US Environmental Protection Agency Office of Pesticide Programs Office of Pesticide Programs Microbiology Laboratory Environmental Science Center, Ft. Meade, MD Standard Operating Procedure for Neutralization Confirmation Assay for Disinfectant Products Tested against Mycobacterium bovis (BCG) SOP Number: MB-11-04 Date Revised: 04-29-13 ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 1 of 12 SOP Number MB-11-04 Title Neutralization Confirmation Assay for Disinfectant Products Tested against Mycobacterium bovis (BCG) Scope Describes the methodology for determining the effectiveness of a neutralizer used when testing the tuberculocidal activity of disinfectants against Mycobacterium bovis (BCG) on hard surfaces using liquid, sprays, or towelettes. Application 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. Approval Date SOP Developer: Print Name: SOP Reviewer Print Name: Quality Assurance Unit Print Name: Branch Chief Print Name: Date SOP issued: Controlled copy number: Date SOP withdrawn: ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 2 of 12 TABLE OF CONTENTS Contents Page Number 1. DEFINITIONS 3 2. HEALTH AND SAFETY 3 3. PERSONNEL QUALIFICATIONS AND TRAINING 3 4. INSTRUMENT CALIBRATION 3 5. SAMPLE HANDLING AND STORAGE 3 6. QUALITY CONTROL 3 7. INTERFERENCES 3 8. NON-CONFORMING DATA 3 9. DATA MANAGEMENT 3 10. CAUTIONS 3 11. SPECIAL APPARATUS AND MATERIALS 4 12. PROCEDURE AND ANALYSIS 4 13. DATA ANALYSIS/CALCULATIONS 9 14. FORMS AND DATA SHEETS 9 15. REFERENCES 10 ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 3 of 12 1. Definitions Additional abbreviations/definitions are provided in the text. 1. "Exposed" Neutralizer: Neutralizer solution which has been used to inactivate the product. 2. "Fresh" Neutralizer: Neutralizer solution which has not been used to inactivate the product. 2. Health and Safety Follow procedures specified in SOP MB-01, Laboratory Biosafety. The Study Director and/or lead analyst should consult the Material Safety Data Sheet for specific hazards associated with products. 3. Personnel Qualifications and Training Refer to SOP ADM-04, OPP Microbiology Laboratory Training. 4. Instrument Calibration Refer to SOPs EQ-01, EQ-02, EQ-03, EQ-04 and EQ-05 for details on method and frequency of calibration. 5. Sample Handling and Storage Refer to SOP MB-22, Disinfectant Sample Preparation, and SOP COC-01, Chain of Custody Procedures. 6. Quality Control Appropriate quality control measures are integrated into each SOP. For quality control purposes, the required information is documented on the appropriate forms (see section 14). 7. Interferences 1. For each assay, use one preparation of each medium for the treatment and control groups. Differences in media quality (media performance) between preparations of media may lead to misleading neutralization results. 2. An interaction between a product's active ingredient and the neutralizer can interfere with the reading of results. 3. Presence of contamination will interfere with the interpretation of results and may necessitate repeat analysis. 8. Non- conforming Data Management of non-conforming data will be specified in the study protocol; procedures will be consistent with SOP ADM-07, Non- Conformance Reports. 9. Data Management Data will be archived consistent with SOP ADM-03, Records and Archives. 10. Cautions 1. Media performance (Subculture Media Control) must be acceptable in order to interpret the neutralization results. 2. There are time sensitive steps in this procedure including the use period of the test chemical. ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 4 of 12 3. Verify the volume of dilution blanks, neutralizer tubes, and subculture tubes in advance and adjust accordingly. 11. Special Apparatus and Materials 1. Refer to section 11 (Special Apparatus and Materials) of MB-07, MB- 23, and MB-24 depending on the type of carrier used in the neutralization assay. 12. Procedure and Analysis 1. For an overview of the setup of this procedure, see Tables 1 and 2. 2. The general procedure for conducting the assay is the same for liquid, spray, and towelette products. Follow the test parameters specified for product testing (e.g., H2O hardness, use dilution, organic soil, neutralizer, contact time, temperature) for the neutralization confirmation assay. 3. This assay is designed to simulate the conditions of the efficacy test; however, sterile carriers are used instead of inoculated carriers. Diluted M bo vis (BCG) inoculum is added directly to the various sets of subculture media tubes. The inoculum is quantified by plating on Middlebrook 7H9 (M7H9) or Middlebrook 7H11 (M7H11) agar; this provides a quantitative assessment of the neutralizer's effectiveness and any bacteriostatic actions resulting from interactions between the neutralizer, subculture media, and/or disinfectant. 4. Each assay requires eight sterile carriers: four for the Subculture Media + Exposed Neutralizer Treatment and four for the Subculture Media + Fresh Neutralizer Treatment. One carrier is used per each inoculum dilution described in 12.1c. Use the carrier type required for the product (i.e. porcelain penicylinders for liquid products or glass slide carriers for spray or towelette products). 5. For spray tests: Before conducting the neutralization assay, practice applying the spray product to sterile glass slides to determine the product's level of dispersion. If the product beads up and rolls off of the slide rather than completely covering the glass slide as it would in a typical efficacy evaluation (with inoculated slides), apply 10 |iL of an organic material (e.g., broth used for culturing test organism, 5% horse serum in sterile deionized water) onto the surface of each sterile glass slide and spread it with a sterile loop and dry the slides for 30 minutes per SOP MB-24. Once drying is complete, apply the spray product again to verify that product dispersion is improved, and conduct the neutralization assay. Based on previous observations in the laboratory, the addition of an organic substance to the surface of the slide increases product dispersion on glass slides. If no organic substance is deemed necessary, no drying of the sterile glass slides is necessary ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 5 of 12 prior to conducting the assay. 6. The Neutralization Confirmation Assay for M bovis (BCG): Processing Sheet (see section 14) must be used for tracking testing activities. 12.1 Preparation of Inoculum a. Prepare standardized inoculum per section 12.1 of MB-07, MB- 23, or MB-24. b. If the test parameters specify the addition of an organic soil load to the inoculum, perform the neutralization assay with the organic soil load added to the inoculum. Otherwise, prepare the inoculum without the addition of an organic soil load. c. Make serial ten-fold dilutions of the standardized inoculum in 9 mL of Modified Proskauer Beck medium (MPB) or phosphate buffered dilution water (PBDW). Use up to four dilutions (e.g., final dilutions of 10"4 through 10"7) to inoculate the subculture media described below. d. Estimate the CFU/mL. Briefly mix each serial dilution tube prior to plating. Plate 0.1 mL aliquots of appropriate dilutions in duplicate on M7H9 or M7H11 agar using surface spread plating. Spread inoculum evenly over the surface of the agar. Plates must be dry prior to incubation. e. Record the dilution and plating information on the Neutralization Confirmation Assay forM bovis (BCG): Inoculum Enumeration Form (see section 14). f. Incubate plates (inverted) concurrently with the neutralization test subculture tubes at 36 ± 1°C for 17-21 days. Count colonies. Plates that have colony counts over 300 will be reported as TNTC. Record the counts on the Neutralization Confirmation Assay for M. bovis (BCG): Inoculum Enumeration Form (see section 14). 12.2 Disinfectant Sample Preparation. a. Follow guidelines for disinfectant sample preparation provided in SOP MB-22. 12.3 Subculture Media + Exposed Neutralizer Treatment a. Expose four sterile carriers to disinfectant according to the instructions specified in the test parameters for efficacy testing. b. Expose carriers to the disinfectant at appropriate intervals (e.g., one minute intervals). Record the carrier transfer information on the Neutralization Confirmation Assay for M. bovis (BCG): Time ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 6 of 12 Recording Sheet for Carrier Transfers (see section 14) c. Allow product to remain in contact with the carrier per the specified contact time. For a contact time of 10 min, the carrier must be exposed to the disinfectant within ± 5 s of the prescribed exposure time. d. After the fourth carrier has been treated with the disinfectant and the contact time is complete, sequentially transfer carriers into tubes containing the specified neutralizer. Transfer carriers according to methods specified in the appropriate efficacy test method SOP. Drain the excess disinfectant from the carrier prior to the transfer. Note: For spray and towelette products, use 20 mL neutralizer per 38 x 100 mm tube. For liquid products use 10 mL neutralizer per 25 x 100 mm tube. e. Shake the tube containing the carrier in neutralizer thoroughly and transfer the carrier to the primary subculture medium tube containing 20 mL MPB broth within 5-10 minutes. f. For liquid products, sequentially transfer 2 mL aliquots from each of the four neutralizer tubes into one tube of each of the 2 additional subculture media (M7H9, K or TB) specified by the test parameters. This portion of the assay is not timed, but the aliquots should be transferred to the subculture media within approximately 30 ± 5 minutes. g. One tube of each medium will be inoculated with one of the four inoculum dilutions. Note: For spray and towelette products, transfer 2 mL aliquots from each of the four neutralizer tubes into two tubes of each of the 2 additional subculture media (M7H9, K or TB). Two tubes of each subculture media will be inoculated with one of the four inoculum dilutions. 12.4 Subculture Media + Fresh Neutralizer Treatment a. Expose four sterile carriers to neutralizer, one carrier per tube of neutralizer (this portion of the assay is not timed). b. Shake the tube containing the carrier in neutralizer thoroughly and transfer the carrier to the primary subculture medium tube containing 20 mL MPB broth within 5-10 minutes. c. For liquid products, sequentially transfer 2 mL aliquots from each of the four neutralizer tubes into one tube of each of the 2 additional subculture media (M7H9, K or TB) specified by the ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 7 of 12 test parameters. This portion of the assay is not timed, but the aliquots should be transferred to the subculture media within approximately 30 ± 5 minutes. d. One tube of each medium will be inoculated with one of the four inoculum dilutions. Note: For spray and towelette products, transfer 2 mL aliquots from each of the four neutralizer tubes into two tubes of each of the 2 additional subculture media (M7H9, K or TB). Two tubes of each subculture media will be inoculated with one of the four inoculum dilutions. 12.5 Subculture Media Controls a. Subculture Media Control. This control contains four tubes of each preparation of subculture media used in the neutralization assay. Do not add neutralizer to the media. Each tube of each medium will be inoculated with one of the four inoculum dilutions. b. Negative (uninoculated) Media Control. Incubate one uninoculated tube of each medium along with the assay. 12.6 Inoculating Subculture Media a. Inoculate the Subculture Media + Exposed Neutralizer treatment, the Subculture Media + Fresh Neutralizer treatment, and the Subculture Media Control with 0.1 mL of the diluted M. bovis (BCG) inoculum (e.g., dilution tubes 10"3 through 10"6). Inoculate the tubes after all carriers and neutralizer have been transferred. Refer to Table 1 and Table 2 for organization of neutralization assays for liquid and spray/towelette products, respectively. 12.7 Incubation and Presumptive Confirmation Testing a. Incubate tubes at 36± 1 °C. b. Beginning at 21 days, monitor tubes for evidence of typical mycobacterial growth. Continue incubating all tubes for 60 days. c. Record results at 60 days as positive (+) or negative (0) as indicated by the presence or absence of growth. If the 60th day of incubation falls on a weekend or holiday, record the results on the first workday following the 60th day. c. For each medium in the Subculture Media + Exposed Neutralizer treatment, the Subculture Media + Fresh Neutralizer treatment, and the Subculture Media Control, select the tube with growth from the highest dilution of inoculum (i.e., fewest CFU/mL delivered) and perform acid fast staining on a sample of the growth. Acid fast rods are typical forM bovis ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 8 of 12 (BCG). d. If necessary, conduct additional confirmation to include isolation streaks on selective media such as M7H9 or M7H11 agar plates. Following the 17-21 day incubation period, evaluate the colony morphology of the organism on M7H9 or M7H11 agar. On M7H9 or M7H11 agar, M. bovis (BCG) typically appears as colorless to buff-colored, raised, rough growth. e. Record confirmation results on the Neutralization Confirmation Assay forM bovis (BCG): Test Microbe Confirmation Sheet (see section 14). 12.8 Interpretation of Results a. Review the plate count data. One of the four dilutions plated should provide counts within the target range, 5-100 CFU/mL. Subculture tubes inoculated from this dilution also received this low level of challenge, an aspect critical to the determination of neutralization effectiveness and bacteriostatic activity. i. The lack of complete neutralization of the disinfectant or bacteriostatic activity of the neutralizer itself may be masked when a high level ofM bovis (BCG) is added to the subculture tubes. b. The Subculture Media Control (media performance) provides a basis for comparing growth in the subculture tubes to growth in the Subculture Media + Exposed Neutralizer treatment and the Subculture Media + Fresh Neutralizer treatment. Growth in Subculture Media Control verifies the performance of the media and the presence of M. bovis (BCG) in the diluted inoculum. Media is considered to be of acceptable quality if growth is observed when challenged with 5-100 CFU. No growth or only growth in tubes which received high levels of inoculum (e.g., a dilution with plate counts which are too numerous to count) indicates poor media performance. c. The occurrence of growth in the Subculture Media + Fresh Neutralizer treatment as compared to the Subculture Media Control tubes is used to assess any bacteriostatic effects attributable to possible interactions between the neutralizer and subculture media. Interactions between the media and neutralizer may result in growth in some media and not others. No growth or growth only in tubes which received a high level of inoculum (e.g., the dilution with plate counts which are too ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 9 of 12 numerous to count) indicates the presence of bacteriostatic properties as a result of the neutralizer-media interaction. d. The occurrence of growth in the Subculture Media + Exposed Neutralizer treatment tubes as compared to the Subculture Media Control tubes is used to determine the effectiveness of the neutralizer to inactivate the disinfectant when used under simulated test conditions. Interactions between the media and neutralizer may result in growth in some media and not others. No growth or growth only in tubes which received a high level of inoculum (e.g., the dilution with plate counts which are too numerous to count) indicates ineffective neutralization and/or the presence of bacteriostatic properties as a result of the neutralizer-media interaction. e. Each Negative (uninoculated) Media Control tube must show no growth. 13. Data Analysis/ None. Calculations 14. Forms and Data Test Sheets. Test sheets are stored separately from the SOP under the Sheets following file names: Neutralization Confirmation Assay for M bovis (BCG): Time Recording Sheet for Carrier Transfers Neutralization Confirmation Assay for M bovis (BCG): Information Sheet Neutralization Confirmation Assay for M bovis (BCG): Results Sheet - Liquid Products Neutralization Confirmation Assay for M MB-1 l-04_F4.docx bovis (BCG): Results Sheet - Spray/Towelette Products Neutralization Confirmation Assay for M bovis (BCG): Inoculum Enumeration Form Neutralization Confirmation Assay for M bovis (BCG): Test Microbe Confirmation Sheet Neutralization Confirmation Assay forM MB-1 l-04_F7.docx bovis (BCG): Processing Sheet MB-11-04F1. docx MB-1 l-04_F2.docx MB-11-04 F3.docx MB-11-04F5. docx MB-11-04 F6.docx ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 10 of 12 Official Methods of Analysis. Revised 2013. 18* Ed., AO AC INTERNATIONAL, Gaithersburg, MD, (Method 965.12 In vitro Test for Determining Tuberculocidal Activity). ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 11 of 12 Table 1: Components of the Neutralization Confirmation Assay for Liquid Products TiViilmcnl/( onirol M. hm-isi IWCi) Iikiciiliiill Dilution (0.1 ml. iiddcd per 1 uhe) Mi'riisi (O = l uho of Modi;i) M 1*15 (20 ml.) \ni«> (20 ml.) k or 11} (20 ml.) Subculture Media + Exposed Neutralizer 10"3 O/Carrier O O 10"4 O/Carrier O O 10"5 O/Carrier o o 10"6 O/Carrier o o Subculture Media + Fresh Neutralizer 10"3 O/Carrier o o 10"4 O/Carrier o o 10"5 O/Carrier o o 10"6 O/Carrier o o Subculture Media Control 10"3 O o o to-4 O o o 10"5 O o o 10"6 O o o Negative Uninoculalcd Control Not inoculated O o o ------- SOP No. MB-11-04 Date Revised 04-29-13 Page 12 of 12 Table 2: Components of the Neutralization Confirmation Assay for Spray/To welette Products TiViilmcnl/( onirol M. hm-isi IWCi) Iikiciiliiill Dilution (0.1 ml. iiddcd per 1 uhe) Mi'riisi (O = l uho of Modi;i) M 1*15 (20 ml.) \ni«) (20 ml.) k or 11} (20 ml.) Subculture Media + Exposed Neutralizer to-3 O/Carrier OO OO 10"4 O/Carrier oo OO 10"5 O/Carrier OO oo to-6 O/Carrier oo oo Subculture Media + Fresh Neutralizer 10"3 O/Carrier oo oo 10"4 O/Carrier oo oo 10"5 O/Carrier oo oo 10"6 O/Carrier oo oo Subculture Media Control to-3 O o o 10"4 O o o 105 O o o 10"6 O o o Negative Uninoculalcd Control Not inoculated O o o ------- |