHOD CALIBRATION 8
9.0 SAMPLE HANDLING AND STORAGE 8
10.0 PROCEDURE AND ANALYSIS 8
11.0 DATA ANALYSIS/CALCULATIONS 19
12.0 DATA MANAGEMENT/RECORDS MANAGEMENT 19
13.0 QUALITY CONTROL 19
14.0 NONCONFORMANCE AND CORRECTIVE ACTION 20
15.0 REFERENCES 20
16.0 FORMS AND DATA SHEETS 21
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1.0 SCOPE AND APPLICATION:
1.1 This SOP describes the Use-dilution methodology used to determine the efficacy
of disinfectants against three organisms, Pseudomonas aeruginosa,
Staphylococcus aureus, and Salmonella enter ica, on hard surfaces. The
methodology is based on AOAC methods 955.15 (Testing Disinfectants against
Staphylococcus aureus), 964.02 (Testing Disinfectants against Pseudomonas
aeruginosa), and 955.14 (Testing Disinfectants against Salmonella cholerasuis)
which have been officially modified (editorial modifications) - see references
15.1 and 15.2.
1.2 For product evaluations under the Antimicrobial Testing Program (ATP), a study
protocol is developed which identifies the specific test conditions for a product
sample such as contact time, dilutions, neutralizers, etc.
2.0 DEFINITIONS:
2.1 AOAC = AOAC INTERNATIONAL
2.2 ATCC = American Type Culture Collection
2.3 TSA = trypticase soy agar
2.4 TSB = trypticase soy broth
2.5 NB = nutrient broth
2.6 NA = nutrient agar
2.7 MSA = mannitol salt agar
2.8 CTA = cystine trypticase agar
2.9 OD = outside diameter
2.10 ID = inside diameter
2.11 CFU = colony forming unit
2.12 References to water mean reagent-grade water, except where otherwise specified.
2.13 Dilution blanks = tubes of phosphate buffered dilution water (PBDW)
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3.0 HEALTH AND SAFETY:
3.1 All manipulations of the test organisms are required to be performed in
accordance to biosafety practices stipulated in SOP MB-01, Lab Biosafety.
3.2 Disinfectants may contain a number of different active ingredients, such as heavy
metals, aldehydes, peroxides, phenol, etc. Personal protective clothing or devices
are recommended during the handling of these items for the purpose of activation,
dilution, or efficacy testing. A chemical fume hood or other containment
equipment is employed when performing tasks with concentrated products. The
study analyst may wish to consult the Material Safety Data Sheet for the specific
product/active ingredient to determine the best course of action.
4.0 CAUTIONS:
4.1 Use aseptic techniques to prevent contamination.
4.2 Media indicated in sections 10.1.1.2 and 10.1.1.3 for rehydrating lyophilized
cultures are specified on the ATCC Product Information Sheet that accompanies
each organism. Upon purchase of new organisms, verify that media requirements
have not changed by checking the new ATCC Product Information Sheet.
4.3 The volume of dilution blanks, neutralizer tubes, and subculture tubes will be
verified in advance and adjusted accordingly.
4.4 Strict adherence to the protocol is necessary for the validity of the test results.
4.5 Use inoculated carriers for determining carrier counts and performing efficacy
testing as soon as possible after drying on the day of preparation to avoid a
reduction in microbial titer. Overnight or long term storage of inoculated carriers
is not allowed.
4.6 Complete dilution plating within 2 hours after the completion of carrier sonication
or vortexing. If the serial dilutions are not made and plated immediately, the
sonicated tubes are kept at 2-5°C until this step can be done.
4.7 For spread plating: ensure that the entire surface of the agar plate is dry before
adding inoculum. If necessary, leave the agar plates uncovered in the biological
safety cabinet (BSC) until the moisture has been completely absorbed into the
medium.
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4.8 To ensure the stability of a diluted product, prepare the dilutions within three
hours of the disinfectant treatment step unless specified otherwise.
4.9 Use appropriate aseptic techniques for all test procedures involving the
manipulation of the test organisms and associated test components.
4.10 These microbiological methods are very technique-sensitive and technique-
oriented; thus, exact adherence to the method, good laboratory practices, and
quality control are required for proficiency and validity of the results.
4.11 Detergents used in washing glassware may leave residues which are
bacteriostatic. Test for inhibitory residues on glassware periodically according to
SOP QC-03, Glass Washing and Detergent Residues Test.
4.12 The primary subculture medium should serve as a suitable neutralizer for the test
substance as well as an adequate growth medium which must be confirmed in
advance or concurrently with the use dilution test.
4.12.1 See SOP MB-17, Neutralization Confirmation, for the procedure to
determine suitability of the neutralizer (primary subculture tube) for
the test substance.
4.12.2 See SOP QC-11, Performance Assessment and Sterility Verification,
to determine whether or not the subculture medium is an adequate
growth medium.
5.0 INTERFERENCES:
5.1 Contamination of stock cultures will negatively impact disinfectant efficacy
testing. It is critical to maintain the highest standards of good laboratory practices
and aseptic technique during all manipulations and handling of stock cultures.
5.2 Avoid touching the interior sides of the medication tube while the carriers are
being lowered into the disinfectant agent and the hook is being removed. Contact
with the interior sides of the medication tube may cause adhesion of bacterial
cells which are not in contact with the disinfectant. This may result in re-
inoculation of the carriers with organism as they are being removed from the
medication tube. Re-inoculation of the carriers with organism can lead to false
positive results.
5.3 Contaminated plates will interfere with the recording of carrier count results.
Visually inspect all agar plates prior to use - discard any plates with evidence of
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contamination. For contamination following the incubation phase, if atypical
colonies or contamination are evident that interfere with the enumeration of the
test organism, record as a contaminant(s). Data from other dilutions, if the CPUs
result in a countable range, may be used to calculate the final CPU/carrier.
6.0 PERSONNEL QUALIFICATIONS:
6.1 Personnel are required to be knowledgeable of the procedures in this SOP.
Documentation of training and familiarization with this SOP can be found in the
training file for each employee.
6.2 The laboratory staff shall confirm (i.e., documentation in the training file of
familiarization with the SOP) that they can properly perform the procedure before
commencing work. If the standard AOAC method changes, confirmation shall be
repeated.
7.0 SPECIAL APPARATUS AND MATERIALS:
7.1 Test organisms. Pseudomonas aeruginosa (ATCC No. 15442), Staphylococcus
aureus (ATCC No. 6538) and Salmonella enterica (ATCC No. 10708) obtained
directly from a reputable supplier (e.g., ATCC).
7.2 Culture media (e.g., nutrient agar). Note: Commercial dehydrated media made to
conform to the recipes provided in AOAC Methods 955.15, 964.02, and 955.14
may be substituted.
NOTE: The use of synthetic broth is stipulated in the official AOAC methods for
test culture preparation; however, it is rarely used by the laboratory and only used
upon request.
7.2.1 Nutrient broth: Boil 5 g beef extract (paste or powder), 5 g NaCl,
and 10 g peptone (anatone) in 1 L H2O for 20 minutes and dilute to
volume with H2O; adjust to pH 6.8 ± 0.1. Filter through paper
(Whatman No. 4, or equivalent), place 10 mL portions in 20 x 150 mm
test tubes, and steam sterilize 20 min at 121°C. Use this broth for
daily transfers of test cultures.
7.3 Subculture media (e.g., letheen broth, fluid thioglycollate medium). Note:
Commercial dehydrated media made to conform to the recipes provided in AOAC
Methods 955.15, 964.02, and 955.14 may be substituted.
7.4 Trypticase soy agar (TSA). Plating medium for carrier enumeration.
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7.5 Sterile water. Use reagent-grade water 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 and SOP QC-01, Quality Assurance of
Purified Water for details on reagent-grade water.
7.6 Carriers. Polished stainless steel cylinders, 8 ± 1 mm OD, 6 ± 1 mm ID, 10 ± 1
mm length; type 304 stainless steel, SS 18-8 (S & L Aerospace Metals, Maspeth,
NY or Fisher Scientific catalog number 07-907-5Q as of December 2008).
7.7 Glassware. For disinfectant, use autoclavable 25 x 100 mm tubes (Bellco Glass
Inc., Vineland, NJ). For cultures/subcultures, use autoclavable reusable or
disposable 20 x 150 mm tubes. For stock cultures, use 16 x 100 mm screw cap
tubes. Cap tubes with closures before sterilizing. Sterilize all glassware in hot air
oven at 180°C or steam sterilize for a minimum of 20 minutes at 121°C with
drying cycle.
7.8 Water bath/chiller unit. Constant temperature for test chemical, capable of
maintaining 20 ± 1°C temperature or specified temperature for conducting the
test.
7.9 Test tube racks. Any convenient style.
7.10 Transfer loops. 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 19380, USA). Fit other end in suitable
holder. Bend loop at 30° angle with stem. Volumetric transfer devices may be
used instead of transfer loops (e.g., micro volume pipet).
7.11 Wire Hook. For carrier transfer. Make 3 mm right angle bend at end of 50-75
mm nichrome wire No. 18 B&S gage. Place other end in suitable holder.
7.12 Timer. For managing timed activities, any certified timer that can display time in
seconds.
7.13 Sonicator (ultrasonic cleaner). For carrier counts.
7.14 Gram stain kit.
7.15 VITEK 2 Compact. For the automated identification of microorganisms.
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7.16 VITEK 2 Compact Identification Cards. Gram negative (GN) and Gram positive
(GP).
8.0 INSTRUMENT OR METHOD CALIBRATION:
8.1 Refer to the laboratory equipment calibration and maintenance SOPs (SOP EQ
series) for details on method and frequency of calibration.
9.0 SAMPLE HANDLING AND STORAGE:
9.1 Follow appropriate chain-of-custody (COC) guidelines during testing as
stipulated in SOP COC-01, Sample Log-in and Tracking.
9.2 Disinfectants are stored according to manufacturers' recommendations or at room
temperature if the product label or testing parameters do not identify a storage
temperature. Those disinfectants requiring activation or dilution prior to use will
only be activated or diluted within three hours of testing unless test parameters
specify otherwise.
10.0 PROCEDURE AND ANALYSIS:
10.1 Culture Initiation, Maintenance and Quality Control.
10.1.1 Culture Initiation.
10.1.1.1 Every 12 months (or sooner if the quality of the stock
culture is compromised) initiate new stock cultures from
lyophilized cultures of Pseudomonas aeruginosa (ATCC
15442), Staphylococcus aureus (ATCC 6538), and
Salmonella enterica (ATCC 10708) from ATCC or other
reputable supplier.
10.1.1.2 Open ampule of freeze dried organism as indicated by
ATCC.
10.1.1.3 Using a tube containing 5-6 mL of TSB, aseptically
withdraw 0.5 to 1.0 mL and rehydrate the pellet for
Staphylococcus aureus and Pseudomonas aeruginosa.
Using a tube containing 5-6 mL of NB, aseptically
withdraw 0.5 to 1.0 mL and rehydrate the pellet for
Salmonella enterica.
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10.1.1.4 Aseptically transfer the entire rehydrated pellet back into
the original tube of broth (TSB for S. aureus and P.
aeruginosa, NB for S. enterica) designated as "TUBE A,"
(see Attachment 2). Mix well.
10.1.1.5 Streak for isolation using a loopful of rehydrated
suspension on duplicate plates - use TSA for S. aureus and
P. aeruginosa, use NA for S. enterica.
10.1.1.5.1 In addition for S. aureus and P. aeruginosa,
streak a loopful of rehydrated suspension
onto both MSA and Cetrimide agar.
Selective media is not used for S. enterica.
10.1.1.6 Incubate broth culture (TUBE A) and plate cultures at 36 ±
1°C for 24 ± 2 hours for S. aureus, P. aeruginosa, and S.
enterica.
10.1.1.7 Record all manipulations on the Organism Culture
Tracking Form (see 16.1).
10.1.2 Culture Identification and Quality Control.
10.1.2.1 Initial confirmation testing for quality control (QC) will be
performed using the 24 ± 2 hour NA or TSA plates from
step 10.1.1.5.
10.1.2.2 Following the incubation period (as stated in 10.1.1.6),
record the colony morphology as observed on the NA or
TSA plates and selective media plates (including the
absence of growth) and stain reaction. See Attachment 1
for details on cell and colony morphology, colony
characteristics on selective media, and stain reactions.
10.1.2.2.1 For*?, aureus, note the organi sm' s growth
characteristics on MSA (colony size, color,
texture, etc.) and Cetrimide (absence of
growth). For P. aeruginosa, note the
organism's growth characteristics on
Cetrimide (colony size, color, texture, etc.)
and MS A (absence of growth). Check for
consistency with the genus and species of
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the organism to be tested (round, shiny, and
yellow for S. aureus on MSA, and flat,
greenish-yellow, and opaque for P.
aeruginosa on Cetrimide).
10.1.2.2.2 For each organism, perform a Gram stain
from growth taken from the TSA or NA
plates. Perform the Gram stain according to
the manufacturer's instructions. Observe
the Gram reaction by using brightfield
microscopy at 1000* magnification (oil
immersion).
10.1.2.3 Perform VITEK™ analysis according to the manufacturers'
instructions.
10.1.2.4 Record all confirmation results on the Test Microbe
Confirmation Sheet (Quality Control) (see 16.2).
10.1.3 Generation of Stock Cultures.
10.1.3.1 Use the 24 ± 2 hour TUBE A (see Attachment 2) broth
culture discussed in 10.1.1.4 to initiate stock cultures.
10.1.3.2 For S. aureus and S. enter ica, streak six nutrient agar slants
each. For P. aeruginosa, stab six CTA tubes.
10.1.3.3 Incub ate the S. aureus and S. enterica si ants and P.
aeruginosa stabs at 36 ± 1°C for 48 ± 2 hours.
10.1.3.4 Following incubation, store the cultures at 2-5°C for 30 ± 2
days. These cultures are identified as the "stock cultures."
Begin stock culture transfers as outlined in section 10.1.5.
Repeat the cycle for a maximum of one year.
10.1.3.5 From a set of six stock cultures, one is used every 30 ± 2
days for QC and to generate new stock cultures, four may
be used per month (one/week) for generation of test
cultures, (see SOP MB-05, Use Dilution Method; and SOP
MB-06, Testing Spray Disinfectants) and one is a back-up
tube.
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10.1.4 Monthly QC of Stock Cultures.
10.1.4.1 Conduct monthly QC of stock cultures. Use one
refrigerated stock culture tube and streak a loopful on a
plate of TS A. For S. aureus and P. aeruginosa, streak a
loopful onto both selective media (MSA and Cetrimide), as
noted in section 10.1.1.5.
10.1.4.2 Incubate the plates at 36 ± 1°C for 24 ± 2 hours (18-24
hours for use in the VITEK 2 Compact). Follow steps
outlined in section 10.1.2.2 to confirm the identity of the
organism.
10.1.5 Culture Maintenance.
10.1.5.1 Every 30 ± 2 days inoculate a new set of stock culture
tubes from a current stock culture tube. Use the same
refrigerated stock culture tube used for Monthly QC
described in 10.1.4.1 to inoculate 6 new stock cultures
tubes as outlined in 10.1.3.2.
10.1.5.2 Incubate the new stock cultures as indicated in 10.1.3.3.
10.1.5.3 Following the incubation period, store the stock cultures at
2-5°C for 30 ± 2 days.
10.2 Test Culture Preparation:
10.2.1 Initiate test culture by inoculating a 10 mL tube (20 x 150 mm) of
nutrient broth from a stock slant or stab culture. Transfer one 4 mm
ID loopful (or use a 10 jiL certified transfer loop) of inoculum from
the stock culture into the broth.
10.2.2 Two sets of cultures (one set as a backup) of the same organism may
be initiated in parallel from the same stock culture and subcultured;
however, only one set of the final cultures is used for actual testing.
10.2.3 Make at least 3 consecutive 24 ± 2 hour transfers (use one 4 mm ID
loopful, or a 10 jiL certified transfer loop, or a calibrated micro
volume pipet to deliver 10 jiL) in 10 mL nutrient broth or synthetic
broth incubated at 36 ± 1°C. For the purposes of conducting the Use-
dilution method, the lab allows 5-7 daily transfers. If only one of the
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consecutive 24 hour transfers has been missed, it is not necessary to
repeat the previous 3-day sequence prior to the inoculation of the 48-
54 hour test culture.
10.2.4 For the final subculture step, inoculate for the test procedure, a
sufficient number of 25 x 150 mm tubes (e.g., eight to ten) containing
20 mL nutrient broth; incubate 48-54 hours at 36 ± FC.
10.2.5 A minimum of five days are required to obtain the culture for
inoculating carriers. For example, the culture sequence must begin on
Thursday for testing to commence on the following Tuesday.
10.2.6 Record all culture transfers on the Organism Culture Tracking Form
(see 16.1).
10.3 Carrier Inoculation for S. aureus, P. aeruginosa, and S. enterica:
10.3.1 A single test involves the evaluation of 60 inoculated carriers (one
organism) against one product sample. In addition to the 60 carriers, 6
carriers are required to estimate carrier bacterial load and a minimum
of 6 more are included as extras. Thus, a minimum of 72 inoculated
carriers are required to perform a single test.
10.3.2 For S. aureus and S. enterica, using a Vortex-style mixer, mix 48-54
hour nutrient broth test cultures 3-4 seconds and let stand 10 minutes
at room temperature before continuing. Remove the upper portion of
each culture (e.g., upper 3A or approximately 15 mL), leaving behind
any debris or clumps, and transfer to a sterile flask; pool cultures in the
flask and swirl to mix. Aliquot 20 mL portions into sterile 25 x 150
mm test tubes. Prepare a minimum of four tubes.
10.3.3 For P. aemginosa, do not shake 48-54 hour test culture. The pellicle
from the 48-54 hour cultures must be removed from the broth before
mixing on a Vortex mixer by gently aspirating the broth away from the
pellicle using a pipette. Any disruption of the pellicle resulting in
dropping, or breaking up of the pellicle in culture before or during its
removal renders that culture unusable in the use-dilution test. Visually
inspect the culture for signs of pellicle. Discard tubes with fragments
of pellicle. Using a vortex-style mixer, mix nutrient broth test cultures
3-4 seconds and let stand 10 minutes at room temperature before
continuing. Remove the upper portion of each culture (e.g., upper 3A
or approximately 15 mL), leaving behind any debris or clumps, and
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transfer to a sterile flask; pool cultures in the flask and swirl to mix.
Aliquot 20 mL portions into sterile 25 x 150 mm test tubes. Prepare a
minimum of four tubes.
10.3.4 If organic burden is required for testing, the appropriate amount of
organic burden is added to the pooled test culture prior to the
inoculation of carriers. For a 5% v/v preparation, add 1 mL organic
burden per 19 mL pooled test culture (e.g., add 5 mL organic burden
to 95 mL pooled test culture). Swirl to mix. Aliquot 20 mL portions
into sterile 25 x 150 mm test tubes. Prepare a minimum of four tubes.
10.3.5 Use only carriers that have been physically screened, have passed
bioscreening, and have been appropriately prepared (see SOP MB-03,
Screening Carriers).
10.3.6 Using a sterile hook, aseptically transfer 20 carriers prepared as
described above into each of the tubes containing the test culture.
Drain the water from the carriers by tapping them against the side of
the tube before transferring. Multiple carriers may be transferred on a
single wire hook. The test culture must completely cover the carriers.
If a carrier is not covered, gently shake the tube, or reposition the
carrier within the tube with a sterile wire hook. Be sure to inoculate a
sufficient number of carriers for the test. (Alternately, the water may
be siphoned off the carriers and the 20 mL test culture added directly
to the carriers without transferring).
10.3.7 After 15 ± 2 min contact period, remove carriers using flamed
nichrome wire hook; shake carrier vigorously against side of the tube
to remove excess culture, and place on end in vertical position in
sterile Petri dish matted with 2 layers of Whatman No. 2 (or
equivalent) filter paper, making sure that carriers do not touch to
prevent improper drying. Place no more than 12 carriers in a Petri
dish. Carriers that touch or fall over cannot be used for testing and
must be removed and recleaned. Once all of the carriers have been
transferred, cover and place in incubator at 36 ± 1°C and let dry 40 ± 2
min.
10.3.8 Record the timed carrier inoculation activities on the Time Recording
Sheet for Carrier Inoculation Steps (see 16.3).
10.4 Enumeration of bacterial inocula (carrier counts):
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10.4.1 Synchronize the carrier count assay and efficacy testing. Assay
carriers for carrier counts (sonicated) within 2 hours of drying. Record
time of sonication on Serial Dilution/Plating Tracking Form (16.8).
10.4.2 One carrier is randomly extracted from each of 6 Petri dishes (12
carriers/dish).
10.4.3 Place each inoculated carrier into a tube containing 10 mL of letheen
broth.
10.4.4 Place all tubes with carriers into an appropriately sized beaker and fill
the beaker with tap water to the level of letheen broth in the tubes.
10.4.4.1 Hold the beaker in the sonicator so that the water level in
the beaker is even with the water level fill line on the
sonicator tank and fill the tank up with tap water to the
water level fill line. Be sure that the water level in the tank
never falls below one inch from the top of the tank.
10.4.4.2 Hold the beaker in the sonicator tank so that it is not
touching the bottom and that all three liquid levels (inside
the test tubes, inside the beaker and the sonicator tank) are
the same.
10.4.4.3 Using an official timer, sonicate carriers for 60 ± 5
seconds.
10.4.5 Serial ten-fold dilutions of the sonicated carrier tubes are made in 9
mL dilution blanks (see 16.8).
10.4.6 If the serial dilutions are not made and plated immediately, the
sonicated tubes are kept at 2-5°C until this step can be done.
Complete the dilutions and plating within 2 hours after sonication.
10.4.7 Plate 0.1 mL aliquots of appropriate dilutions in duplicate on ISA
using pour or surface spread plating. Briefly vortex (1-3 sec.) each
serial dilution tube prior to plating.
Note: Dilutions 10"2 through 10"4 should produce plates with CPU in
the appropriate range.
10.4.8 If the spread plate method is used for bacterial enumeration, ISA
plates are prepared in advance and are refrigerated until needed.
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10.4.8.1 Allow refrigerated plates to come to room temperature
prior to use. To spread dilutions evenly over the dry
surface of the agar, use a glass, autoclavable or disposable
spreading rod and plate spinner until the surface is
completely dry.
10.4.9 If the pour plate method is used for bacterial enumeration, the ISA is
prepared and tempered after autoclaving (approx. 1 hr) to 45-50°C in a
water bath prior to use. Tempered agar is added to each plate after the
addition of the appropriate dilution and swirled to uniformly disperse
the inoculum.
10.4.10 Incubate plates at 36 ± 1°C for 24-48 hrs.
10.4.11 Colonies may be counted by hand or with the aid of a plate counter.
Plates that have colony counts over 300 will be reported as TNTC.
Record counts on the Carrier Count Data Sheet (see 16.9). See section
11 for data analysis.
10.5 Disinfectant Sample Preparation.
10.5.1 Prepare disinfectant sample per SOP MB-22.
10.5.2 Equilibrate the water bath and allow it to come to 20 ± 1°C or the
temperature specified (±1°C). Prepare the disinfectant dilutions within
3 hours of performing the assay unless test parameters specify
otherwise. Ready-to-use products are tested as received; no dilution is
required.
10.5.3 Dispense 10 mL aliquots of the diluted disinfectant or ready-to-use
product into 25 x 100 mm test tubes, one tube per carrier. Place tubes
in the equilibrated water bath for approximately 10 minutes to allow
test solution to come to specified temperature. Record the temperature
of the water bath and recirculating chiller before and after testing on
the AOAC Use-Dilution Test Information Sheet (see 16.5).
10.6 Test Procedure:
10.6.1 After the required drying time, the carriers are sequentially transferred
from the Petri dish to the test tubes containing the disinfectant at 30
second intervals or at the pre-determined drop interval for products
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with contact times less than 10 minutes. When lowering the carriers
into the disinfectant tubes, neither the carrier itself nor the tip of the
wire hook can touch the interior sides of the tube. (Individual
manipulation of the carriers is required.) Use a certified timer to time
the transfers. Modify intervals to accommodate exposure times other
than 10 min.
Note: Above step is one of the most critical, technique-sensitive areas
of method. False positives can result from transfer of live organisms
to sides of tubes due to contact or aerosol formation. If the side is
touched, mark or note the tube; the tube is not counted if it yields a
positive result.
10.6.2 One carrier is added per tube. Immediately after placing carrier in the
test tube, briefly swirl tube before placing it back in the bath. For a
contact time often minutes, the carrier must be deposited in the tube
within ± 5 seconds of the prescribed drop time. For contact times of
less than ten minutes, the analyst will work with the team leader and
senior scientist to identify an appropriate (i.e., shorter) drop interval.
Using alternating hooks, flame-sterilize the hook and allow it to cool
after each carrier transfer.
10.6.3 After the carriers have been deposited into the disinfectant, and the
exposure time is complete, the carriers are then transferred in a
sequentially timed fashion into the primary subculture tubes
containing the appropriate neutralizer (10 mL in 20 x 150 mm tubes).
As with the transfers to the disinfectant tubes, transfers into subculture
tubes are to be done within ± 5 seconds (see section 10.6.2) of the
actual transfer. The carrier is removed from the disinfectant tube with
a sterile hook, tapped against the interior sides of the tube to remove
the excess disinfectant and transferred into the subculture tube. Avoid
contact of the carrier to the interior sides of the subculture tube during
transfer. Flame-sterilize the hook after each carrier transfer.
10.6.4 After the carrier is deposited in the subculture tube, recap the
subculture tube and shake thoroughly. Place subculture tubes into 36 ±
1°C incubator.
10.6.5 After a minimum of 30 ± 5 minutes from the end of the transfer into
primary subculture tubes, remove tubes from the incubator and
transfer carrier from the primary tube to a secondary tube of sterile
medium.
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SOP No. MB-05-07
Date Revised 08-18-09
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10.6.5.1 Transfer the carriers using a sterile wire hook to a second
subculture tube containing 10 mL of the appropriate
subculture medium which may contain a suitable
neutralizer. Move the carriers in order but the movements
do not have to be timed. Thoroughly shake the subculture
tubes after all of the carriers have been transferred.
10.6.6 Check all test tube racks for proper transfer of carriers (i.e., carriers
are in the correct tubes, no tubes have two carriers) before completing
the testing day.
10.6.7 Incubate both the primary and secondary subculture tubes 48 ± 2 hours
at36± 1°C.
10.6.8 Record timed events on the Time Recording Sheet for Carrier Transfer
Form (see 16.4).
10.7 Viability controls. On testing day, place a dried inoculated carrier into a tube
containing 10 mL primary subculture medium and a second dried, inoculated
carrier into a tube containing 10 mL secondary subculture medium. Incubate
tubes for 48 ± 2 hours at 36 ± 1°C. Positive growth in both tubes validates the
test system. Failure to have growth in either of the tubes invalidates the test.
10.8 Results:
10.8.1 Report results as + (growth), or 0 (no growth) as determined by
presence or absence of turbidity, on the AOAC Use-dilution Results
Sheet (see 16.6). A positive result is one in which the broth culture
appears turbid. A negative result is one in which the broth appears
clear. Each tube is shaken prior to recording results to determine the
presence or absence of turbidity. The primary and secondary
subculture tubes for each carrier represent a "carrier set."
10.8.2 A positive result in either the primary or secondary subculture tube is
considered a positive result for a carrier set.
10.8.3 In the event that there are positive carriers present in the test, the test
may be repeated in order to confirm the outcome.
10.8.4 Once the results are recorded, it is important that the carriers be
reprocessed before use in another study.
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SOP No. MB-05-07
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10.9 Confirmation Steps:
10.9.1 Confirm a minimum of three positive carrier sets per test, if available,
using Gram staining, solid media, and VITEK™ analysis. If there are
less than three positive carrier sets, then each carrier set will be
confirmed. If both tubes are positive in a carrier set, only one tube is
selected for confirmatory testing (preferably the secondary subculture
tube with carrier).
10.9.2 For a test with greater than 20 positive carrier sets, confirm at least
20% by Gram stain, and a minimum of 4 positive carrier sets by Gram
staining, solid media, and VITEK™ analysis (see SOP QC-22, VITEK
2 Compact) to ensure the identity of the organism. Again, if both
tubes are positive in a carrier set, only one tube (preferably the
secondary subculture tube with carrier) is selected for confirmatory
testing.
10.9.3 See Attachment 1 for Gram stain reactions, cell morphology, and
colony characteristics on solid media.
10.9.4 Gram stains are performed on smears taken from the positive culture
tubes. For the additional confirmatory tests, a loopful of broth from
each selected culture tube is streaked on both TSA and selective media
appropriate for the test organism and incubated for 18-24 hours at 36 ±
1°C. The selective agar is checked for the correct reaction and the
culture on the TSA plate is used for preparing the inoculum for the
VITEK™ analysis.
10.9.5 Perform the VITEK™ analysis according to the manufacturer's
instructions.
10.9.6 If confirmatory testing determines that the identity of the organism
was not the test organism, the positive entry (+) on the results sheet
must be annotated to indicate a contaminant was present.
10.10 Re-use of Stainless Steel Carriers: After use, all carriers are autoclaved. Carriers
for which test results were negative may be reused after cleaning. Carriers that
are positive are re-cleaned and screened biologically (see SOP MB-03, Screening
Carriers) before re-use. These carriers may be reused if the biological screening
test results in no growth. The extra carriers that were inoculated but not used are
autoclaved, re-cleaned, and used again.
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SOP No. MB-05-07
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11.0 DATA ANALYSIS/CALCULATIONS:
11.1 Calculations will be computed using a Microsoft Excel spreadsheet (see 16.10).
Electronic copies of the spreadsheet as well as hard copies will be retained.
11.2 To calculate CFU/mL per carrier when 3 serial dilutions are plated, use the
following calculation scheme where 10"x, 10"y, and 10"z are the dilutions plated:
(avg CPU forWx) + (avg CPU forWy)+(avg CPU/orl(T)
KT + KF+KT
11.3 Counts from 0 through 300 and their associated dilutions will be included in the
calculations.
11.3.1 Sample calculation: For average CFU of 115 at the 10"3 dilution, 15 at
the 10"4 dilution, and 0 at the 10"5 dilution, the CFU/mL per carrier
would be 1.2 x 105 CFU/mL per carrier.
11.4 To calculate CFU/carrier, multiply the CFU/mL per carrier by the volume of
media used to suspend carrier for sonication (10 mL) and round numbers to 2
significant figures for reporting the final data.
11.4.1 Sample calculation: 1.2 x 105 CFU/mL per carrier x 10 mL = 1.2 x
106 CFU/carrier.
11.5 Calculate the log density for each carrier by taking the logio of the density (per
carrier).
11.6 Calculate the mean log density across carriers for each test. Let M denote the
mean log density. The 10M is the geometric mean density for the test.
12.0 DATA MANAGEMENT/RECORDS MANAGEMENT:
12.1 Data will be recorded promptly, legibly, and in indelible ink on the appropriate
forms (see 16.0). Completed forms are archived in notebooks kept in secured file
cabinets in room D217. Only authorized personnel have access to the secured
files. Archived data is subject to OPP's official retention schedule contained in
SOP ADM-03, Records and Archives.
13.0 QUALITY CONTROL:
13.1 For quality control purposes, the required information is documented on the
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SOP No. MB-05-07
Date Revised 08-18-09
Page 20 of 3 3
appropriate form(s) (see 16.0).
14.0 NONCONFORMANCE AND CORRECTIVE ACTION:
14.1 If the results of quality control do not verify the identity of the test organism, then
the culture is discarded and a new culture is initiated. New stock cultures are
established as outlined in section 10.0 of this SOP.
14.2 Strict adherence to the protocol is necessary for the validity of the test results.
Any deviation from the standard protocols must be recorded on the form and an
explanation for the deviation given.
14.3 The mean log density for carriers inoculated with S. aureus and P. aeruginosa
must be at least 6.0 (corresponding to a geometric mean density of 1.0 x 106); a
mean log density below 6.0 invalidates the test.
14.3.1 Values below 1.0 x 106 may be indicative of a dilution error, poor
media quality, interference by environmental parameters (e.g., carrier
drying and culture incubation conditions), contamination, or lack of
adherence to the method.
14.3.2 The prescribed minimum will also account for the addition of 5%
organic soil to the inoculum.
14.4 Carrier counts for S. enterica are expected to be comparable to counts for S.
aureus and P. aeruginosa.
14.5 A product test exhibiting passing results (i.e., 0 or 1 positive carrier sets for the
test microbe) will be repeated if contamination is present for more than one
carrier set. For a failing product test (i.e., 2 or more positive carrier sets for the
test microbe), no contamination is permissible in any of the carrier sets and the
test is deemed invalid and must be repeated.
15.0 REFERENCES:
15.1 Official Methods of Analysis. 2009. 18th Ed., AOAC INTERNATIONAL,
Gaithersburg, MD, (Methods 955.15 and 964.02).
15.2 Official Methods of Analysis. 2006. 18th Ed., AO AC INTERNATIONAL,
Gaithersburg, MD, (Method 955.14).
15.3 Holt, J., Krieg, N., Sneath, P., Staley, J. and Williams, S. eds. 1994. Bergey's
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SOP No. MB-05-07
Date Revised 08-18-09
Page 21 of 33
Manual of Determinative Bacteriology, 9th Edition. Williams & Wilkins,
Baltimore, MD.
15.4 Krieg, Noel R. and Holt, John G. 1984. Sergey's Manual of Systematic
Bacteriology Volume 1. Williams & Wilkins, Baltimore, MD.
15.5 Sneath, P., Mair, N., Sharpe, M.E., and Holt, J. eds. 1986. Sergey's Manual of
Systematic Bacteriology Volume 2. Williams & Wilkins, Baltimore, MD.
16.0 FORMS AND DATA SHEETS:
16.1 Organism Culture Tracking Form
16.2 Test Microbe Confirmation Sheet (Quality Control)
16.3 AOAC Use-Dilution Test: Time Recording Sheet for Carrier Inoculation Steps
16.4 AOAC Use-Dilution Test: Time Recording Sheet for Carrier Transfers
16.5 AOAC Use-Dilution Test Information Sheet
16.6 AOAC Use-Dilution Test Results Sheet
16.7 Test Microbe Confirmation Sheet
16.8 AOAC Use-Dilution Test Serial Dilution/Plating Tracking Form
16.9 AOAC Use-Dilution Test Carrier Count Data Sheet
16.10 Sample Carrier Count Spreadsheet
MS Excel spreadsheet: Carrier Count Template_UDT_v2
Attachment 1: Typical Growth Characteristics of strains of P. aeruginosa, S.
aureus, and S. enterica
Attachment 2: Culture Initiation Flow Chart for S. aureus, P. aeruginosa, and S.
enterica
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SOP No. MB-05-07
Date Revised 08-18-09
Page 22 of 3 3
16.1
ORGANISM CULTURE TRACKING FORM
OPP Microbiology Laboratory
Organism:
Source and Strain no.:
MRME Number:
Supply Control Number:
Lot Number:
Date
Lime
Init.
Subculture Source
Lransfer*
Monthly
Daily
Media Inoculated
(and # inoc.)
Media Prep No.
Incubation
Conditions
Comments
"Monthly" indicates the monthly transfers for culture and "Daily" indicates a 24/48 hr serial transfer (added to control number)
NR = None Required, TC = Test Culture, applied after daily transfer number
TSB = Tryptic soy broth, NB = nutrient broth
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SOP No. MB-05-07
Date Revised 08-18-09
Page 23 of 3 3
16.2
TEST MICROBE CONFIRMATION SHEET (Quality Control)
OPP Microbiology Laboratory
Organism:
Source and Strain no.:
MRME*** Number:
Notes:
Source:
Tube/Plate
ID
Date/
Initials
Staining
Results*
Media Information
Name
Prep. No.
Inc. Time/
Temp.
Results
Date/
Initials
Colony Characteristics
Vitek#**
Record Gram stain results: GPC = Gram Positive Cocci; GNR = Gram Negative Rods
Vitek tracking number
Use MRME notation for all organisms (refer to MB-02).
ISA = trypticase soy agar, MSA = mannitol salt agar, NA = nutrient agar
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Date Revised 08-18-09
Page 24 of 3 3
16.3
AOAC Use-Dilution Test: Time Recording Sheet for Carrier Inoculation Steps
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
Test Date
Product Reg. No.
Product Name
Sample No.
Organism
Initials/Date
Test ID
Inoculum Settle Time*
Start Time
/
/
/
End Time
/
/
/
Carrier Seeding Time*
Start Time
/
/
/
End Time
/
/
/
Carrier Dry Time*
Start Time
/
/
/
End Time
/
/
/
* Recorded from laboratory clock/and timer.
Comments:
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SOP No. MB-05-07
Date Revised 08-18-09
Page 25 of 33
16.4
AOAC Use-Dilution Test: Time Recording Sheet for Carrier Transfers
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
Test Date
Product Reg. No.
Product Name
Sample No.
Organism
Initials/Date
Set
Drop
Interval
Carrier Drop Start Time
(into the disinfectant)
Clock
Timer
Carrier Drop End Time
(into the primary subculture/neutralizer media)
Clock
Timer
Carrier Transfer
(into secondary subculture)
Start Time1
Comments:
1 Carrier transfer into secondary subculture (time elapsed after last carrier dropped in primary); taken from clock
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SOP No. MB-05-07
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16.5
AOAC Use-Dilution Test Information Sheet
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
EPA Reg. No.
Name
Sample No.
Lot No.
Expiration Date
SOP
Test Date
Comments:
TEST PARAMETERS/Confirmed by:
H2O Hardness (CaCO3) ppm
Specified
Specified
Specified
Titrated (Buret)/Date/Init.
HACH/Date/Init.
As Prepared/Date/Init.
As Prepared/Date/Init.
Specified
Specified
Specified
Chiller Unit Display
Before:
After:
Test Tube Water Bath
Before:
After:
As Tested
Specified
TEST MICROBE INFORMATION/Confirmed by:
Test Microbe
Org. Control No
Avg. CPU/Carrier
48-54 Hour Culture
Date/Time
Initiated
Harvested
REAGENT/MEDIA INFORMATION/Confirmed by:
Reagent/Media
Prep. No.
Reagent/Media
Prep. No.
Neutralizer volume verified: n Yes
n No
Subculture volume verified: n Yes
n No
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SOP No. MB-05-07
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16.6
AOAC Use-Dilution Test Results Sheet
OPP Microbiology Laboratory
PRODUCT INFORMATION/Confirmed by:
EPA Reg. No.
Name
Sample No.
Test Date
Test Organism
CARRIER INFORMATION (to be completed by Analyst)
Carrier Drop Time Interval
Carrier Set
Analyst
TEST RESULTS
Date Recorded/Initials
Primary Subculture / Secondary Subculture (carrier)
1
/
11
/
21
/
31
/
41
/
51
/
2
/
12
/
22
/
32
/
42
/
52
/
3
/
13
/
23
/
33
/
43
/
53
/
Results Summary
4
/
14
/
24
/
34
/
44
/
54
/
5
/
15
/
25
/
35
/
45
/
55
/
6
/
16
/
26
/
36
/
46
/
56
/
7
/
17
/
27
/
37
/
47
/
57
/
8
/
18
/
28
/
38
/
48
/
58
/
Number of Carrier Sets with Growth
Number of Carrier Sets without Growth
Viability Controls (Record growth as "+", no growth as "0
"): Primary Sec
;ondary Ac
9
/
19
/
29
/
39
/
49
/
59
/
10
/
20
/
30
/
40
/
50
/
60
/
ceptable: Yes No
Modifications/Comments :
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SOP No. MB-05-07
Date Revised 08-18-09
Page 28 of 33
16.7
Test Microbe Confirmation Sheet
OPP Microbiology Laboratory
TEST INFORMATION/Confirmed by:
EPA Reg. No.
Name
Sample No.
Test Date
Test Organism
Comments:
Source:
Tube/Plate ID
Date/
Initials
Stain
Results1
Media Information
Type
Prep. No.
Inc. Time/
Temp.
Results
Date/
Initials
Colony Characteristics
Vitek ID
(if applicable)
Record Gram Stain results as GPC=Gram positive cocci or GNR=Gram negative rods.
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16.8
AOAC Use-Dilution Test Serial Dilution/Plating Tracking Form
OPP Microbiology Laboratory
TEST INFORMS
EPA Reg. No.
Name
Sample No.
Test Date
Organism
SOP
iTION/Confirmed bv:
DILUTION/PLATING SCHEME/Confirmed t
Sonication Start Time (clock):
^^^^^
Starting volume of diluent
Volume added to serial dilution tube (1 mL)
Volume plated (0.1
mL)
Final dilution (used for calculations)1
Number of plates per dilution
Plating medium
Number of carriers evaluated
Comments: N/A
>v:
~
Dilution Tube
10°* 10"1 10"2 10"3 10"4
N/A
= not applicable
Dilution blank volume verified: n Yes n No Subculture medium volume verified: n Yes n No
* Volume of medium in the tube with the carrier will be accounted for in the CPU/carrier calculation.
Adjusted for volume plated.
REAGENT/MEDIA INFORMATION/Confirmed by:
Reagent/Media
Prep. No.
Reagent/Media
Prep. No.
16.9
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AOAC Use-Dilution Test Carrier Count Data Sheet
OPP Microbiology Laboratory
TEST INFOR1V
EPA Reg. No.
Name
Sample No.
Test Date
Organism
SOP
Test Type
lATION/Confirmed bv:
RESULTS
Date/Initials
Plating method
Volume of media in initial tube receiving carrier
Carrier Noxx/^
.s^ Dilution
1
2
3
4
5
6
/
/
/
/
/
/
CPU per Dilution Plate (2)
/
/
/
/
/
/
/
/
/
/
/
/
Comments: N/A = not applicable
16.10
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Sample Carrier Count Spreadsheet (v2)
OPP Microbiology Laboratory
Carrier Count Spreadsheet
OPP Microbiology Lab oratoiy
TEST INFORMATION/Confinnedby:
EPA Reg. No.
Name
Sample No.(s)
Test Date
Organism
SOP
Test Type
Volume of media m tube with earner (mL):
^v^Canier No.
Dilution ^^x^
1
2
3
4
5
6
CFU per Pkte
CFU/mL
per carrier
Mean per carrier for all carriers tested:
CFU/caniar
LD/camer
Comments: LD = log density
Attachment 1
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SOP No. MB-05-07
Date Revised 08-18-09
Page 32 of 33
Typical Growth Characteristics of strains of P. aeruginosa, S. aureus, and S. enterica (see ref.
15.2, 15.3 and 15.4).
Gram stain reaction
P. aeruginosa*
(-)
S. aureus*
(+)
S. enterica*
(-)
Typical Growth Characteristics on Solid Media
Mannitol Salt
Cetrimide
ISA
No Growth
circular, small, initially opaque,
turning fluorescent green over
time; agar fluorescent yellowish
green
flat, opaque to off-white, round
spreading (1)
circular, small, yellow colonies,
agar turning fluorescent yellow
No Growth
small, circular, yellow or white,
glistening
N/A
N/A
entire, glistening, circular,
smooth, translucent, low convex
Typical Microscopic Characteristics
Cell dimensions
Cell appearance
0.5-1.0 um in diameter by 1.5-
5.0 um in length*
straight or slightly curved rods,
single polar flagella, rods
formed in chains
0.5-1.5 um in diameter*
spherical, occurring singly, in
pairs and tetrads, sometimes
forming irregular clusters
0.7-1 .5 um in diameter by 2.0-
5.0 um in length*
straight rods, peritrichous
flagella
*After 24±2 hours
(1) Plates from dilution plating. The agar plate may show three different colony types: a) circular, undulate edge, convex, rough
and opaque; b) circular, entire edge, convex, smooth and translucent; c) irregular, undulate edge, convex, rough, spreading, and
translucent. Colony c) reverts to colony type a) after 24 hour incubation. Pyocyanin is not produced.
Attachment 2
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SOP No. MB-05-07
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Culture Initiation and Stock Culture Generation Flow Chart for S. aureus, P. aeruginosa, and S.
enterica
(D Rehydrate ampule.
® Transfer entire rehydrated
pellet to TUBE A.
I
TSB
or
NB
Ampule
I
® Culture ID & Quality Control
MSA & Cetrimide
TSA
TUBE A
(pre-incubation)
Gram VITEK
Stain
TUBE A
(post-incubation)
CULTURE INITIATION
® Obtain lyophilized cultures annually from ATCC. Using a tube containing 5-6 mL of TSB aseptically withdraw
0.5 to 1.0 mL and rehydrate the pellet for S. aureus and P. aemginosa. Using a tube containing 5-6 mL of NB,
aseptically withdraw 0.5 to 1.0 mL and rehydrate the pellet for S. enterica.
® Aseptically transfer the entire rehydrated pellet back into the original tube of broth (TSB for S. aureus and P.
aemginosa, NB for S. enterica) designated as "TUBE A." Mix well. Use suspension in TUBE A for CULTURE ID
& QUALITY CONTROL. Incubate TUBE A for & aureus, P. aemginosa, and S. enterica for 24 hours at 36 ± 1°C.
CULTURE ID & QUALITY CONTROL
® Using a loopful of rehydrated suspension from TUBE A, streak for isolation on duplicate plates (TSA for S.
aureus, P. aemginosa, and NA for S. enterica). In addition for S. aureus and P. aemginosa, streak a loopful of
rehydrated suspension onto both MSA and Cetrimide agar. Selective media is not used for S. enterica. Incubate
plates for S. aureus, P. aemginosa, and S. enterica for 24 hours at 36 ± 1°C. Record results on the Test Microbe
Confirmation Sheet.
STOCK CULTURE GENERATION
© Using the 24 ± 2 hour TUBE A broth culture: initiate stock cultures. For S. aureus and S. enterica, streak-
inoculate six NA slants. For P. aemginosa, stab-inoculate six CTA tubes. Incubate the S. aureus and S. enterica
slants and P. aemginosa stabs at 36 ± 1°C for 48 ± 2 hours. Record all manipulations on the Organism Culture
Tracking Form.
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