US Environmental Protection Agency Office of Pesticide Programs Office of Pesticide Programs Microbiology Laboratory Environmental Science Center, Ft. Meade, MD Standard Operating Procedure for Media and Reagents Preparation and Quality Evaluation SOP Number: MB-10-04 Date Revised: 05-07-13 ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 1 of 15 SOP Number MB-10-04 Title Media and Reagents Preparation and Quality Evaluation Scope Describes the procedures used to log-in, prepare, and evaluate the quality of media and reagents used in microbiological assays by the Microbiology Laboratory Branch (MLB). Application For use in the quality evaluation of media and reagents used by MLB. 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Name: Quality Assurance Unit Print Name: Branch Chief Print Name: Date SOP issued: Controlled copy number: Date SOP withdrawn: ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 2 of 15 TABLE OF CONTENTS Contents Page Number 1.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 4 13. DATA ANALYSIS/CALCULATIONS 10 14. FORMS AND DATA SHEETS 10 15. REFERENCES 11 ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 3 of 15 1. Definitions Additional abbreviations/definitions are provided in the text. 1. General growth media = Media which support the growth of a broad range of organisms and are used for the general cultivation and maintenance of microorganisms. General growth media are non- selective. Examples include nutrient agar and tryptic soy agar. 2. Selective media = Media that permit the growth of one type of bacterium while inhibiting the growth of other types. Selective media facilitate the isolation of a desired species. Examples include mannitol salt agar and cetrimide agar. 3. CFU = Colony forming unit. 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 chemicals. 3. Personnel Qualifications and Training Refer to SOP ADM-04, OPP Microbiology Laboratory Training. 4. Instrument Calibration Refer to SOP EQ-01-06, EQ-02-06, EQ-03-05, and EQ-08-05 for details on method and frequency of calibration. 5. Sample Handling and Storage Not applicable 6. Quality Control 1. Document the required information on the appropriate forms (see section 14). 2. Process re-usable glassware in Miele or Lancer dishwashers and check glassware for detergent residues as noted in SOP-QC-03, Glass Washing and Detergent Residues Test. 3. Use de-ionized water to prepare media. Water must meet the quality indicators noted in SOP-QC-01, Quality Assurance of Purified Water. 7. Interferences 1. Discard media if a media preparation number or sterilization batch number is illegible or missing and cannot be determined from the Media/Reagent Preparation Sheet or the Daily Sterilization Record Information Log Form (see 14). 2. Inspect all pre-sterilized laboratory supplies upon receipt for damage or torn packaging; if supplies are damaged they will be discarded. 3. Initiate routine sterility checks of pre-sterilized items if contamination in a test is due to pre-sterilized items. 8. Non- conforming Management of non-conforming data will be specified in the standard test method; procedures will be consistent with SOP ADM-07, Non- ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 4 of 15 Data Conformance Reports. 9. Data Management Data will be archived consistent with SOP ADM-03, Records and Archives. 10. Cautions 1. Do not use products past the manufacturer's recommended expiration date. 2. Use volumetric glassware as indicated on Media Preparation Sheets. 3. Allow hot autoclaved media to equilibrate for a minimum of 30 minutes prior to plating or addition of heat-sensitive additives. 4. Reassessment of the sterility of media and reagents not used in a timely manner after preparation and/or have been in storage for more than one month is strongly recommended prior to use. See guidance in section 12.5. 11. Special Apparatus and Materials 1. Autoclaves (refer to QC-13) 2. Water baths (refer to Attachment 1) 3. Thermometers (refer to EQ-02) 4. Inoculating loops 5. Cultures of Pseudomonas aeruginosa (ATCC # 15442), Staphylococcus aureus (ATCC #6538), and Salmonella enterica (ATCC #10708). 6. Culture of Mycobacterium bovis (BCG) from Organon Teknika. 7. Spore suspension of Bacillus subtilis (ATCC #19659); a commercial preparation of spores can be purchased. One source for the commercial preparation is Presque Isle Cultures, 3804 West Lake Rd., P.O. Box 8191, Erie PA 16505. In addition a B. subtilis spore suspension can be generated in-house. 8. Spore suspension of Clostridium sporogenes (ATCC # 3584) prepared in-house. 9. Any additional microbes used in the laboratory. 10. Pre-sterilized filtration unit with pore size of 0.2 |im or 0.45 |im such as Nalgene Analytical Filter Units. 12. Procedure and Analysis 1. Utilize approved media preparation sheets. Approved media preparation sheets are archived on the G-drive. 2. Select appropriate preparation sheet and the required volume. 12.1 Preparation of Media and Reagents a. Assign a media preparation number for all media, reagents, and carriers prepared in the laboratory. Enter this number into the Media/Reagent Preparation Log Form and the Media/Reagent Preparation Sheet (see 14). The media preparation number consists ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 5 of 15 of two parts: i. The first seven digits represent the date the medium or reagent was prepared: P-MMDDYY where P=Prepared, MM=month, DD=day and YY=the last two digits of the calendar year. ii. The suffix, where the digits after the dash act as a counter for the number of preparations made on the same date. For example, the first preparation made on January 8, 2013 would have the media preparation number P-010813-01. The next item prepared on that same day would have a suffix of -02; the third preparation made on that same day would have a suffix of-03, etc. b. Label each preparation of media or reagent clearly with the name of the preparation and the media preparation number. c. Strictly follow the specific directions for preparation of media and reagents as listed on the Preparation/Modifications/Notes section of the Media/Reagent Preparation Sheet. d. Complete all fields on the media preparation sheet with the appropriate information for that section. If a section is not applicable to the item being prepared, place N/A (not applicable) in that section. e. Instructions for sterilization of media and reagents as listed on the Media/Reagent Preparation Sheet. i. For media and reagents that are sterilized by autoclaving, assign a sterilization batch number. The sterilization batch number consists of two parts: the first seven digits represent the date the batch was sterilized: S-MMDDYY where S=Sterilization, MM=month, DD=day and YY=the last two digits of the calendar year. ii. The suffix where the first digit after the dash indicates the autoclave used and the next two digits act as a counter for the number of preparations made on the same date. iii. For example, the first batch sterilized on January 8, 2013 in autoclave 1 (Room B206) would have the sterilization batch number S-010813-101. The next batch sterilized on that same day and same autoclave would have a suffix of-102, the third batch sterilized would have a suffix of-103; etc. iv. Record the sterilization batch number in the Sterilization section on the Media/Reagent Preparation Sheet as well as in the Daily Sterilization Record Information Log Form (see ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 6 of 15 14). v. For media and reagents that are filter sterilized, record the sterilization mechanism in the Sterilization section on the Media/Reagent Preparation Sheet. vi. For media and reagents that do not require sterilization, record N/A in the Sterilization section on the Media/Reagent Preparation Sheet. f After autoclaving, record the pH of the media or reagent at room temperature (20°C to 25 °C) unless otherwise specified on the Media/Reagent Preparation Sheet. When the pH reading is stable, record the final pH on the sheet with the temperature in the Final pH After Autoclaving section. g. Discard the batch of media if the pH falls outside of the desired range as specified on the Media/Reagent Preparation Sheet. 12.2 Storage, Shelf Life and Inspection of Media and Reagents The storage and shelf life requirements for the media or reagent are listed at the bottom of the Media/Reagent Preparation Sheet. See Attachment 2 for detailed instructions. 12.3 Media Performance Assessment Conduct the performance verification of media prior to or concurrently with use, preferably with the organism that corresponds to the anticipated use of the medium. Media controls used in neutralization confirmation studies or titer assays may also be used in place of specific media performance tests. a. Culture Preparation: Use appropriate daily culture, test culture or standardized spore suspension for the purpose of inoculating media for performance assessment. For media prepared for use in research projects, stock cultures or test cultures generated for use in that project may be used for media performance. Refer to the relevant procedures for culture preparation. b. Inoculation and Incubation of Solid Media: For each preparation of solid media in plates or tubes (e.g, tryptic soy agar, nutrient agar, Middlebrook 7H9), use 6-8 plates to assess media performance. Spread plate, in duplicate, 3-4 serial ten-fold dilutions of the test microbe. i. Target counts of 30-300 CFU/plate are desirable and should result from at least one of the plated dilutions - this dilution will serve as the basis for determining media performance. The dilutions should result in plate counts which are too numerous to count (TNTC) through extinction or near ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 7 of 15 extinction levels. ii. For solid media in tubes (e.g., nutrient agar slants, Middlebrook 7H9 slants), inoculate a minimum of2 tubes per batch with an undiluted culture of the test microbe. iii. For selective media in plates or tubes (e.g., mannitol salt agar, cetrimide agar), streak (for plates) or stab (for tubes) inoculate a minimum of 2 tubes or plates with an undiluted culture of the appropriate target organism (i.e., the organism the media is designed to identify). Perform an isolation streak on selective media in plates to aid in the assessment of the medium's reaction and organism's colony characteristics. iv. See Attachment 3 for detailed instructions. c. Inoculation and Incubation of Liquid Media: For liquid media (e.g., letheen broth, nutrient broth, Modified Proskauer Beck medium), evaluate 6-8 tubes of each batch for performance testing. Inoculate tubes in duplicate with 0.1 mL aliquots of 3-4 serial ten- fold dilutions of the appropriate culture. i. Verily CFU/tube by spread plating in duplicate on the appropriate medium. See Attachment 3 for detailed instructions. 12.4 Performance Results for Solid and Liquid Media a. Performance Results for Solid Media: i. Enumerate the colonies per plate, determine the average CFU/plate and CFU/mL of diluted inoculum, and assess the colony morphology. ii. Record findings on the Performance and Sterility Assessment of Media in Plates form (see 14). iii. If growth occurs and exhibits typical morphology, record a "+". If no growth is apparent, record a "0". iv. If atypical growth is observed (not the test microbe), record as contaminant. v. If the inoculum titer is significantly below the target of 30- 300 CFU, then either the media performance is unsatisfactory or the starting inoculum was substandard; media performance assessment should be repeated. vi. For selective media, verify the performance per the appropriate media reactions (e.g. agar turning fluorescent green for cetrimide agar) or colony characteristics, see corresponding method SOPs MB-05 and MB-07. Complete ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 8 of 15 the form (see 14) under the "Performance Assessment" caption by checking either Satisfactory or Unsatisfactory per the observations. vii. For media in tubes, record the presence or absence of growth on the Performance and Sterility Assessment of Media in Tubes form (see 14). viii. For plates, enumerate the colonies per plate, determine the average CFU/plate and CFU/mL of diluted inoculum, and assess the colony morphology (refer to SOP MB-02: Tracking of Test Microorganisms for colony characteristics). Record findings on the Performance and Sterility Assessment of Media in Plates form (see 14). ix. For tubes, record the presence or absence of growth. Record findings on the Performance and Sterility Assessment ofMedia in Tubes form (see 14). x. For tubes inoculated withM bay is (BCG), determine the presence or absence of growth by assessing each tube for typical growth in tubes. Growth on solid media in tubes is represented by the presence of rough, raised, thick colonies with a nodular or wrinkled surface and an irregular thin margin, off-white to faint buff or even yellow. b. Performance Results for Liquid Media: i. Record findings on the Performance and Sterility Assessment of Liquid Media form (see 14). ii. For each tube, record a "+"if growth is observed (indicated by turbidity or growth) or a "0" if growth is not observed. iii. If atypical growth is ob served (not the test microbe), record as contaminant. iv. Following incubation, assess the performance of each medium and record observations on the Performance and Sterility Assessment of Media in Tubes form (see 14). Read plate counts to determine the CFU/tube. v. Performance is judged to be satisfactory when at least one of the two tubes in a dilution set that received a sufficiently low challenge (1-50 CFU/tube) of the test microbe shows growth. The number of CFUs delivered to each tube in a set is based on the corresponding averaged plate counts for that dilution. All tubes in dilution sets receiving greater than the targeted challenge should show growth as well. Based on this criterion under the "Performance ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 9 of 15 Assessment" caption on the form, check either Satisfactory or Unsatisfactory. 12.5 Sterility Verification of Solid and Liquid Media Verify sterility on a minimum of 2% of each preparation of solid and liquid media. a. Place plates or tubes of media, for use with organisms other than M. bovis (BCG), in a 36 ± 1°C incubator for 3-10 days. Evaluate sterility for media purchased from a vendor as well. b. Incubate solid and liquid media used for the growth ofM bovis (BCG) at 36 ± 1°C for 17-21 days. c. Following incubation if no growth on solid media or in liquid media is observed, record a "0" (satisfactory). If growth is observed, record a "+" (unsatisfactory) on the appropriate Performance and Sterility Assessment form (see 14). On each form, fill in "Sterility Assessment" by indicating either Satisfactory or Unsatisfactory per the observations. d. For solid media, a satisfactory result for sterility is typically based on no microbial growth observed in the plates or tubes following incubation. Although infrequent, an occasional bacterial or fungal colony may appear on the surface of the agar; in these instances, examine the remaining plates of the preparation in question prior to use to determine the extent of the contamination, and perform an additional sterility assessment. If any growth is observed in the second assessment, discard the medium. 12.6 Sterility Verification of Reagents (not used to support microbial growth) a. If a reagent is dispensed into multiple bottles, evaluate only one bottle per preparation for sterility. b. Use a pre-sterilized 0.45 |imor0.2 |im filter unit and aseptically filter approximately 2% of the volume from one bottle of reagent. c. Do not place anything such as a pipette into the reagent bottle. d. Aseptically transfer the filter to a TSA or NA plate, incubate for 3- 10 days at 36 ± 1°C, and assess filter for presence of microbial growth. e. For reagents that are dispensed in tubes, aseptically filter the number of tubes that represents approximately 2% of the total volume prepared through one pre-sterilized 0.45 |am or 0,2 |im filter unit, plate the filter on TSA or NA, and incubate for 3-10 days at 36 ± 1°C. f For example, suppose 1 L of dilution water is prepared in 9 mL aliquots. 2% of 1 L is 20 mL; therefore filter the contents of 3 tub es (27 mL). ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 10 of 15 g. Following incubation of filter, if no growth is observed, record a "0" (satisfactory). If growth is observed, record a "+" (unsatisfactory). Record the assay results and observations on the Sterility Verification for Reagents form (see 14). 12.7 Sterility of Carriers a. For stainless steel and porcelain carriers or other small carriers (e.g., 5x5x1 mm glass coupons), place one carrier per autoclaved preparation into a 10 mL tube ofFluid Thioglycollate Medium (FTM) or Letheen Broth (LB) and incubate for 3-10 days at 36 ± rc. b. For glass slide carriers, place one carrier per autoclaved batch into a 20 mL tube of FTM or LB and incubate for 3-10 days at 36 ± 1°C. c. Following incubation, if no growth is observed, record a "0" and if growth is observed record a "+" on the appropriate form. Record the assay results and observations on the Sterility Verification of Official Carriers form (see 14). 13. Data Analysis/ Calculations 1. Verify that the amounts of ingredients specified in the recipe are accurate. For example, if 2L of FTM are made, the prep sheet should show the calculation of 29.8 g for 1 liter multiplied by 2: 29.8 x 2 = 59.6 g dehydrated medium required to prepare 2 liters of FTM. 2. Calculate the percent difference due to evaporation or loss during autoclaving. The percent difference is determined using the formula: Percent Difference = 100% - [(Measured Volume/Target Value) x 100] 14. Forms and Data Sheets 1. Attachment 1: List of Water Baths 2. Attachment 2: Storage and Inspection of Media and Reagents 3. Attachment 3: Inoculation and Incubation of Solid and Liauid Media 4. Test Sheets. Test sheets are stored separately from the SOP under the following file names: Media/Reagent Preparation Log Form MB-10-04 Fl.docx Blank Media/Reagent Preparation Sheet MB-10-04 F2.xlsx Daily Sterilization Record Log Form MB-10-04 F3.docx Water Bath Temperature Record MB-10-04 F4.docx Performance and Sterility Assessment of Solid MB-10-04 F5.xlsx Media in Plates Performance and Sterility Assessment of Liquid MB-10-04 F6.xlsx and Solid Media in Tubes ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 11 of 15 Sterility Verification of Reagents MB-10-04 F7.xlsx Sterility Verification of Official Carriers MB-10-04 F8.xlsx 15. References 1. Official Methods of Analysis. Revised 2013. 18th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, (Methods 955.14, 955.15, 964.02, 961.02, and 966.04). 2. Official Methods of Analysis. Revised 2012. 18th Ed., AOAC INTERNATIONAL, Gaithersburg, MD, (Method 965.12). ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 12 of 15 Attachment 1 List of Water Baths a. Precision Scientific Series 180: Water bath# 1, Serial Number 698091238. b. Precision Scientific Series 183: Water bath # 2, Serial Number 9505426. c. Precision Scientific Series 180: Water bath# 3, Serial Number 698101642. d. Scientific Products (S/P): Water bath # 4, Serial Number 0678. e. VWR Model 89032-206: Water bath #5, Serial Number WF0823006. ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 13 of 15 Attachment 2. Storage and Inspection of Media and Reagents Media Type Storage Inspection Liquid Media in Tubes with Morton Closures Store at room temperature for up to 2 months in plastic bags. Prior to use, inspect liquid media in tubes with Morton Closures stored longer than one month. Check the fluid level in approximately 5% per batch and/or per rack of media to be used in testing to determine if more than 5% of the fluid has evaporated. If less than 5% of the solution has evaporated, the media can be used; otherwise discard the media. Liquid Media in Capped Tubes or Bottles Store for up to 3 months (tightly capped). Prior to use, inspect liquid media in tubes with screw caps stored longer than two months. Check the fluid level in approximately 5% per batch and/or per rack of media to be used in testing to determine if more than 5% of the fluid has evaporated. If less than 5% of the solution has evaporated, the media can be used; otherwise discard the media. Reagents If not labile or harmed by long storage periods, use for up to one year from the date of preparation. If a recommended shelf life is known, use within that time. Do not use commercially prepared reagents beyond the manufacturer's expiration date. Prior to use, inspect reagents for any sign of contamination and discard appropriately if necessary. For dilution blanks, determine if more than 2% of the fluid level has evaporated. If more than 2% of the fluid has evaporated, adjust the volume in each tube prior to use or prepare a new batch as necessary. ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 14 of 15 Media Type Storage Inspection Solid Media in Plates Store in plastic bags at 2-5°C or at room temperature, agar-side up. Puncture the plastic bags in a few areas to allow air exchange and to reduce excessive condensation. Store both selective and non-selective media for up to 3 months. Prior to use, look for evidence of contamination, uneven filling or bubbles on the surface of agar, color changes, hemolysis and signs of dehydration such as shrinking, cracking and loss of volume. Discard any defective plates. Solid Media in Tubes Store both selective and non-selective media at 2-5°C for up to 3 months. Prior to use, look for evidence of contamination, uneven filling, color changes, hemolysis and signs of dehydration such as shrinking, cracking and loss of volume. Discard any defective tubes. Commercially Purchased Media Do not use commercially prepared media beyond the manufacturer's expiration date. Prior to use, look for evidence of contamination, uneven filling, color changes, hemolysis and signs of dehydration such as shrinking, cracking and loss of volume. Discard any defective media tubes or plates. ------- SOP No. MB-10-04 Date Revised 05-07-13 Page 15 of 15 Attachment 3. Inoculation and Incubation of Solid and Liquid Media Test Microbe Inoculation of Solid and Liquid Media Incubation of Solid and Liquid Media S. aureus, P. aeruginosa, S. enterica and B. subtilis Inoculate tubes and plates with 0.1 mL of the 10"5 to 10"7 dilutions. The dilutions plated assume an initial titer of approximately 10 to 109 CFU/mL. Incubate plates and/or tubes at 36 ± 1°C for 24- 48 h. C. sporogenes Inoculate tubes and plates with 0.1 mL of the 10° to 10"7 dilutions. The dilutions plated assume an initial titer of approximately 10 to 109 CFU/mL. Incubate plates and/or at 36 ± 1°C for 24-48 h under anaerobic conditions (e.g. Gas-Pak anaerobic environment generation system). M. bovis (BCG) Inoculate tubes and plates with 0.1 mL of the 10" 3, 104,10"5 and 10"6 dilutions of the standardized culture. The dilutions plated assume an initial culture titer of 107 to 108 CFU/mL of standardized culture. Note: The lowest dilutions (10"3 and 10"4) will likely show CFU levels which are TNTC. Incubate plates and/or tubes at 36 ± 1°C for 17- 21 days. ------- |