mm s r4 US Environmental Protection Agency Office of Pesticide Programs Office of Pesticide Programs Microbiology Laboratory Environmental Science Center, Ft. Meade, MD Standard Operating Procedure for Germicidal Spray Products as Disinfectants: Testing of Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica SOP Number: MB-06-06 Date Revised: 01-15-13 ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 1 of 18 SOP Number MB-06-06 Title Germicidal Spray Products as Disinfectants (GSPT): Testing of Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica Scope Describes the germicidal spray products as disinfectants methodology (see 15.1) used to determine the efficacy of disinfectants against Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica on hard surfaces. 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. Although the default growth medium specified in this SOP is synthetic broth, other growth media (e.g., nutrient broth) may be specified by the study sponsor. 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Name: Quality Assurance Unit Print Name: Branch Chief Print Name: Date SOP issued: Controlled copy number: Date SOP withdrawn: ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 2 of 18 TABLE OF CONTENTS Contents Page Number 1.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 4 10. CAUTIONS 4 11. SPECIAL APPARATUS AND MATERIALS 4 12. PROCEDURE AND ANALYSIS 5 13. DATA ANALYSIS/CALCULATIONS 10 14. FORMS AND DATA SHEETS 10 15. REFERENCES 11 ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 3 of 18 1. Definitions Abbreviations/definitions are provided in the text. 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 SOP 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 For quality control purposes, the required information is documented on the appropriate form(s) (see section 14). 7. Interferences 1. Any disruption of the Pseudomonas aeruginosa pellicle resulting in the dropping or breaking of the pellicle in culture before or during its removal renders that culture unusable. 2. The carriers inside the Petri dishes should be dry prior to inoculation. Moisture can interfere with the concentration and drying of the inoculum on the glass slide carrier. 3. Any inoculated carrier that is wet at the conclusion of the carrier drying period should not be used. 8. Non- conforming Data 1. Sterility and/or viability controls do not yield expected results. 2. The mean log density for control carriers falls outside the specified range. Note: The prescribed minimum and maximum carrier counts also account for the addition of 5% organic soil to the inoculum. a. The mean TestLD for carriers inoculated with S. aureus and P. aeruginosa must be at least 5.0 (corresponding to a geometric mean density of 1.0 x 105) and not above 6.5 (corresponding to a geometric mean density of 3.2 x 106); a mean TestLD below 5.0 and above 6.5 invalidates the test, except for two retesting scenarios (outlined in the study protocol). b. The mean TestLD for carriers inoculated with S. enterica must be at least 4.0 (corresponding to a geometric mean density of 1.0 x 104) and not above 5.5 (corresponding to a geometric mean density of 3.2 x 105); a mean TestLD below 4.0 and above 5.5 invalidates the test, except for two retesting scenarios (outlined in the study protocol). 3. If contamination is present in the test system for more than one carrier for ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 4 of 18 a passing product, retesting is required. No contamination is acceptable for a product test in which the product fails to meet the standard. 4. 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. There are time sensitive steps in this procedure including the use periods of the inoculated carriers and the test chemical. 2. Verify the volume of dilution blanks, neutralizer tubes, and subculture tubes in advance and adjust accordingly. 11. Special Apparatus and Materials 1. Subculture media (e.g., letheen broth, fluid thioglycollate medium, and Dey/Engley broth). Note: Commercial media made to conform to the recipes provided in AO AC Method 961.02 may be substituted. 2. Test organisms. Pseudomonas aeruginosa (ATCC No. 15442), Staphylococcus aureus (ATCC No. 6538) and Salmonella enterica (ATCC No. 10708) obtained directly from ATCC. 3. Culture media. Note: Commercial media (e.g., synthetic broth) made to conform to the recipes provided in AO AC Method 961.02 may be substituted. a. Synthetic broth. Use for (10 mL) daily transfers and (10 mL) final test cultures of S. aureus, P. aeruginosa and S. enterica. b. Nutrient broth. Alternatively, use for (10 mL) daily transfers and (10 mL) final test cultures of P. aeruginosa. 4. Trypticase soy agar (TSA). For use in propagation of the test organism to generate frozen cultures and as a plating medium for carrier enumeration. Alternately, TSA with 5% sheep blood (BAP) may be used. 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. 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. 6. Carriers. Glass Slide Carriers, 25 mm x 25 mm (or comparable size) borosilicate glass cover slips with number 4 thickness. Refer to SOP MB- 03, Screening of Stainless Steel Cylinders, Porcelain Cylinders and Glass Slide Carriers Used in Disinfectant Efficacy Testing. 7. Specialized glassware. For cultures/subcultures, use autoclavable 38 x 100 mm medication tubes (Bellco Glass Inc., Vineland, NJ). Cap tubes with closures before sterilizing. For glassware used to prepare test ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 5 of 18 chemical, refer to SOP MB-22. 8. Spray Disinfectant Apparatus. Refer to Attachment 4. 9. Micropipettes. For performing culture transfers and serial dilutions. 10. Positive displacement pipette. With corresponding sterile tips able to deliver 0.01 mL. 11. Timer. For managing timed activities, any certified timer that can display time in seconds. 12. Electronic Plate Scanning Device. 3M™ Petrifilm™ Plate Reader, 3M Food Safety, St. Paul, MN, USA, Cat. No. 6499, or equivalent. 12. Procedure and Analysis Prior to testing, perform the neutralization assay to determine if secondary subculture tubes are necessary (refer to SOP MB-17, Neutralization Confirmation). The AO AC Germicidal Spray Products Test Processing Sheet (see section 14) must be used for tracking testing activities. 12.1 Test Culture Preparation a. Defrost a single cryovial at room temperature and briefly vortex to mix. Add 10 |iL of the thawed frozen stock (single use) to a tube containing 10 mL of growth medium (e.g., synthetic broth or nutrient broth), vortex, and incubate at 36 ± 1°C for 24 ± 2 h. One daily transfer is required prior to the inoculation of a final test culture. Daily cultures may be subcultured for up to 5 days; each daily culture may be used to generate a test culture. For S. aureus and S. enterica only, briefly vortex the 24 h cultures prior to transfer. b. For the final subculture transfer, inoculate a sufficient number of 20 x 150 mm tubes containing 10 mL growth medium (e.g., synthetic broth or nutrient broth) with 10 |iL per tube of the 24 h culture then vortex to mix. Incubate 48-54 h at 36 ± 1°C. Do not shake the 48-54 h test culture. Record all culture transfers on the Organism Culture Tracking Form (see section 14). 12.2 Carrier Inoculation for S. aureus, P. aeruginosa, and S. enterica a. Inoculate approximately 80 carriers; 60 carriers are required for testing, 6 for control carrier counts, and 1 for the viability control. b. For P. aeruginosa, remove the pellicle from the broth either by decanting the liquid aseptically into a sterile tube, by gently aspirating the broth away from the pellicle using a pipette, or by vacuum removal. Avoid harvesting pellicle from the bottom of the tube. Transfer test culture after pellicle removal into sterile 25 x 150 mm test tubes (up to approximately 20 mL per tube) and visually inspect for pellicle fragments. Presence of pellicle in the final culture makes it unusable for testing. Proceed as below in 12.2c. ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 6 of 18 c. For S. aureus and S. en/erica., using a vortex-style mixer, mix 48-54 h test cultures 3-4 s and let stand 10 min at room temperature before continuing. Remove the upper portion of each culture (e.g., upper 3/4 or approximately 7.5 mL), leaving behind any debris or clumps, and transfer to a sterile flask; pool cultures in the flask and swirl to mix. d. To achieve mean carrier counts within the appropriate range (see section 8), the final test culture may be diluted or concentrated. Dilution of the final test culture (e.g., one part culture plus one part sterile broth) should be made prior to the addition of the OSL to the inoculum. Concentration may be achieved using centrifugation (e.g. 5000g for 20 min.) and re-suspending the pellet in the appropriate volume of the sterile final test culture medium necessary to meet the carrier count range. Note: The use of a spectrophotometer to measure optical density (optical density at 650 nm) is recommended as a tool for assessing the need to dilute the final test culture. Always use sterile broth medium to calibrate the spectrophotometer. e. 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. Swirl to mix. Aliquot a sufficient volume of culture into sterile test tubes. f. Use a calibrated positive displacement pipette to transfer 0.01 mL of the test culture onto approximately 1 square inch of the sterile test carrier in the Petri dish. Vortex-mix the inoculum periodically during the inoculation of carriers. Immediately spread the inoculum uniformly over the majority of the carrier surface using a sterile loop. Do not allow the inoculum to contact the edge of the glass slide carriers during the inoculation process. Cover dish immediately. g. Dry carriers in incubator at 36 ± 1°C for 30-40 min. Record the timed carrier inoculation activities on the AO AC Germicidal Spray Products Test Processing Sheet (see section 14). Perform efficacy testing within two hours of drying. 12.3 Enumeration of viable bacteria from carriers (control carrier counts) a. Assay dried carriers in 2 sets of three carriers, one set immediately prior to conducting the efficacy test and one set immediately following the test. Randomly select 6 inoculated carriers for carrier count analysis prior to efficacy testing. b. Place each of the inoculated, dried carriers in a 38 x 100 mm culture tube or sterile 50 mL polypropylene conical tube containing 20 mL of letheen broth. Vortex immediately - 60 ± 5 seconds fori5, aeruginosa or 120 ± 5 seconds for S. aureus and S. enterica. Record the time of sonication on the AO AC Germicidal Spray Products Test Processing ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 7 of 18 Sheet (see section 14). c. After vortexing, briefly mix and make serial ten-fold dilutions in 9 mL dilution blanks of PBDW. Refer to the AO AC Germicidal Spray Products Test Carrier Counts Form (see section 14). If the serial dilutions are not made and plated immediately, keep the tubes at 2-5°C until this step can be done. Complete the dilutions and plating within 2 h after vortexing. Alternatively, pool the letheen broth from the tubes with the carriers after vortexing. Briefly vortex after pooling. Serially dilute and plate an aliquot of the pooled media (60 mL). The average carrier count per set will be calculated. d. Briefly vortex each serial dilution tube prior to plating. Plate 0.1 mL aliquots of appropriate dilutions in duplicate on TSA or BAP using spread plating. Plate appropriate dilutions to achieve colony counts in the range of 30-300 colony forming units (CFU) per plate. Spread inoculum evenly over the surface of the agar. Plates must be dry prior to incubation. e. Incubate plates (inverted) at 36 ± 1°C for up to 48 ± 2 h. f. Count colonies. Plates that have colony counts over 300 will be reported as TNTC. Record counts on the AO AC Germicidal Spray Products Test Carrier Counts Form (see section 14). See section 13 for data analysis. g. Alternatively, Petrifilm may be used for enumeration of bacterial organisms. Follow manufacturer's instructions for preparation and incubation of Petrifilm cards. Note: A culture purity check should be conducted on one dilution of one carrier. 12.4 Disinfectant Sample Preparation a. Prepare disinfectant sample per SOP MB-22. 12.5 Test Procedure a. After the required carrier drying time, spray the slides sequentially for a specified time, distance, and number of pumps at timed intervals (typically 30 seconds) with the carriers in a horizontal position. Use a certified timer to time the spray interval. b. Spray the slide within ±5 seconds of the specified time for a 10 minute contact time or within ±3 seconds for contact times less than 1 minute. After spraying, maintain the carriers in a horizontal position. Treated carriers must be kept undisturbed during the contact time. c. After the last slide of a set (typically 20 slides) has been sprayed with ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 8 of 18 the disinfectant, and the exposure time is complete, sequentially transfer each slide into the primary subculture tube containing the appropriate neutralizer within the ±5 second time limit. Drain the excess disinfectant from each slide prior to transfer into the neutralizer tube. Drain carriers without touching the Petri dish or filter paper. Perform transfers with flame sterilized forceps. Alternatively, autoclaved forceps may be used. d. The slide can touch both the interior sides of the Petri dish and the subculture tube during the transfer, but avoid this contact as much as possible. e. After the slide is deposited, recap the subculture tube and shake culture thoroughly. f. Incubate at 36 ± 1°C for 48 ± 2 h. g. If a secondary subculture tube is deemed necessary to achieve neutralization, then transfer the carrier from the primary tube to a secondary tube of sterile medium after a minimum of 30 ± 5 min at room temperature from the end of the initial transfer. Within 25-60 min of the initial transfer, transfer the carriers using a sterile forceps to a second subculture tube containing 20 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. Incubate both the primary and secondary subculture tubes 48 ± 2 h at 36 ± 1°C. Record the results for both tubes (a carrier set) after this time. h. Record timed events on the AO AC Germicidal Spray Products Test Time Recording Sheet for Carrier Transfers (see section 14). 12.6 Sterility and viability controls a. Viability controls. Place 1 (or 2) dried inoculated untreated carrier(s) into separate tubes of the neutralizing subculture broth (if primary and secondary media are different). Incubate tubes with the efficacy test. Report results as + (growth) or 0 (no growth) as determined by presence or absence of turbidity. Growth should occur in both tubes. Record results on AO AC Germicidal Spray Products Test Results Sheet (see section 14). b. Sterility controls. Place one sterile, uninoculated carrier into a tube of neutralizing subculture broth. Incubate tube with the efficacy test. Report results as + (growth), or 0 (no growth) as determined by presence or absence of turbidity. Growth should not occur in the tube. Record results on AO AC Germicidal Spray Products Test Results ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 9 of 18 Sheet (see section 14). 12.7 Results a. Gently shake each tube prior to recording results. Record results as + (growth) or 0 (no growth) as determined by presence or absence of turbidity, on the AO AC Germicidal Spray Products Test Results Sheet (see section 14). b. If secondary subculture tubes are used, the primary and secondary subculture tubes for each carrier represent a "carrier set." A positive result in either the primary or secondary subculture tube is considered a positive result for a carrier set. c. Specialized neutralizedsubculture medium such as Dey/Engley broth will not show turbidity; rather the presence of pellicle at the surface of the medium (for P. aeruginosa) or a color change to the medium (yellow for growth of S. aureus or S. enterica) must be used to assess the results as a positive or negative outcome. i. Use viability controls for comparative determination of a positive tube. ii. If the product passes the performance standard, a minimum of 20% of the remaining negative tubes will be assayed for the presence of the test microbe using isolations streaks on TSA or BAP. Record preliminary results and conduct isolation streaks at 48 ± 2 h; however, continue to incubate negative tubes for up to an additional 24 hours to confirm the results.1 12.8 Confirmatory Steps for Test Microbes a. Confirm a minimum of three positive carrier sets per test. If there are less than three positive carriers, then confirm each carrier. If secondary subculture tubes are used and both tubes are positive in a carrier set, select only the tube with the carrier for confirmatory testing. b. For a test with greater than 20 positive carrier sets, confirm at least 20% by Gram staining, and a minimum of 4 positive carrier sets by Gram staining, solid media, and appropriate biochemical and antigenic analyses to ensure the identity of the organism. c. See Attachment 1 for Gram stain reactions, cell morphology, and colony characteristics on solid media. d. For additional confirmation steps refer to the appropriate Confirmation Flow Chart for S. aureus, P. aeruginosa, and S. enterica (see Attachment 3). e. If confirmatory testing determines that the identity of the unknown 1 Step not contained in the AO AC standard method 961.02. ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 10 of 18 was not the test organism, annotate the positive entry (+) on the results sheet to indicate a contaminant was present. 13. Data Analysis/ Calculations Calculations will be computed using a Microsoft Excel spreadsheet (see section 14). Both electronic and hard copies of the spreadsheet will be retained. Counts from 0 through 300 and their associated dilutions will be included in the calculations. 14. Forms and Data Sheets 1. Attachment 1: Typical Growth Characteristics of strains of P. aeruginosa, S. aureus, and S. enterica. 2. Attachment 2: Culture Initiation Flow Chart for S. aureus, P. aeruginosa, and S. enterica. 3. Attachment 3: Confirmation Flow Charts for S. aureus, P. aeruginosa and S. enterica. 4. Attachment 4: Photographs of spray apparatus. 5. Test Sheets. Test sheets are stored separately from the SOP under the following file names: Physical Screening of Carriers Record MB-03-05 Fl.docx Organism Culture Tracking Form (Frozen Stock MB-06-06 F2.docx Cultures) Test Microbe Confirmation Sheet (Quality MB-06-06 F3.docx Control) AOAC Germicidal Spray Products Test Carrier MB-06-06_F4.docx Counts Form AOAC Germicidal Spray Products Test Time MB-06-06_F5.docx Recording Sheet for Carrier Transfers AOAC Germicidal Spray Products Test MB-06-06_F6.docx Information Sheet AOAC Germicidal Spray Products Test Results MB-06-06 F7.docx Sheet (172°) AOAC Germicidal Spray Products Test Results MB-06-06 F8.docx Sheet (1°) Test Microbe Confirmation Sheet MB-06-06 F9.docx Carrier Count Spreadsheet MS Excel spreadsheet: MB-06-06_F10.xlsx Carrier Count Template_GSPT_v3 AOAC Germicidal Spray Products Test Carrier MB-06-06 F1 l.docx Counts Form (Pooled Carriers) AOAC Germicidal Spray Products Test MB-06-06_F12.docx Processing Sheet ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 11 of 18 15. References 1. Official Methods of Analysis. Revised 2012, publication pending. AO AC INTERNATIONAL, Gaithersburg, MD, (Method 961.02). 2. Krieg, Noel R. and Holt, John G. 1984. Bergey's Manual of Systematic Bacteriology Volume 1. Williams & Wilkins, Baltimore, MD. P. aeruginosa p. 164, S. enterica p. 447. 3. Sneath, P., Mair, N., Sharpe, M.E., and Holt, J. eds. 1986. Bergey's Manual of Systematic Bacteriology Volume 2. Williams & Wilkins, Baltimore, MD. S. aureus p. 1015. 4. Package Insert - Gram Stain Kit and Reagents. Becton, Dickinson and Company. Part no. 882020191JAA. Revision 07/2011. 5. Package Insert - Catalase Reagent Droppers. Becton, Dickinson and Company. Part no. L001237. Revision 06/2010. 6. Package Insert - Staphaurex Plus*. 11/23/07. Remel. Part no. R30950102. Revised 7. Package Insert - Oxidase Reagent Droppers. Becton, Dickinson and Company. Part no. LOO 1133. Revision 06/2010. 8. Package Insert-Wellcol ex* Colour Salmonella. Remel. Part no. R30858301. Revised 10/17/07 ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 12 of 18 Attachment 1 Typical Growth Characteristics of strains of P. aeruginosa, S. aureus, and S. enterica (see ref. 15.2 and 15.3). P. aeruginosa* S. aureus* S. enterica* Gram slain reaction (-) (+) (-) Typical Growth Characteristics on Solid Media Mannitol Salt No Growth circular, small, yellow colonies, agar turning fluorescent yellow N/A Cclrimide circular, small, initially opaque, turning fluorescent green over time; agar fluorescent yellowish green No Growth N/A Xylose lysine dcoxycholale (XLD) agar N/A N/A Round, clear red colonies with black centers Blood agar (BAP) flat, opaque to off-white, round spreading (1), metallic sheen, slightly beta hemolytic small, circular, yellow or white, glistening, beta hemolytic entire, glistening, circular, smooth, translucent, low co nvcx. no n-hc mo lytic Typical Microscopic Characteristics Cell dimensions 0.5-1.0 nm in diameter by 1.5-5.0 nm in length* 0.5-1.5 |im in diameter* 0.7-1.5 nm in diameter by 2.0-5.0 nm in length* Cell appearance straight or slightly curved rods, single polar flagella, rods formed in chains spherical, occurring singly, in pairs and tetrads, sometimes forming irregular clusters straight rods, peritrichous flagella *After 24±2 hours (1) Test organism may display three 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. Pyocyanin is not produced. ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 13 of 18 Attachment 2 Culture Initiation and Stock Culture Generation Flow Chart for S. aureus, P. aeruginosa, and S. enterica © Rehydrate ampule. © Transfer entire rehydrated pellet to TUBE A. Ampule Incubate TSB TUBE A TUBE A (pre-incubation) (post-incubation) ©Stock Culture Generation Inoculate TSA plates with 100 pL culture from TUBE A: incubate. Harvest inoculum from plates. ill Prepare frozen stock cultures © Culture ID & Quality Control BAP A Selective media Gram Stain Additional confirmation steps (see Attachment 3) Al. Preparation of Frozen Stock Cultures. Refer to SOP MB-02 for establishment of the organism control number. a. Initiate new stock cultures from lyophilized cultures of Pseudomonas aeruginosa (ATCC 15442), Staphylococcus aureus (ATCC 6538), and Salmonella enterica (ATCC 10708) from ATCC within 18 months. b. Open ampule of freeze dried organism as indicated by ATCC. Using a tube containing 5-6 mL of TSB, aseptically withdraw 0.5 to 1.0 mL and rehydrate the lyophilized culture. Aseptically transfer the entire rehydrated pellet back into the original tube of broth designated as "TUBE A". Mix well. c. Incubate broth culture (TUBE A) at 36 ± 1°C for 24 ± 2 hours. Record all ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 14 of 18 manipulations on the Organism Culture Tracking Form (see section 14). d. Using a sterile spreader, inoculate a sufficient number of TSA plates (e.g., 5 to 10 plates per organism) with 100 |iL each of the culture. Incubate plates at 36 ± 1°C for 24 ± 2 h. e. Following incubation, add 5 mL cryoprotectant solution (TSB with 15% v/v glycerol) to the surface of each agar plate. Re-suspend the cells in this solution using a sterile spreader or a sterile swab and aspirate the cell suspension from the surface of the agar. Transfer the suspension into a sterile vessel. Repeat by adding another 5 mL of cryoprotectant to the agar plates, re-suspend the cells, aspirate the suspension and pool with the initial cell suspension. f. Mix the pooled contents of the vessel thoroughly. Immediately after mixing, dispense approximately 1.0 mL aliquots into cryovials (e.g., 1.5 mL cyrovials). Perform QC of stock cultures concurrently with freezing (see section A2: QC of Stock Cultures). g. Place and store the cryovials at -70°C or below; these are the frozen stock cultures. Store stock cultures up to 18 months. These cultures are single-use only. A2. QC of Stock Cultures. a. Conduct QC of the pooled culture concurrently with freezing. Streak a loopful on a plate of BAP. In addition, for S. aureus and P. aeruginosa, streak a loopful onto both selective media (MSA and Cetrimide); for S. enterica, streak a loopful onto XLD. Incubate all plates at 36 ± 1°C for 24 ± 2 hours. b. Following the incubation period, record the colony morphology as observed on the BAPs and selective media plates (including the absence of growth) and Gram stain. See Attachment 1 for details on cell and colony morphology, colony characteristics on selective media, and stain reactions. c. For each organism, perform a Gram stain (refer to 15.5) from growth taken from the BAPs according to the manufacturer's instructions. Observe the Gram reaction by using brightfield microscopy at 1000X magnification (oil immersion). d. For additional confirmation steps refer to the appropriate Confirmation Flow Chart for S. aureus, P. aeruginosa, and S. enterica (see Attachment 3). Refer to 15.6-15.9 for instructions. e. Record all confirmation results on the Test Microbe Confirmation Sheet (Quality Control) (see section 14). ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 15 of 18 Attachment 3 Confirmation Flow Chart for S. aureus S. aureus Identification Yes No Positive Negative Positive Not S. aureus Identified as S. aureus Perform Catalase test Perform Staphaurex test BAP - Yellow colonies, (3 hemolytic MSA — Yellow zone around colonies Streak isolate from positive tube or stock cultures onto BAP and MSA. Perform Gram Stain and confirm GPC ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 16 of 18 Attachment 3 (cont.) Confirmation Flow Chart for P. aeruginosa P. aeruginosa Identification \ > es No Negative Positive Not P. aeruginosa Perform Oxidase from BAP colony Identified as P. aeruginosa Streak isolate from positive tube or stock cultures onto BAP and Cetrimide. Perform Gram Stain and confirm GNR BAP - Spreading colonies, "metallic sheen", slightly p hemolytic Cetrimide - Growth with yellow/green pigmentation (pyoverdin +) ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 17 of 18 Attachment 3 (cont.) Confirmation Flow Chart for S. enterica S. enterica Identification Yes No Positive Negative Perform Wellcolex Color Salmonella Test from BAP colony Streak isolate from positive tube or stock cultures onto BAP and XLD. Perform Gram Stain and confirm GNR BAP - Round colonies, non-hemolytic XLD - Red colonies with black centers (H2S positive) Not Salmonella Identified as S. enterica ------- SOP No. MB-06-06 Date Revised 01-15-13 Page 18 of 18 Attachment 4 ------- |