LENTICULE discs Certified Reference Materials for Microbiology Produced in double accredited laboratory fulfilling ISO/IEC 17025 and ISO Guide 34 Escherichia coli CRM09001M This certificate should not be parted from the certified reference material with batch number: BCBS3252V Uncertified data and guidance for use can be found at the end of this certificate. 1. Public Health England (PHE) - Manufacturing of Reference Material (Bulk) Sigma-Aldrich - Filling, Packaging and Distribution This certificate is designed and the reference material values and uncertainties are determined in accordance with: ISO Guide 31:2000 Reference materials contents of certificates and labels ISO Guide 34:2009 General requirements for the competence of reference materials producers and ISO Guide 35:2006 Reference materials general and statistical principles for certification. Manufacturing and testing of the Bulk reference material is done by Public Health England (PHE). Preparation and retesting of the final product ready for use for customers, which includes filling, packaging and distribution, is done by Sigma-Aldrich. 2. Description of Reference Material Bacteria in a pure culture preserved in a tablet format (LENTICULE disc) with a self-indicating silica gel desiccant. The micro-organisms provided in LENTICULE disc format are classified as Hazard Group 2 according to the UK Advisory Committee on Dangerous Pathogens using the Approved List of Biological Agents http://www.hse.gov.uk/pubns/misc208.pdf. A Group 2 organism may cause human disease and may be a hazard to laboratory workers, but is unlikely to spread to the community. These products are dispatched under the Dangerous Goods Classification: UN 3373 Biological substances, Category B. Refer to the Safety Data Sheet for further information: http://www.sigmaaldrich.com/msds The silica gel self-indicating inserts are not classified as dangerous material. Page 1 of 10
Catalogue number: Lot number: CRM09001M BCBS3252V Expiry date: May 2017 Starting material: NCTC 9001 - Escherichia coli (Freeze-dried microorganism in a glass ampoule) Starting Material Batch: NCTC 9001 Batch 16 3. Certified Values and Uncertainties Organism name: Escherichia coli NCTC number: 9001 Conditions: Columbia blood agar / aerobic / 30 o C / 48 hrs Geometric Mean Value: 5.1 x 10 3 colony forming units (cfu) Geometric Mean +3SD 1.5 x 10 4 colony forming units (cfu) Geometric Mean +2SD 1.2 x 10 4 colony forming units (cfu) Geometric Mean -2SD 2.2 x 10 3 colony forming units (cfu) Geometric Mean -3SD 1.8 x 10 3 colony forming units (cfu) Expanded uncertainty (log 10 value): 0.031 (95% CI; coverage factor = 2) Expected range: 2.2 x 10 3-1.2 x 10 4 cfu per LENTICULE disc The measurement of uncertainty (MoU) originates from the standard deviation (SD) resulting from the geometric mean value obtained during homogeneity testing. It takes into account media batch to batch variability, which is done by multiplying within batch standard deviation by 1.6. 4. PHE (FEPTU) Quality Control Data for Geometric Mean Value QC batch size: 45 LENTICULE discs Date of testing: 6 June 2016 Conditions: Columbia blood agar / aerobic / 30 o C / 48 hrs Page 2 of 10
The counts were undertaken by surface inoculation and incubation as described above. This is in accordance with the technique described in PHE s Food and Environmental Proficiency Testing Unit (FEPTU) Method Manual for Food Microbiology Section 12: Method for the Enumeration of Microorganisms - Colony Count Technique at 22 ºC and 30 ºC (aerobic colony counts) which is based on ISO 4833: Part 2. Microbiology of food and animal feeding stuffs - Horizontal method for the enumeration of microorganisms - colony count at 30 ºC by the surface plating technique. 5. Traceability to NCTC culture by Independent Means Metrological traceability chain - biological (qualitative) This strain is traceable to seed stock for Escherichia coli NCTC 9001, batch NCTC 9001 Batch 16 and is manufactured from one sub-culture ensuring that the characteristics are retained. This strain has been verified by PHE s Applied and Functional Genomics (AFG) Unit at Microbiology Services (Colindale) using fluorescent amplified fragment length polymorphism (FAFLP) analysis, and is traceable back to the NCTC seed stock (refer to page 2 for Starting Material Batch details). PHE s AFG Unit is accredited by Clinical Pathology Accreditation (UK) Ltd (CPA) and is audited by FEPTU to ISO/IEC 17025:2005 - General requirements for the competence of testing and calibration laboratories. Reference: Desai, M; Russell J.E; and Gharbia S. (2006) Comparison of the genetic stability of paired reference strains preserved by freeze-drying and on LENTICULE discs with fluorescent amplified fragment length polymorphism. FEMS Microbiology Letters 257:1 158. Page 3 of 10
Metrological traceability to a measurement unit (quantitative) The viable organism level per LENTICULE disc is measured by PHE as colony forming units (cfu). This is carried out using plate culturing techniques based on ISO 4833 (see section 4). Where the reference material contains a high level of organisms (>5 x 10 3 ), a Spiral plater (Don Whitely WASP2) is used to enumerate the seed stock and the reference material produced as LENTICULE discs. The cfu are counted per plate and the cfu per disc is calculated using Don Whitley Counting Tables (WASP 2 user manual Section 15: appendices). NCTC ampoules used for the starting material are not quantified. The viable organism level per LENTICULE disc is reconfirmed as colony forming units (cfu) by Sigma-Aldrich after packaging. 6. Instructions for Correct Storage of this Reference Material On receipt store the mylar bag containing the plastic vials with the LENTICULE discs unopened at 20 ± 5 ºC. 7. General Instructions for Intended Uses of this Reference Material Remove the plastic vials from the freezer and allow the LENTICULE discs to reach ambient temperature (5-10 minutes) before use. Once removed from the freezer, do not refreeze the LENTICULE discs. Use within one hour of transfer to ambient temperature. Intended Uses: i) Quality control of culture media ii) Microbiology process control for internal quality control iii) Spiking samples for internal quality control http://www.sigmaaldrich.com/mibi-crm Refer also to the uncertified data and guidance for use at the end of this certificate. Use of diluents: We recommend using Pages saline and peptone saline (MRD) for the rehydration diluent and recovery of organisms. Certified reference materials should not be placed directly into distilled water (DW). They should be rehydrated in another diluent before DW is inoculated. Certified reference materials containing Legionella spp. should be rehydrated in Pages saline or 1/40 Ringers. Page 4 of 10
i) Quality Control of Culture Media If the LENTICULE discs are to be used for media other than those stipulated for the geometric mean value(s) you are advised to calibrate the LENTICULE discs for the conditions in your own laboratory. You are advised to use a minimum of 20 LENTICULE discs for calibration. Direct Inoculation - Solid Media Invert the plastic container over the centre of the agar plate until the LENTICULE disc drops directly onto the agar. Allow the LENTICULE disc to rehydrate for approximately 10 minutes (until a drop of liquid forms) then spread the resultant drop over the entire plate. Incubate under routine conditions for the organism and count the number of colonies obtained. Inoculation Through Liquid Media Invert the plastic container until the LENTICULE disc drops into the liquid medium (e.g. 1 ml, 10 ml, 100 ml). Allow the liquid culture to stand for approximately 10 minutes to rehydrate the LENTICULE disc, and then mix thoroughly. Inoculate the agar plates using the routine enumeration methods. Incubate under routine conditions and count the number of colonies obtained. Quality Control of Liquid Media Invert the plastic container until the LENTICULE disc drops into an appropriate amount of the broth medium (e.g. 10 ml, 225 ml) allow the liquid culture to stand for approximately 10 minutes to rehydrate the LENTICULE disc, and then mix thoroughly. Incubate and sub-culture in accordance with your routine procedures. ii) Process Controls Invert the plastic container until the LENTICULE disc drops into an appropriate amount of the primary broth (e.g. buffered peptone water) allow the liquid medium to stand for 10 minutes to rehydrate the LENTICULE disc, and then mix thoroughly. Continue in accordance with your routine procedures, preparing dilutions as appropriate. Incubate and sub-culture in accordance with your routine procedures. iii) Spiked Samples Confirm that the sample to be spiked does not contain the reference material organism. Weigh/measure an appropriate sample size and prepare the sample in the diluent used routinely for the relevant sample type (e.g. buffered peptone water). Invert the plastic container until the LENTICULE disc drops into the sample, e.g. homogenised food / broth medium (e.g. 10 ml, 225 ml) or water sample allow the liquid to stand for 10 minutes to rehydrate the LENTICULE disc, and then mix thoroughly. Process in accordance with your routine procedures for the target organism. 8. Hazardous Information Refer to the Safety Data Sheet for reference materials in LENTICULE disc format. http://www.sigmaaldrich.com/msds Page 5 of 10
9. Homogeneity A sample size of 45 LENTICULE discs was used to confirm batch homogeneity. This reference material was prepared in accordance with PHE / FEPTU Standard Operating Procedures that have been shown to produce a homogeneous product. Homogeneity calculations include (but are not limited to): The geometric mean: the average using the reverse log of the arithmetic mean of the first 15 and last 15 discs log values Expanded uncertainty using 95 % Confidence Intervals (CI): batch is accepted if the value is < 0.05 Within batch variation is calculated using the 2SD of the log values: batch is accepted if the value is < 0.2 T-test (two-sample assuming equal variance): is used to check that there is no difference between the first 15 and the last 15 discs produced. A batch is accepted if the P value returned is > 0.05 or if the overall mean log variation of the counts between the first and last 15 is 0.1 Anova (one way): is used to test that there is no difference of the means and variance of the first 15, the last 15 and a random set of discs (3 x 5) produced. A batch is accepted if the P value returned is > 0.05 or if the overall mean log variation of the counts between the five groups is 0.15 10. Quality Standard Documentation Public Health England: Accredited by the United Kingdom Accreditation Service (UKAS) to ISO Guide 34:2009 General requirements for the competence of reference material producers through assessment against this Guide and the relevant requirements of ISO/IEC 17025:2005. Sigma-Aldrich: Accredited by the Swiss Accreditation Service (SAS) to ISO Guide 34:2009 General requirements for the competence of reference material producers and ISO/IEC 17025:2005. Page 6 of 10
11. Signatures and Issue Date Certified by Certificate issue date Quality system (PHE) 08 Jun 2016 Zak Prior (General Manager and Project Lead) 4003 Reviewed by Quality systems (Sigma-Aldrich) K. D. Schmidt, Ph.D. ISO Guide 34 ISO 17025 16368 ISO 9001 Page 7 of 10
Commutability Data and Guidance for Use For general information about LENTICULE discs certified reference materials (CRMs) go to: www.sigmaaldrich.com/mibi-crm These CRMs are designed for use as controls in food and water microbiology testing laboratories and most of the products have multiple uses. CRMs show commutability when a mathematic ratio can be obtained from comparing different testing procedures to that in the certificate of analysis. The CRMs may be inoculated into real or simulated food and/or water matrices to provide process controls for traditional and molecular methods. CRMs with lower levels are particularly useful for determining the sensitivity of a detection method or the filtration methods used for water testing. Some of the lower level CRMs are useful for quality control of culture media. However, the certified values provided for any CRM batch are specific for those conditions indicated in Section 4 of the certificate. Results may be different under conditions other than those indicated in Section 4. Table 1 on the following page provides guidance for consideration when using LENTICULE discs. The data in Table 1 is not certified data and may require validation in the testing laboratories that use these materials. It is provided for guidance when selecting the CRM that will be most likely to fit the purpose for which it is required. The data in Table 1 was derived from studies undertaken in the PHE Food and Environmental Proficiency Testing Unit (FEPTU). Tables 2 and 3 provide commutability information when the CRMs are used as a process control for membrane filtration. Page 8 of 10
Table 1: Guidance on commutability information for use of LENTICULE discs certified reference materials when directly inoculated onto different media and incubation conditions Organism name NCTC no. Culture medium Incubation conditions (temp /time) Potential difference from certified data Bacillus cereus 7464 PCA 30 C/48 h 30-35% reduction compared with CBA Bacillus cereus 7464 PEMBA 30 C/48 h 30-35% reduction compared with CBA Bacillus cereus 7464 MYP agar 30 C/48 h 35-40% reduction compared with CBA Cronobacter sakazakii 11467 PCA 30 C/48 h 20% reduction compared with CBA Escherichia coli 9001 PCA 30 C/48 h 15-40% reduction compared with CBA Escherichia coli 9001 Brilliance E. coli 37 C/24 h 20-45% reduction compared with CBA Escherichia coli 9001 TTC agar 37 C/24 h 20-45% reduction compared with CBA Escherichia coli 9001 TBX agar 37 C/24 h 37 C/24 h 55-80% reduction compared with CBA Escherichia coli 9001 VRBG agar 37 C/24 h 55-90% reduction compared with CBA Escherichia coli 9001 MacConkey agar 37 C/24 h 70-95% reduction compared with CBA Escherichia coli O157 12900 PCA 30 C/48 h 65% reduction compared with CBA Enterococcus faecalis 775 PCA 30 C/48 h 2-12% reduction compared with CBA Raoultella planticola formerly Klebsiella aerogenes 9528 PCA 30 C/48 h 0-33% reduction compared with CBA Listeria monocytogenes 11994 PCA 30 C/48 h None compared with CBA Listeria monocytogenes 11994 Listeria monocytogenes 11994 Chromogenic Listeria agar Brilliance Listeria agar 37 C/48 h 37 C/48 h None compared with CBA None compared with CBA Listeria monocytogenes 11994 Oxford agar 37 C/48 h None compared with CBA Listeria monocytogenes 11994 PALCAM agar 37 C/48 h None compared with CBA Pseudomonas aeruginosa 10662 PCA 30 C/48 h 0-42% reduction compared with CBA Salmonella Nottingham 7832 PCA 30 C/48 h None compared with CBA Salmonella Nottingham 7832 Brilliant green agar 37 C/24 h 15% reduction compared with CBA Salmonella Nottingham 7832 XLD agar 37 C/24 h 25% reduction compared with CBA Salmonella Nottingham 7832 XLT4 agar 37 C/24 h 25% reduction compared with CBA Salmonella Nottingham 7832 Brilliance Salmonella 37 C/24 h 25% reduction compared with CBA Salmonella Typhimurium 12023 PCA 30 C/48 h 17% reduction compared with CBA Salmonella Typhimurium 12023 Brilliant green agar 37 C/24 h 32% reduction compared with CBA Salmonella Typhimurium 12023 XLD agar 37 C/24 h 42% reduction compared with CBA Salmonella Typhimurium 12023 XLT4 agar 37 C/24 h 52% reduction compared with CBA Salmonella Typhimurium 12023 Brilliance Salmonella 37 C/24 h 37% reduction compared with CBA Staphylococcus aureus 6571 PCA 30 C/48 h 7-26% reduction compared with CBA Staphylococcus aureus 6571 Baird Parker agar 30 C/48 h 17-36% reduction compared with CBA Staphylococcus aureus 6571 Chapman agar 30 C/48 h 17-36% reduction compared with CBA Page 9 of 10
Table 2: Guidance on commutability information for use of LENTICULE discs certified reference materials when used as a process control for potable and surface water testing for MEMBRANE FILTRATION LENTICULE disc added to 90mL Maximum Recovery Diluent, rehydrated and filtered onto 47mm Sartorius membranes (pore size 0.45mm) Organism name Product Culture medium Incubation conditions (temp/time) Potential difference from certified data (geometric mean) including 95% Confidence limits Clostridium perfringens CRM13170L TSC 37 C/24 h/ anaerobic 35 42% reduction Enterobacter aerogenes CRM10006L MLSB 37 C/24 h/ aerobic 63 67% reduction Enterococcus faecalis CRM00775L SB 37 C/48 h/ aerobic 43 51% reduction Escherichia coli CRM09001L MLSB Escherichia coli CRM09001L MLSB Escherichia coli CRM09001M MLSB Escherichia coli CRM09001M MLSB Pseudomonas aeruginosa 30-37 C cyclic/24 h/ aerobic 30-44 C cyclic/24 h/ aerobic 30-37 C cyclic/24 h/ aerobic 30-44 C cyclic/24 h/ aerobic 41 48% reduction 49 58% reduction 65 69% reduction 75 79% reduction CRM10662L PCN 37 C/24-48 h/ aerobic 32 41% reduction Staphylococcus aureus CRM06571L MSA 37 C/24-48 h/ aerobic 65 71% reduction Table 3: Guidance on commutability information for use of LENTICULE discs certified reference materials when used as a process control for environmental water testing - for MEMBRANE FILTRATION LENTICULE disc added to 1 L Pages saline, rehydrated and filtered onto 47mm Sartorius membranes (pore size 0.22mm) following ISO 11731:1998 Part 1. Organism name Product Culture medium Incubation conditions (temp/time) Potential difference from certified data including 95% Confidence limits Legionella pneumophila CRM12821M GVPC 36 C / 6 days 76 81% reduction Legionella bozemanii CRM11368M GVPC 36 C / 6 days 70 78% reduction Page 10 of 10