US Environmental Protection Agency Office of Pesticide Programs Office of Pesticide Programs Microbiology Laboratory Environmental Science Center, Ft. Meade, MD Standard Operating Procedure for Tracking of Test Microorganisms SOP Number: MB-02-05 Date Revised: 02-23-12 ------- SOP No. MB-02-05 Date Revised 02-23-12 Page 1 of 10 Initiated By: Technical Review: QA Review: Approved By: EPA/OPP MICROBIOLOGY LABORATORY ESC, Ft. Meade, MD Standard Operating Procedure for Tracking of Test Microorganisms SOP Number: MB-02-05 Date Revised: 02-23-12 Print Name: Print Name: Technical Staff Print Name: QA Officer Print Name: Branch Chief Date: / / Date: / / Date: / / Date: / / Effective Date: / / Controlled Copy No.: Withdrawn By: Date: / / ------- SOP No. MB-02-05 Date Revised 02-23-12 Page 2 of 10 TABLE OF CONTENTS Contents Page Number 1.0 SCOPE AND APPLICATION 3 2.0 DEFINITIONS 3 3.0 HEALTH AND SAFETY 3 4.0 CAUTIONS 3 5.0 INTERFERENCES 3 6.0 PERSONNEL QUALIFICATIONS 3 7.0 SPECIAL APPARATUS AND MATERIALS 3 8.0 INSTRUMENT OR METHOD CALIBRATION 3 9.0 SAMPLE HANDLING AND STORAGE 4 10.0 PROCEDURE AND ANALYSIS 4 11.0 DATA ANALYSIS/CALCULATIONS 6 12.0 DATA MANAGEMENT/RECORDS MANAGEMENT 6 13.0 QUALITY CONTROL 6 14.0 NONCONFORMANCE AND CORRECTIVE ACTION 6 15.0 REFERENCES 6 16.0 FORMS AM) DATA SHEETS 6 ------- SOP No. MB-02-05 Date Revised 02-23-12 Page 3 of 10 1.0 SCOPE AND APPLICATION: 1.1 This protocol provides guidance for establishing receipt and expiration dates for microorganisms used in the Microbiology Laboratory Branch (MLB) as well as denotation and tracking of those microorganisms. 2.0 DEFINITIONS: 2.1 ATCC = American Type Culture Collection 3.0 HEALTH AND SAFETY: 3.1 All manipulations of the test organisms are required to be performed in accordance with biosafety practices stipulated in SOP MB-01, Lab Biosafety. 4.0 CAUTIONS: 4.1 Expired ampules of freeze-dried organisms are not used and will be discarded appropriately. 5.0 INTERFERENCES: None. 6.0 PERSONNEL QUALIFICATIONS: 6.1 Personnel are required to be knowledgeable about and to comply with the laboratory's culturing and disinfectant testing procedures. Documentation of training and familiarization with these procedures can be found in the training file for each employee. 7.0 SPECIAL APPARATUS AND MATERIALS: 7.1 Pseudomonas aeruginosa (ATCC 15442), Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 19659), and Salmonella enterica (ATCC 10708); ordered annually and received directly from ATCC. 7.2 Mycobacterium bovis (BCG); ordered and received directly from Organon Teknika. 7.3 Other microorganisms used in the microbiology laboratory may be purchased from appropriate vendors when necessary. 8.0 INSTRUMENT OR METHOD CALIBRATION: ------- SOP No. MB-02-05 Date Revised 02-23-12 Page 4 of 10 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: Not applicable. 10.0 PROCEDURE AND ANALYSIS: 10.1 Supply Control Number 10.1.1 All freeze-dried ampules are given a supply control number upon receipt (see SOP QC-09, Control Numbers). 10.1.2 The supply control number for ampules consists of the date the ampule was received (R) and the date the ampule expires (E). For example: For a dehydrated ampule of S. aureus received on 01-05- 12 and expiring on 03-12-13, the supply control number would be R010512E031213. For a dehydrated ampule ofM bovis (BCG) received on 07-24-11 and expiring on 09-04-13, the supply control number would be R072411E090413. 10.2 Organism Control Number. 10.2.1 The organism control number consists of the date the reconstituted microbe expires (ME), a two-letter suffix denoting the organism, and any culture transfer notation (refer to section 10.3 for culture transfer notation). 10.2.1.1 The two-letters in the suffix of the organism control number denote the genus and species of the organism (for example, Pa fori5, aeruginosa, Sa for S. aureus, Bs forB. subtilis, and Se for S. enterica). Other microorganisms used in the microbiology laboratory will be given two-letter suffixes consistent with the notation listed above. 10.2.2 P. aeruginosa, S. aureus, B. subtilis, and S. enterica. 10.2.2.1 A culture of S. aureus reconstituted on 02-03-12 would receive an organism control number of ME020313-Sa followed by any culture transfer notations. 10.2.2.2 Once reconstituted, P. aeruginosa, S. aureus, B. subtilis, ------- SOP No. MB-02-05 Date Revised 02-23-12 Page 5 of 10 and S. enterica may only be subcultured for a period of one year. Any cultures, including stock cultures, daily transfers, and test cultures, are valid for up to 12 months after reconstitution of the lyophilized culture (the ME date). In the case provided (section 10.2.1.1), stock cultures and any resulting test cultures are valid through 02-03-13. 10.2.2.3 Once expired, the stock cultures must be autoclaved and discarded and a new culture initiated from a new unexpired lyophilized lot from ATCC. 10.2.3 M. bovis (BCG). 10.2.3.1 M. bo vis (BCG) is not required to be replaced annually and does not have an expiration date for stocks (based on AO AC Method 965.12). A re-ordering cycle for new cultures has not been established. Therefore, the culture notation consists of MEOOOOOO-Mb followed by any culture transfer notations. Continuous transfers of M. bovis (BCG) may be made unless the organism has been compromised. 10.3 Culture Transfer Notation of Test Microbes. 10.3.1 See footnotes for Organism Culture Tracking Form for culture transfer notations for S. aureus, P. aeruginosa, and S. enterica (see section 16.0). For example, a Staphylococcus test culture notation would be MEXXXXXX-Sa-1104TC, where 11 is the monthly culture transfer (number of months since the month of initiation) and 04 is the daily culture transfer (4th daily culture transfer); TC is applied to indicate a 48 hour test culture. 10.3.2 See footnotes for Organism Culture Tracking Form tor Mycobacterium bovis (BCG) culture transfer notations (see section 16.0). For M. bovis (BCG), the test culture notation would be MEXXXXXX-Mb-1103SL, where 11 represents the month of culture transfer (the month of the year) and 03 represents the week of the month for that transfer (the 3rd week of the month). 10.3.2.1 SC is applied to identify a stock culture (e.g., a transfer from a slant to a slant). SL is applied to identify the test culture (this indicates a transfer from a slant culture to a ------- SOP No. MB-02-05 Date Revised 02-23-12 Page 6 of 10 liquid culture). LL can also be applied to identify a test culture (this indicates a transfer from a liquid MPB culture to another liquid culture). 10.3.2.2 The weeks of each month are numbered consecutively starting with the 1st Monday of the month (as 01) and ending with the last Monday of the month (depending on the number of Mondays in the month, as either 04 or 05). 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 Test Microbe Conformation Sheets and the Organism Culture Tracking Forms. 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 appropriate record form(s) (see section 16.0 of this SOP as well as MB-05, MB- 06, MB-07, MB-09, MB-23 and MB-24). 14.0 NONCONFORMANCE AND CORRECTIVE ACTION: 14.1 Entry errors will be corrected upon discovery. 15.0 REFERENCES: None. 16.0 FORMS AND DATA SHEETS: 16.1 Organi sm Culture Tracking Form 16.2 Test Microbe Confirmation Sheet (Quality Control) 16.3 Organism Culture Tracking Form tor Mycobacterium bovis (BCG) 16.4 Test Microbe Confirmation Sheet (Quality Control) for Mycobacterium bovis (BCG) ------- SOP No. MB-02-05 Date Revised 02-23-12 Page 7 of 10 16.1 ORGANISM CULTURE TRACKING FORM OPP Microbiology Laboratory Organism/ATCC #: Organism Control No.: Supply Control Number (for ampule): Dale Microbe Initialed: Lot Number (for ampule): Notes: Dale Time Init. Subculture Source Transfer* Media Inoculated (and # inoculated) Media Prep No. Incubation Conditions Comments Monthly Daily * "Monthly" indicates the monthly transfers for culture and "Daily" indicates a 24/48 hr serial transfer (added to organism control number) NR = None Required, TC = Test Culture, applied after daily transfer number TSB = Tryptic soy broth, NB = nutrient broth ------- SOP No. MB-02-05 Date Revised 02-23-12 Page 8 of 10 16.2 TEST MICROBE CONFIRMATION SHEET (Quality Control) OPP Microbiology Laboratory Organism/ATCC #: Organism Control No.: Supply Control Number (for ampule): Dale Microbe Initialed: Lot Number (for ampule): Notes: Source: Tube/Plate ID Dale/ Initials Staining Results* Media Information Results Name Prep. No. Inc. Time/ Temp. Date/ Initials Colony Characteristics Vitek #** * Record Gram stain results: GPC = Gram Positive Cocci; GNR = Gram Negative Rods ** Vitek tracking number and organism identification TSA = trypticase soy agar, MSA = mannitol salt agar, NA = nutrient agar ------- SOP No. MB-02-05 Date Revised 02-23-12 Page 9 of 10 16.3 ORGANISM CULTURE TRACKING FORM for Mycobacterium bovis (BCG) OPP Microbiology Laboratory Organism/ATCC #: Organism Control No.: Supply Control Number (for ampule): Date Microbe Initialed: Lot Number (for ampule): Notes: Date Time Init. Subculture Source Transfer Media Inoculated (and # inoculated) Media Prep No. Incubation Conditions Comments Month* Week** * Indicates the month of the year the culture was transferred; added to organism control number ** Indicates the week of the month for the transfer (week 1-5); added to organism control number. Note: Weekly (SL or LL) cultures may be used on days 21-25. SL = slant to liquid (indicates a test culture), LL = liquid to liquid (indicates a test culture), applied after weekly transfer number; NR = None Required; N/A = Not Applicable; SC = Stock Culture; QC = Quality Control MPB = Modified Proskauer Beck broth, M7H9 = Middlebrook 7H9, M7H11 = Middlebrook 7H11 ------- SOP No. MB-02-05 Date Revised 02-23-12 Page 10 of 10 16.4 TEST MICROBE CONFIRMATION SHEET (Quality Control) for Mycobacterium bovis (BCG) OPP Microbiology Laboratory Organism/ATCC #: Organism Control No.: Supply Control Number (for ampule): Date Microbe Initiated: Lot Number (for ampule): Notes: Source: Tube/Plate ID Date/Initials Staining Results* Media Information Results Name Prep. No. Inc. Time/ Temp. Date/ Initials Colony Characteristics *Record Acid Fast staining results (AFR = Acid Fast rods) M7H9 = Middlebrook 7H9, M7H11 = Middlebrook 7H11 ------- |