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
                                                             Date Revised 08-18-09
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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Withdrawn By: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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                                                   SOP No. MB-05-07
                                                   Date Revised 08-18-09
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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 2-5C 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 121C. 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 180C or steam sterilize for a minimum of 20 minutes at 121C with
       drying cycle.

7.8    Water bath/chiller unit. Constant temperature for test chemical, capable of
       maintaining 20  1C 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 
                    1C 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  1C for 48  2 hours.

          10.1.3.4   Following incubation, store the cultures at 2-5C 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  1C 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
                           2-5C 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  1C. 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  1C 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 2-5C 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 45-50C 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   1C 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  1C or the
                 temperature  specified (1C). 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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                                                      SOP No. MB-05-07
                                                      Date Revised 08-18-09
                                                      Page 16 of 33

          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  
          1C 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
                                                            Page 17 of 33
                 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 1C.

       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  1C.  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
                                                            Date Revised 08-18-09
                                                            Page 18 of 33
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 
                 1C.  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
                                                                 Date Revised 08-18-09
                                                                 Page 19 of 33
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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                                                           SOP No. MB-05-07
                                                           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
                                                                     Date Revised 08-18-09
                                                                     Page 26 of 3 3
 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
                                                        Date Revised 08-18-09
                                                        Page 27 of 3 3
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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                                                               SOP No. MB-05-07
                                                               Date Revised 08-18-09
                                                               Page 29 of 3 3
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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                                                       SOP No. MB-05-07
                                                       Date Revised 08-18-09
                                                       Page 30 of 33
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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                                                        SOP No. MB-05-07
                                                        Date Revised 08-18-09
                                                        Page 31 of 33
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 242 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
                                                                           Date Revised 08-18-09
                                                                           Page 33 of 33
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  1C.
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  1C. 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  1C for 48  2 hours. Record all manipulations on the Organism Culture
Tracking Form.

-------