US Environmental Protection Agency Office of Pesticide Programs Microbiology Laboratory Environmental Science Center, Ft. Meade, MD Determining the Presence of Microbial Contamination in Disinfectant Products Standard Operating Procedures (SOPs) Office of Pesticide Programs SOP Number: QC-21-01 Date Revised: 03-05-09 ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 1 of 18 EPA/OPP MICROBIOLOGY LABORATORY ESC, Ft. Meade, MD Standard Operating Procedure for Determining the Presence of Microbial Contamination in Disinfectant Products SOP Number: QC-21-01 Date Revised: 03-05-09 Initiated By: Technical Review: Print Name: Print Name: Technical Staff QA Review: Print Name: QA Officer Approved By: Print Name: Branch Chief Date: / / Date: / / Date: / / Date: / / Effective Date: / / Controlled Copy No.: Withdrawn By: Date: / / ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 2 of 18 TABLE OF CONTENTS Contents Page Number 1.0 SCOPE AND APPLICATION 2 2.0 DEFINITIONS 3 3.0 HEALTH AND SAFETY 3 4.0 CAUTIONS 3 5.0 INTERFERENCES 4 6.0 PERSONNEL QUALIFICATIONS 4 7.0 SPECIAL APPARATUS AND MATERIALS 4 8.0 INSTRUMENT OR METHOD CALIBRATION 5 9.0 SAMPLE HANDLING AND STORAGE 5 10.0 PROCEDURE AND ANALYSIS 6 11.0 DATA ANALYSIS/CALCULATIONS 8 12.0 DATA MANAGEMENT/RECORDS MANAGEMENT 8 13.0 QUALITY CONTROL 8 14.0 NONCONFORMANCE AND CORRECTIVE ACTION 8 15.0 REFERENCES 9 16.0 FORMS AND DATA SHEETS 9 1.0 SCOPE AND APPLICATION: ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 3 of 18 1.1 The assay provides data on the occurrence of microbial contamination of liquid and spray antimicrobial products. This assay is consistent with methodology for conducting the AO AC Use Dilution Method or the AO AC Germicidal Spray Products as Disinfectants Test, but with sterile carriers. 1.2 The assay is qualitative and designed to recover and culture bacterial contamination (i.e., spores of Bacillus sp.) from a hospital disinfectant sample. In most instances, the assay will be deemed necessary due to historical evidence of bacterial contamination associated with the sample itself, a previously-collected sample of the same product, or a registrant's product line. 1.3 Products that show no evidence of product-borne contamination when tested by this procedure are considered adequate for efficacy testing; however, samples with confirmed contamination will be deemed unsuitable for conducting efficacy tests. 2.0 DEFINITIONS: 2.1 AO AC = AO AC INTERNATIONAL 2.2 UDM = AO AC Use Dilution Method 2.3 GSPT = AOAC Germicidal Spray Products as Disinfectants Test 3.0 HEALTH AND SAFETY: 3.1 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 The volume of neutralizer tubes and subculture tubes will be verified in advance and adjusted accordingly. ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 4 of 18 4.3 Strict adherence to the protocol is necessary for the validity of the test results. 4.4 Follow appropriate chain-of-custody guidelines during testing as stipulated in SOP-COC-Ol, Sample Log-in and Tracking. Sample handling and custody procedures are considered the same as for efficacy testing. 4.5 To avoid introduction of contaminating organisms into the test system and cross- contamination from tube to tube during the assay, it is recommended that hooks be autoclaved prior to use, that one hook be used for a predetermined number of tubes and then be replaced with an autoclaved, unused hook, that there is sufficient time between transfers (from disinfectant to neutralizer and neutralizer to subculture medium) to thoroughly flame (to red-hot) the wire hook, and that the use of a water bath be avoided (see section 10.1.3). In addition, the ultraviolet light (UV) in the biological safety cabinet should be left on overnight prior to performing the assay (see section 10.1.4). 5.0 INTERFERENCES: 5.1 Exposing the product to external contaminants during opening and dispensing, and the use of non-sterile laboratory supplies may interfere with the outcome of this analysis. 5.2 Quality control measures for media and reagents used in this evaluation must be followed as outlined in SOP QC-11, Performance and Sterility of Media and Reagents. 6.0 PERSONNEL QUALIFICATIONS: 6.1 Personnel are required to be knowledgeable about the procedures in this SOP. Documentation of training and familiarization with this SOP can be found in the training file for each employee. 7.0 SPECIAL APPARATUS AND MATERIALS: 7.1 Carriers. Polished stainless steel cylinders, 8 ± 1 mm OD, 6 ± mm ID, 10 ± 1mm length; type 304 stainless steel, SS 18-8 (S& L Aerospace Metals, Maspeth, NY or Fisher Scientific, catalog number 07-907-5Q). 7.2 Glass Slide Carriers. Bellco 25 mm x 25 mm (or comparable size) borosilicate glass cover slips with number 4 thickness (Bellco Glass, Inc., item number: 1916- S0131). ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 5 of 18 7.3 Appropriate Spray Disinfectant Apparatus. 1A Glassware. For disinfectant (UDM), use autoclavable 25 x 100 mm tubes (Bellco Glass Inc., Vineland, NJ). For neutralizer and subculture media, use autoclavable reusable or disposable 20 x 150 mm tubes. For the GSPT, use 38 mm x 100 mm tubes (Bellco Glass Inc.) for neutralizer and subculture media. 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. 7.5 Test tube racks. Any convenient style. 7.6 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. 7.7 Positive displacementpipet (to deliver 10|il to glass slides) and sterile tips. 7.8 Timer. For managing timed activities, any certified timer that can display time in seconds. 7.9 Gram stain kit. 7.10 VITEK 2 Compact. For the automated identification of microorganisms. 7.11 VITEK 2 Compact Identification Cards. 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. 9.3 Prepare disinfectant sample per SOP MB-22, Disinfectant Sample Preparation. ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 6 of 18 10.0 PROCEDURE AND ANALYSIS: 10.1 To conduct the assay, follow OPP Microbiology Laboratory SOP MB-05, AO AC Use Dilution Method for Testing Disinfectants for liquid products, and SOP MB- 06, Germicidal Spray Products as Disinfectants Test for spray products, including liquid products to be applied as a spray. The following exceptions/comments will apply: 10.1.1 No inoculum will be generated as test microbes are not used in this assay. 10.1.2 If test parameters call for an organic soil load, disregard this requirement. However, see section 10.1.7 of this SOP for a recommendation to apply an organic substance to glass slides in the event that the spray product beads up and flows off of a glass slide in the AO AC Germicidal Spray Products as Disinfectants Test. The addition of an organic substance (e.g., 5% horse serum in sterile deionized water, letheen broth) in this case is to increase dispersion of the spray product across the entire slide. 10.1.3 For liquid products, the recirculating chiller and water bath specified in SOP MB-05 to maintain the temperature of the disinfectant will not be used. Elimination of the water bath will reduce any potential for introducing water-borne contaminants into the test system. Place racks of disinfectant-containing tubes directly onto the surface of the biological safety cabinet. Exposure of carriers to disinfectant will be performed at room temperature. 10.1.4 The ultraviolet light (UV) in the biological safety cabinet should be left on overnight prior to performing the test. Necessary test equipment (i.e., vortex, hooks, timer) will be placed into the biological safety cabinet prior to turning on the UV light. 10.1.5 A single assay involves the evaluation of 60 sterile carriers for one product sample. Approximately 65-70 sterile carriers (60 plus extras) are prepared. 10.1.6 For liquid products: Sterile stainless steel carriers are removed from the water, placed onto filter paper in sterile glass Petri dishes, and dried for 40 minutes per SOP MB-05. Once drying is complete, the AO AC Use Dilution Method is conducted using the dried, ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 7 of 18 uninoculated carriers according to the test parameters (i.e., product dilution, neutralizer, subculture media, contact time). Alternately, the drying step may be eliminated, and sterile stainless steel carriers may be transferred from the tube of sterile water directly to tubes of disinfectant according to the test timing scheme (e.g., 30 second drop interval). 10.1.7 For spray products: Conduct the AO AC Germicidal Spray Products as Disinfectants Test method using the sterile glass carriers according to the test parameters (i.e., product dilution, neutralizer, subculture media, contact time). Recommendation: • Before conducting the spray test, the analyst must practice applying the spray product to sterile glass slides to determine the product's level of dispersion. • If the product beads up and flows off of the slide rather than completely covering the glass slide as it would in a typical efficacy evaluation (with inoculated slides), the analyst should apply 10 |iL of an organic material (e.g., 5% horse serum in sterile deionized water, letheen broth) onto the surface of each sterile glass slide and spread it with a sterile loop. • Dry the slides for 40 minutes per SOP MB-06. • Once drying is complete, apply the spray product again to determine if product dispersion is improved. • Based on previous observations in our laboratory, the addition of an organic substance to the surface of the slide increases product dispersion on glass slides. • If the application of an organic substance is deemed necessary to perform the test, apply the substance, dry the slides for 40 minutes as per SOP MB-06, and conduct the modified AO AC Germicidal Spray Products as Disinfectants Test method using the dried, sterile carriers according to the test parameters (i.e., product dilution, neutralizer, subculture media, contact time). 10.1.8 Carrier counts are not to be performed. 10.1.9 Report results as (+) for microbial growth or (0) for no growth on the results sheet. In the "Comments" section of the results form, describe the growth in positive tubes (e.g., uniformly turbid, string-like, etc.). Note: Shaking tubes prior to recording results may disrupt the unique ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 8 of 18 physical appearance of typical Bacillus sp. growing in liquid media (i.e., string-like, fibrous, not producing uniformly turbid media). 10.1.10 A minimum of three positive (showing microbial growth) carrier sets, if available, should be confirmed using Gram staining, growth on trypticase soy agar (TSA; for initial identification and isolation), and VITEK. 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. 10.1.11 For a test with greater than 20 positive carrier sets, confirm at least 20% by Gram staining, TSA, and VITEK analysis. Again, if both tubes are positive in a carrier set, only one tube is selected for confirmatory testing. 11.0 DATA ANALYSIS/CALCULATIONS: None 12.0 DATA MANAGEMENT/RECORDS MANAGEMENT: 12.1 Data will be recorded promptly, legibly, and in indelible ink on the appropriate forms. Completed forms are archived in notebooks kept in secured file cabinets in the file 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. 12.2 A performance report will be compiled as stipulated in SOP ADM-01, Performance Reports. 13.0 QUALITY CONTROL: 13.1 For quality control purposes, the required information is documented on the appropriate form(s) (see 16.0). 14.0 NONCONFORMANCE AND CORRECTIVE ACTION: 14.1 If a product is confirmed to be contaminated, no further efficacy testing will be initiated. 14.2 Strict adherence to the protocol is necessary for the validity of the test results. Any deviation from the standard protocol must be brought to the Study Director's attention and recorded in the raw data along with an explanation for the deviation given. The deviation and reason for it must be documented on the GLP ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 9 of 18 Compliance form in the final report. 15.0 REFERENCES: 15.1 Official Methods of Analysis. 2006. 18th Ed., AO AC INTERNATIONAL, Gaithersburg, MD, (Methods 955.14, 955.15, and 964.02). 15.2 Official Methods of Analysis. 1990. 15th Ed., AO AC INTERNATIONAL, Gaithersburg, MD, (Method 961.02). 16.0 FORMS AND DATA SHEETS: 16.1 Determining Microbial Contamination in Liquid Disinfectants: Time Recording Sheet for Carrier Inoculation Steps 16.2 Determining Microbial Contamination in Liquid Disinfectants: Time Recording Sheet for Carrier Transfers 16.3 Determining Microbial Contamination in Liquid Disinfectants: Information Sheet 16.4 Determining Microbial Contamination in Liquid Disinfectants: Results Sheet 16.5 Determining Microbial Contamination in Spray Disinfectants: Time Recording Sheet for Carrier Inoculation Steps 16.6 Determining Microbial Contamination in Spray Disinfectants: Time Recording Sheet for Carrier Transfers 16.7 Determining Microbial Contamination in Spray Disinfectants: Information Sheet 16.8 Determining Microbial Contamination in Spray Disinfectants: Results Sheet 16.9 Test Microbe Confirmation Sheet ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 10 of 18 16.1 Determining Microbial Contamination in Liquid Disinfectants: Time Recording Sheet for Carrier Inoculation Steps OPP Microbiology Laboratory TEST INFORMATION/Confirmed by: Tcsl Dale Product Reg. No. Product Name Sample No(s). Organism Not Applicable Initials/Date Test ID Carrier Dry Time* Start Time End Time UDT * Recorded from laboratory clock/and timer. ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 11 of 18 16.2 Determining Microbial Contamination in Liquid Disinfectants: Time Recording Sheet for Carrier Transfers OPP Microbiology Laboratory TEST INFORMATION/Confirmed by: Tcsl Dale Product Reg. No. Product Name Sample No(s). Organism Not Applicable Initials/Dale Set Drop Interval Carrier Drop Start Time (into the disinfectant.) Carrier Drop End Time (into the primary subcullurc/ncutralizcr media) Carrier Transfer (into secondary subculture) Clock Tinier Clock Timer Start Time' 1-20 21-40 41-60 Comments: Carrier transfer into secondary subculture (time elapsed after last carrier dropped in primary); taken from clock ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 12 of 18 16.3 Determining Microbial Contamination in Liquid Disinfectants: Information Sheet OPP Microbiology Laboratory TEST INFORMATION/Confirmed by: EPA Reg. No. SOP Name Test Date Sample No. Comments: Assay will be performed without test microbes. Lot No. Expiration Date TEST PARAMETERS/Confirmed by: H20 Hardness (CaCOi) ppm Specified Use Dilution Nculralizcr Specified Specified Titrated (Burct)/Datc/Inil. HACH/Datc/Init. As Preparcd/Date/Init. Contact Time Other Parameters Specified As Tested Specified REAGENT/MEDIA INFORMATION/Confirmed by: Reagent/Media Prep. No. Reagent/Media Prep. No. Neutralizer volume verified: ~ Yes ~ No Subculture volume verified: ~ Yes ~ No ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 13 of 18 16.4 Determining Microbial Contamination in Liquid Disinfectants: Results Sheet OPP Microbiology Laboratory PRODUCT INFORMATION/Confirmed by: EPA Reg. No. Test Date Name Test Organism Not Applicable Sample No. CARRIER INFORMATION (to be completed by Analyst) Carrier Drop Time Interval Carrier Set Analyst TEST RESULTS Dale Recorded/Initials Primary Subculture / Secondary Subculture (carrier) 1 2 3 4 5 6 7 8 9 10 / / / / / / / / / / 11 12 13 14 15 16 17 18 19 20 / / / / / / / / / / 21 22 23 24 25 26 27 28 29 30 / / / / / / / / / / 31 32 jj 34 35 36 37 38 39 40 / / / / / / / / / / 41 42 43 44 45 46 47 48 49 50 / / / / / / / / / / 51 52 53 54 55 56 57 58 59 60 / / / / / / / / / / Results Summary Number of Carrier Sets with Growth Number of Carrier Sets without Growth Modifications/Comments: ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 14 of 18 16.5 Determining Microbial Contamination in Spray Disinfectants: Time Recording Sheet for Carrier Inoculation Steps OPP Microbiology Laboratory TEST INFORMATION/Confirmed by: Test Dale Product Reg. No. Product Name Sample No(s). Test Organism Not Applicable Initials/Dale Test ID Carrier Dry Time* Start Time End Time GSPT * Recorded from laboratory clock/and timer. ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 15 of 18 16.6 Determining Microbial Contamination in Spray Disinfectants: Time Recording Sheet for Carrier Transfers OPP Microbiology Laboratory TEST INFORMATION/Confirmed by: Test Dale Product Reg. No. Product Name Sample No(s). Test Organism Not Applicable Initials/dale Set Spray Interval Carrier Spray Start Time (into the disinfectant) Carrier Spray End Time (into the neutralizcr/primarv subculture)' Carrier Transfer (into secondary subculture) Clock Timer Clock Timer Start Time 1-20 21-40 41-60 Comments: Carriers sprayed by: . Carriers transferred by: Carrier transfer into secondary subculture; taken from clock. ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 16 of 18 16.7 Determining Microbial Contamination in Spray Disinfectants: Information Sheet OPP Microbiology Laboratory TEST INFORMATION/Confirmed by: EPA Reg. No. SOP Name Test Date Sample No. Comments: Assay will be performed without test microbes. Lot No. Expiration Date TEST PARAMETERS/Confirmed by: H20 Hardness (CaCOO ppm Specified Use Dilution Ncutralizer Specified Specified Titrated (Buret)/Dale/Init. HACH/Date/Init. As Prepa red/Da te/I nil. Contact Time Other Parameters Specified As Tested Specified REAGENT/MEDIA INFORMATION/Confirmed by: Reagent/Media Prep. No. Reagent/Media Prep. No. Neutralizer volume verified: ~ Yes ~ No Subculture volume verified: ~ Yes ~ No ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 17 of 18 16.8 Determining Microbial Contamination in Spray Products: Results Sheet OPP Microbiology Laboratory PRODUCT INFORMATION/Confirmed by: EPA Reg. No. Test Dale Name Test Organism Not Applicable Sample No. TEST RESULTS Dale Recorded/Initials Primary Subculture / Secondary Subculture (carrier) 1 2 3 4 5 6 7 8 9 10 / / / / / / / / / / 11 12 13 14 15 16 17 18 19 20 / / / / / / / / / / 21 22 23 24 25 26 27 28 29 30 / / / / / / / / / / 31 32 33 34 35 36 37 38 39 40 / / / / / / / / / / 41 42 43 44 45 46 47 48 49 50 / / / / / / / / / / 51 52 53 54 55 56 57 58 59 60 / / / / / / / / / / Results Summary Number of Carrier Sets with Growth Number of Carrier Sets without Growth Modifications/Comments: ------- SOP No. QC-21-01 Date Revised 03-05-09 Page 18 of 18 16.9 Test Microbe Confirmation Sheet OPP Microbiology Laboratory TEST INFORMATION/Confirmed by: EPA Reg. No. Test Dale Name Test Organism Not Applicable Sample No. Comments Source: Tubc/Plalc ID Dale/ Initials Slain Results* Media Information Results Name Prep. No. Inc. Time/ Temp. Date/ Initials Colony Characteristics VITEK ID** (if applicable) * GPC=Gram positive cocci, GNR=Gram negative rods, GPR=Gram positive rods ** VITEK numerical profile number ------- |