{"Affiliation":[{"label":"Affiliation","value":"Non UBC","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","classmap":"vivo:EducationalProcess","property":"vivo:departmentOrSchool"},"iri":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","explain":"VIVO-ISF Ontology V1.6 Property; The department or school name within institution; Not intended to be an institution name."}],"AggregatedSourceRepository":[{"label":"Aggregated Source Repository","value":"DSpace","attrs":{"lang":"en","ns":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","classmap":"ore:Aggregation","property":"edm:dataProvider"},"iri":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","explain":"A Europeana Data Model Property; The name or identifier of the organization who contributes data indirectly to an aggregation service (e.g. Europeana)"}],"Contributor":[{"label":"Contributor","value":"Vancouver Coastal Health Authority. 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This could be a full linked open date URI or an internal identifier"}],"FullText":[{"label":"Full Text","value":"Copper in Transit Study - Phase IIMicrobial Testing ProtocolVancouver Coastal Health, Version 2 May 18, 2022Contents(Version 2 includes Appendix 1 \u2013 this appendix gives a summary (following the one-year in-transit study)of our findings in use of 3M\u2122 Petrifilm plates used directly on antimicrobial surfaces as bacterial recoverymethod.)1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 General Evaluation Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Antimicrobial Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Vehicle Auditing - Westech Cleaning Audit Systems, Inc. . . . . . . . . . . . . . . . . . . . . . 65 Supplies for In-Transit Microbial Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65.1 Laboratory Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65.2 Westech Auditing Inc. are responsible for having the following supplies for the Micro-bial Audits: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Laboratory Preparation of Environmental Audit Kits . . . . . . . . . . . . . . . . . . . . . . . . 86.1 3M Petrifilm\u2122 Aerobic Count Plates (Standard) . . . . . . . . . . . . . . . . . . . . . . 86.2 Preparation of Letheen Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86.3 6.3 Petrifilm Plate QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96.4 Petrifilm Preparation: Hydration of Petrifilm Aerobic Count Plates . . . . . . . . . . . . 106.5 Specimen QR Coding System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126.6 Audit Kit Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Laboratory - Receipt of samples and plate incubation . . . . . . . . . . . . . . . . . . . . . . . 147.1 Receipt of samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147.2 Instructions for Evening Lab Staff for Petrifilm cultures . . . . . . . . . . . . . . . . . . 148 Petrifilm Aerobic Plate Reading and Enumeration . . . . . . . . . . . . . . . . . . . . . . . . . 158.17 Plate Counting Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178.18 Export of Plate Count Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Laboratory - Organism Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1919.1 Petrifilm Plate Randomization and Coding System . . . . . . . . . . . . . . . . . . . . 199.2 Imprinting Petrifilm to BAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219.3 Identification of colonies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229.4 Transit Identification Spreadsheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2210 Sample Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2311 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2312 Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242Prepared and authored by Tracey Woznow, Infection Prevention and Control and Medical Microbiology, Vancouver Coastal Health1 IntroductionBeginning in October 2020, TransLink (TL) and Vancouver Coastal Health (VCH) partnered with UBC, VGHand UBC Hospital Foundation, Coalition Healthcare Acquired Infection Reduction (CHAIR) and Teck Re-sources Ltd. (Teck) to do a two stage project. The pilot study (Phase I) was completed in early 2021, wherevarious antimicrobial surfaces were evaluated over a five-week period for their anti-microbial efficiency. Thispilot study also examined the antimicrobial surfaces for durability, maintenance, cost, transit rider impact andoperational feasibility for use by transit authorities. UBC Department of Materials Engineering provided ex-pertise on assessing the durability of the antimicrobial products.Phase I (Pilot):In the pilot study, two buses and two sky train cars were selected to be retrofitted and each vehicle hadfour antimicrobial products added to select high touch surfaces. Specific areas included stanchions, doorhandles and seat armrests within the vehicles \u2013 each product applied to four areas within each vehicle. Theanti-microbial efficiency of the products was compared against standard sky train\/bus \u2018control\u2019 surfaces;therefore, the vehicles were acting as their own controls throughout the study.The four antimicrobial surfaces were tested at various times during each week of the study, both for micro-biocidal efficacy and total organic load (measured by ATP Bioluminescence testing). Vehicles were alsotested \u2018After Cleaning\u2019 (before vehicle shift) and \u2018Before Cleaning\u2019 (after vehicle shift).Following the in-field (in-transit) study, the select high touch surface stanchions and handles were removedfrom vehicles and delivered to VCH Microbiology Laboratory for in-lab (in-vitro) antimicrobial testing, fol-lowed by durability testing at UBC Department of Materials Engineering.Phase II:Phase II Study was initiated in March 2021 and the site trial launched on September 28, 2021. In partnershipwith Teck Resources Limited (Teck), as well as with the original partners from Phase I, Toronto TransitCommission (TTC) and Sinai Health (Mount Sinai Hospital) will be involved with Phase II of the study.Phase II site trial will run for 12 months in both cities (Vancouver and Toronto), from September 2021 toOctober, 2022.Phase II involves the following vehicles:\u2022 Vancouver (TL) - 3 buses and 4 SkyTrain cars\u2022 Toronto (TTC) \u2013 3 buses, 2 subway cars, 2 streetcarsEach vehicle will be retrofitted with 3 different Copper products on various high touch stanchion locationswithin vehicles. The anti-microbial efficiency of the products will be compared against the standard vehicle3\u2018control\u2019 surfaces, therefore the vehicles will act as their own controls throughout the study.The copper products will be tested for their long-term antimicrobial effect within the public transit settingsas well as their long-term operability and durability.The objective of the Phase II Study is to determine whether selected copper products installed on transithigh-touch surfaces will maintain their durability and anti-microbial efficacy over 12 months.The study aims to gain understanding in the following technical areas:\u2022 Antimicrobial efficacy of copper products against bacteria and viruses over the 12-month period.\u2022 Macroscopic and microscopic changes of copper products over the 12-month period.\u2022 Operations and maintenance requirements of copper products in transit systems.\u2022 Impact of cleaning procedures and disinfection products on antimicrobial efficacy of copper products.\u2022 Relationship of chemical and physical properties of copper products, such as copper content andsurface roughness, and their antimicrobial efficacy.\u2022 Durability and antimicrobial efficacy (bacteria and viruses) after 12 months of usage in the transitsystem.The surfaces will be tested bi-monthly during the year long period of the study for microbiocidal efficacy andtotal organic load (measured by ATP Bioluminescence testing). Durability will be tested on a monthly basis.2 General Evaluation GuidelinesThe following parameters will be used to assess the antimicrobial efficacy of surfaces:In-Transit Testing:\u2022 Culture testing for microbial kill \u2013 total aerobic bacterial counts of environmental surfaces will beassessed using 3M Aerobic Petrifilm\u2122 plates.\u2022 ATP Bioluminescence testing for total organic load \u2013 total organic load on surfaces will be eval-uated. Surfaces will be tested using an assay to detect for the presence of adenosine triphosphate(ATP), an organic compound in the cells of all living (and once-living) organic organisms. Presence ofATP is an indicator of organic contamination and potential microbial surface contamination.\u2022 Identification of organisms and pathogens of interest \u2013 one third of the Petrifilm Plates fromboth copper and control surfaces will be randomly selected for identification of organisms, includingpathogens of interest, following plating and enumeration.4In-Vitro Testing:(Full procedures for In-Vitro Testing in separate document)In-vitro testing will be conducted at the VCH Medical Microbiology Lab.Small discs (coupons) of the various copper products as well as control surfaces will be subjected to200 rounds of simulated use with various disinfectants and artificial sweat using the Wiperator. Afterthat time, they will be assessed for antimicrobial efficacy. (Methodology as outlined in In-vitro evaluationof antimicrobial efficacy and durability of three copper surfaces in healthcare Bryce E, Velapatino, B,Khorami H et al used. https:\/\/avs.scitation.org\/doi\/10.1116\/1.5134676)Organisms used in-vitro to assess the surfaces for antimicrobial efficacy following Wiperator treatments:\u2022 a gram negative bacteria - Pseudomonas aeruginosa\u2022 a gram positive bacteria - Staphylococcus aureus\u2022 HCoV- 229E as a surrogate for COVID-19\u2022 Murine\/Feline calicivirus as surrogate for NorovirusIn-vitro test work main steps include:\u2022 Preparation of microbes.\u2022 Spreading of microbes on the surfaces of copper samples and controls.\u2022 Collecting microbes from copper samples and controls at set time periods.\u2022 Microbe broth dilution, plating, incubation overnight at 37 \u00b0C. Viable counts at 24hr and 48hr.\u2022 Calculation of microbial reduction.Upon completion of test work, all surface samples and controls to be sent to UBC for material charac-terization as per VCH instructions.3 Antimicrobial ProductsTypes of Copper Products:1. Copper decals2. Solid copper covers3. Copper spray application54 Vehicle Auditing - Westech Cleaning Audit Systems, Inc.Westech Cleaning Audit Systems, Inc. have been contracted to perform all vehicle surface specimen col-lection for the study, both in Vancouver and Toronto, as well as develop and maintain database system forstudy results.Each vehicle audit will be performed by a team of two Westech auditors.Westech auditors were trained in-class and on-site in surface specimen collection prior to the start of pilotstudy. For Phase II of the study, Westech developed an in-depth PowerPoint presentation for auditors toview for further training, prior to the second phase of the study. Following the presentation, auditors weregiven written tests to evaluate their learning.5 Supplies for In-Transit Microbial Testing*Detailed auditing procedures and supplies outlined in Transit Environmental Auditing Protocol5.1 Laboratory Supplies\u2022 3M Petrifilm\u2122 Aerobic Count (Standard) Plates (pre-hydrated with 1.0mL bisulphite free LetheenBroth by lab staff just prior to audit)\u2022 Letheen Broth dehydrated media, bisulphite free\u2022 Circular plastic impression discs from 3M for hydration of plates\u2022 3M Petrifilm Plate Reader Advanced (PPRA)\u2022 Laptop with 3M PPRA software pre-installed\u2022 30\u00b0C incubator\u2022 Autoclavable bottles (100mL) with screw caps for autoclaving and storage of prepared Letheen broth\u2022 Pipettors, pipette tips\u2022 Biohazardous bags\u2022 Brown paper bags\u2022 Labels and QR stickers\u2022 Blood Agar Plates, 5% Sheep blood6\u2022 MALDI supplies for organism ID (including target sample plates, 70% Formic Acid, MALDI Matrix,Controls, wooden sticks)\u2022 Gram stain reagents, glass slides, sterile culture loops, oil immersion light microscope\u2022 Stock organisms for QC \u2013 Pseudomonas aeruginosa, Staphylococcus aureus\u2022 Microbank\u2122 tubes for organism freezer storage\u2022 Eppendorf tubes5.2 Westech Auditing Inc. are responsible for having the following supplies forthe Microbial Audits:\u2022 hygiena ATPB SuperSnaps\u2022 \u2018Ensure V2\u2019 ATPB Luminometers\u2022 Spare batteries for Luminometers\u2022 Luminometer calibration rods\u2022 iPod\/iPhone for ATP RLU entry\u2022 *Petrifilm kits prepared by lab staff (See Section 6.0)\u2022 Pre-cut and labelled templates for Culture testing (3 \u00d7 circle cut outs slightly larger than 20cm\u00b2) and1 circle cut out for ATPB testing (20 cm\u00b2 area)\u2022 Coolers, icepacks\u2022 Large storage\/duffle bag for all supplies\u2022 Sharpie, pen\u2022 Clip board with Field sheets, instruction sheets\u2022 Measuring stick\u2022 Wipe able mobile table\u2022 Hand sanitizer\u2022 Accel Intervention 1 minute wipes\u2022 Gloves\u2022 Small container with garbage bag76 Laboratory Preparation of Environmental Audit KitsThe lab Research Assistant (RA) prepares the Petrifilm kits for Westech auditors for microbial culture testing.These kits consist of one set of labelled, hydrated Petrifilm Plates per vehicle. Two sets of paper instructionsare also included in each vehicle kit:\u2022 Instructions and address for sample (Petrifilm Plate) delivery to laboratory by auditors\u2022 Instructions for Evening Lab Staff for incubation of plates on arrival in lab.Petrifilm plates and kits should be prepared by RA a few days prior to the audit cycle. Date and time to bescheduled with Westech to deliver the audit kits just prior to the audit.6.1 3M Petrifilm\u2122 Aerobic Count Plates (Standard)3M\u2122 Petrifilm Aerobic plates are a culture medium system containing a water soluble gel requiring pre-hydration with a neutralizing solution. Plates also contain a red indicator dye allowing for ease of enumera-tion of aerobic bacteria.6.1.1 Storage temperature of plates:\u2022 Before hydration - store in refrigerator (2-8\u00b0C) (*if a package of Petrifilm plates has beenopened, store the remaining non-hydrated plates at room temperature for up to one month.)\u2022 Following hydration, plates to sit for 1 hour prior to use, then are stored in refrigerator (2-8\u00b0C)for up to a maximum of 14 days.\u2022 Following inoculation of plates \u2013 store and transport plates at room temperature until arrivalat lab, where plates are immediately incubated at 30\u00b0C6.1.2 Incubation: Incubate plates for 48 hours at 30\u00b0C. Stack plates face-up in incubator in stacks no morethan 20 plates high. Leave air space around stacks of plates.6.2 Preparation of Letheen Broth*Alternately, pre-made Letheen broth can be ordered.Letheen broth is used as the hydration broth for our Petrifilm plates.6.2.1 Suspend 25.7g of dehydrated Letheen medium in Erlenmeyer flask.6.2.2 Add 1000 mL demineralized H2O to flask.6.2.3 Add stir bar and heat to boiling with stirring to completely dissolve.86.2.4 Measure and adjust pH to 7.0 \u00b1 0.2 at 25\u00b0C using calibrated pH meter.6.2.5 Record details of broth preparation in Media Log, including final pH.6.2.6 Dispense into 10 \u00d7 100mL glass autoclavable bottles with screw caps.6.2.7 Place bottles into a wire basket. Place caps loosely on top of the glass bottles and autoclave at121\u00b0C for 15 minutes.6.2.8 Cool to room temperature, then fully screw on caps.6.2.9 Label, date and store at 2-8\u00b0C.6.2.10 Following preparation of a new batch of Letheen broth, prepare several Petrifilm plates using newbroth and perform QC on plates.6.3 6.3 Petrifilm Plate QCQC is performed on each new batch of Letheen broth and on new lots of Petrifilm plates. Stock gramnegative and gram positive organisms are used for QC.Stock Organisms:\u2022 Create McFarland 0.5 standards (approx. 1.5 \u00d7 108 CFU\/mL) for Staphylococcus aureus and Pseu-domonas aeruginosa from weekly stock culture plates \u2013 approx. OD of 0.08.\u2022 Make serial dilutions to approx. 1.0 \u00d7 103 CFU\/mL\u2022 Add 50uL and 100uL of the 1.0 \u00d7 103 CFU\/mL dilution to two hydrated Petrifilm plates using pipette:lift up top clear film and use pipette to drop the inoculum into the centre of the circular growth areaof plates. Use pipette tip to spread out the inoculum slightly, staying within the circular zone, thenreplace the top clear film and gently rub up and down the top clear film outside of circular growth areawith pads of fingers.\u2022 Label plates with organism and volume and incubate at 30\u00b0C for 48 hours.\u2022 Examine and count plates and record results in QC logTouch QC plates:\u2022 Incubate non-inoculated hydrated plate(s) as negative QC controls\u2022 Use several plates to touch various \u2018dirty\u2019 surfaces (such as floors, used bench surfaces, etc.) aspositive QC controls9\u2022 Label and incubate plates at 30\u00b0C for 48 hours.\u2022 Examine plates and record results in QC log6.4 Petrifilm Preparation: Hydration of Petrifilm Aerobic Count PlatesPrepare and hydrate plates a few days prior to the audit cycle date to allow time to set up kits.6.4.1 3M\u2122 Petrifilm aerobic plate storage: prior to hydration, plates are stored in refrigerator between 2 and8 \u00b0C away from light. (Plates in packages that have been opened are to be stored at room temperatureup to one month to avoid humidity.)6.4.2 Petrifilm SafetyCanadian SDS link:https:\/\/multimedia.3m.com\/mws\/mediawebserver6.4.3 Plates require hydration with 1mL Letheen broth prior to use \u2013 see section 6.2 for instructions onpreparation of Letheen broth.6.4.4 Determine the number of plates required for hydration and remove from refrigerator in small batches.6.4.5 Number of Plates Required for each vehicle:Vancouver Audits:\u2022 CM 2108 \u2013 20 stanchions \u00d7 3 = 60\u2022 CM 2124 \u2013 20 stanchions \u00d7 3 = 60\u2022 CM 2156 \u2013 20 stanchions \u00d7 3 = 60\u2022 ST 301 \u2013 21 stanchions \u00d7 3 = 63\u2022 ST 302 \u2013 21 stanchions \u00d7 3 = 63\u2022 ST 303 \u2013 21 stanchions \u00d7 3 = 63\u2022 ST 304 \u2013 21 stanchions \u00d7 3 = 63Total for all 7 vehicles for one audit in Vancouver = 432 Petrifilm PlatesAdd approx. 3 spare plates per vehicle kit = 7 \u00d7 3 = 21 Therefore, for one Vancouver audit, approx.453 Petrifilm plates will be required.Prepare several extra plates for QC.Toronto Audits:10\u2022 TTC Bus 8310 \u2013 18 stanchions \u00d7 3 = 54\u2022 TTC Bus 8311 \u2013 18 stanchions \u00d7 3 = 54\u2022 TTC Bus 8312 \u2013 18 stanchions \u00d7 3 = 54\u2022 TTC Streetcar 4422 \u2013 20 stanchions \u00d7 3 = 60\u2022 TTC Streetcar 4550 \u2013 20 stanchions \u00d7 3 = 60\u2022 TTC Streetcar 5272 \u2013 21 stanchions \u00d7 3 = 63\u2022 TTC Streetcar 5273 \u2013 21 stanchions \u00d7 3 = 63Total for all 7 vehicles for one audit set in Toronto = 408 Petrifilm PlatesAdd approx. 3 spare plates per vehicle kit = 7 \u00d7 3 = 21 Therefore, for one Toronto audit, approx. 429Petrifilm plates will be required.Prepare several extra plates for QC6.4.6 In laboratory room designated for clean media preparation, disinfect large bench area and lay out aseries of dry Petrifilm plates in rows.6.4.7 Use alcohol wipe to wipe down exterior of pipettor. To hydrate plate, lift up the top clear film ofPetrifilm plate using one hand \u2013 touch only the very bottom overlapping edge of film while lifting toprevent contamination of central growth area, and using the other hand, pipette 1000uL of preparedLetheen broth onto centre of lower portion of plate (grid side) in one large drop.6.4.8 Drop the top clear film back down gently onto lower grid portion on top of broth.6.4.9 Within several seconds, before gel starts to set, take the circular plastic 3M Petrifilm impression tooland place gently (with circular ridge facing down) onto the top centre of the Petrifilm plate. Placethumb in centre of plastic disc and press firmly downwards, holding for a few seconds until circleimpression is made. Press down on the impression tool slowly and carefully to prevent liquidfrom spreading out beyond the circumference of the circle.6.4.10 It is possible to prepare approx. 3-4 plates at a time by adding 1000uL broth to 3-4 plates, then makingimpressions with plastic disc prior to gel forming.6.4.11 After making the impression, examine the plate to ensure there is a clean circle and the broth has notspread outside growth circle area. If this is the case, discard the plate.6.4.12 Leave plates still on bench for 5-10 minutes until gel has fully set.6.4.13 Label with QR code stickers on top edge of plates. Attach labels as follows:When applying QR code labels to the top of Petrifilm Plates, make sure they are centred on plate, and11the top of the QR sticker should be just touching the bottom of the\u2019 3M\u2019 logo (approx. 7mm from topof plate).Follow procedures in Sect. 6.6 to package up plates into vehicle kits.6.4.14 Plates must be hydrated a minimum of 1 hour prior to use.6.4.15 Hydrated plates can be stored in refrigerator up to 14 days, away from light.6.5 Specimen QR Coding SystemEach Petrifilm plate is pre-labelled with stickers containing a unique QR code as well as information forauditors: City, Audit #, Vehicle #, Stanchion location #, Culture #The QR code is designed to allow plates to be automatically read and counted when fed into 3M PetrifilmPlate Reader Advanced (PPRA) automatic plate counter.QR Code Example: AC CM01 \u2013 17 \u2013 T102AC = aerobic plate countCM01 = CM 2108 = vehicle ID17 = Stanchion #17T102 = Culture #212Image courtesy of Westech Cleaning Audit Systems, Inc.6.6 Audit Kit PreparationMedical Microbiology RA will prepare the packages for each vehicle after plates are hydrated and labelled.6.6.1 After hydrating and labelling all Petrifilm plates, separate them out by vehicle number and in order ofstanchion and culture number.6.6.2 For each vehicle, place large labels and small pink labels on two biohazardous bags and on onebrown paper bag. Record audit number and vehicle number on large labels. Auditors can add dateand time.6.6.3 Separate out the plates for each vehicle into two stacks and place them into the two labelled biohaz-ardous bags.136.6.4 Place the two Biohazardous bags into the brown paper bag.6.6.5 Place 3 spare, unlabelled Petrifilm Plates into a biohazardous bag for each vehicle. Label the bag:\u2018Spare Plates\u2019 and place into brown paper bag.6.6.6 Add two instruction sheets to brown bag:\u2022 Sample Transport and Delivery instructions (see Appendix 4)\u2022 Instructions for Evening Lab Staff for Petrifilm cultures (see Appendix 5)6.6.7 Brown bags are then sealed with tape and packaged into boxes.6.6.8 Place boxes in refrigerator until delivery to Westech Staff.6.6.9 Westech staff to place Petrifilm Kits into refrigerator on receipt.Petrifilm Audit Kits7 Laboratory - Receipt of samples and plate incubation7.1 Receipt of samplesAfter completion of audits, Petrifilm plates are transported by auditors directly to Medical Microbiology Lab-oratory. (Refer to Transit \u2018Environmental Auditing Protocol\u2019 for detailed instructions and addresses fortransport and delivery of Petrifilm plates to laboratories.)7.2 Instructions for Evening Lab Staff for Petrifilm culturesLaboratory staff receive the samples directly from auditors and incubate the plates on receipt according tostep-by-step instructions below.14(See Appendix 2 for Instructions for Evening Laboratory Staff on receipt of Petrifilm cultures - for Vancouverand Toronto labs).7.2.1 Starting on Tuesday, October 5th for Vancouver and Tuesday, October 19th for Toronto, Transit StudyPetrifilm plate samples will be delivered to the Medical Microbiology Labs by Westech auditors as partof the Transit study of antimicrobial surfaces.7.2.2 Microbial sample audits will take place every two months for one year. See Transit \u2018EnvironmentalAuditing Protocol\u2019 for detailed schedules.7.2.3 For each bi-monthly audit, samples will be delivered to Vancouver and Toronto labs over four days.Over the 4-day periods, seven vehicles will be tested in both Vancouver and Toronto.7.2.4 Over the four days of each audit cycle, Petrifilm plates will be delivered for the 7 vehicle audits.For Vancouver, there will be a total of approximately 432 Petrifilm Plates delivered and for Toronto,approximately 408 plates over the four day audit period.7.2.5 For Evening Lab Staff instructions on receipt of Petrifilm plates from Westech \u2013 Petrifilm Plate Incu-bation, see instructions sheet in brown paper bag.\u2022 Wash and dry hands. Don gloves. Very carefully remove the sets of culture films from bags andplace them label-side-up into designated 30\u00b0C incubator.\u2022 Be very careful that the top film does not lift off the lower part of culture film.\u2022 Stack up to max. 20-high in incubator\u2022 Ensure there is adequate space for airflow between stacks of films\u2022 Record time and date of incubation on post-it note and place beside films.PLATES REQUIRE 48 HRS INCUBATION AT 30\u00b0C\u2022 Save any sheets from bag into box on top of incubator.\u2022 Discard bags8 Petrifilm Aerobic Plate Reading and EnumerationPlates are ready to be enumerated after 48hrs incubation. Plate counting will be done by lab RA using 3M\u2019sPetrifilm Plate Reader Advanced (PPRA) automated plate counter.Refer to this link for PPRA User Manual:3m-petrifilm-plate-reader-advanced-instructions.pdf158.1 PPRA needs to be connected to a laptop to operate. Laptop is required to have 3M PPRA specificsoftware previously downloaded and installed prior to use. Set-up of software requires QR codemanagement, Countable Range selection (set as 0-300 colonies\/plate), and customization of columnsfor results listing export.8.2 Wash hands and don gloves.8.3 Remove plates from incubator after 48 hrs incubation at 30\u00b0C.8.4 Set up the PPRA by plugging into wall outlet and into laptop USB port to warm up. A circular whitelight on the top of the PPRA will appear when the machine is initially plugged in. This light will turnfrom white to blue and then green. When green circular light is displayed on the top of PPRA, machineis ready to use.8.5 Organize plates into separate stacks by vehicle number.8.6 8.6 Arrange plates in a pile in order of stanchion number and culture number, with stanchion #1 plateson the top.8.7 For example, for vehicle CM 2108: the first plate to be inserted into PPRA and enumerated will be\u2018CM 2108 - Stanchion #1 -culture #1\u2019, followed by \u2018CM 2018 - Stanchion #1 - culture #2\u2019, etc. Thiswill allow us to export results where plates are in the correct order for spreadsheets, and subsequentsend out to Westech database.8.8 When PPRA is ready to use, start with the plates for first vehicle and feed the first plate into the frontintake on PPRA. The machine will automatically pull plate inside counter.8.9 PPRA will read sample number from QR code and enumerate all red-coloured colonies on plate.8.10 The laptop display will list on one row: Sample ID number from QR code, date\/time, Edited total colonycount. The laptop display will also show a photograph of the plate.8.11 RA will be able to zoom in on the photo of plate to check the accuracy of automated count. On closerexamination of plate, if RA finds that PPRA plate count needs to be edited, for example, if the PPRAhas counted particles that do not look like colonies, RA can edit the count made by PPRA directly onthe display. They must validate their reasons for the edited count by selecting a statement describingthe discrepancy from a drop down menu.8.12 There is also an area for comments to be added to the plate information.168.13 Continue enumerating plates in PPRA and checking plate photos and counts regularly as you go. Editcounts and add comments, as required.8.14 In the event that there is a big discrepancy in the count or if the RA has a query about the plate count,RA can press on the \u2018Save\u2019 icon above the photo of plate, and the photo will be saved onto laptop andcan be sent to 3M technical representative for further inquiries if required8.15 Count Comparison: Select approximately 10% of Petrifilm plates following automated PPRA enumer-ation is complete and set aside for manual counting. Manual counts can be compared to automatedcount results to ensure there is no statistically significant difference between the counts. Both sets ofcounts, manual and automated, can be added to Plate Count Comparison Spreadsheet.8.16 The PPRA requires routine cleaning following approximately every 1000 plate counts. Detailed clean-ing procedures are outlined in the PPRA Manual, which can be reached through the Petrifilm PlateManager software by clicking on the \u2018Help\u2019 icon in the top, right side of the screen.8.17 Plate Counting Guidelines(see Appendix 1):\u2022 Enumerate plates after 48 hours of incubation at 30\u00b0C17\u2022 Report colony counts as Colony Forming Units (CFU)\/plate - each Petrifilm plate has a surface areafor growth of 20 cm\u00b2.\u2022 Plates are enumerated using 3M Petrifilm Plate Reader Advanced (PPRA) automated plate reader.\u2022 Preferable counting range of Petrifilm plate is \u2264 300 CFU\/plate, therefore, set countable range onPlate reader to 0-300 CFU\/plate.\u2022 If count is greater than 300, plate count is considered an \u2018estimate\u2019. In these cases, the numberrecorded in the results spreadsheet is \u2018300 CFU\/plate\u2019, however the actual estimate count will berecorded in the \u2018Comments\u2019 section of spreadsheet with an \u2018E\u2019 following the count, represent-ing an \u2018estimate\u2019 count.\u2022 In cases where the plate has a colony that releases an enzyme causing liquefaction of the agar, thePPRA will estimate the % of plate gel area that has been liquefied, and will give an estimate of thecount.In such case, recommend doing a manual count of the plate where no liquefaction of agar is presentand then calculate the Total count for entire plate using this calculation:Total plate count estimate = \u2018count on non-liquefied area of plate\u2019 \u00d7 (100 %\/ (100% - % of platearea liquefied)For example, if 28% of plate area is liquefied and count for remaining area of plate is 60 colonies,Total Estimated Plate Colony Count: 60 CFU \u00d7 (100\/72) = 83.3 CFU\/plate\u2022 Count approximately 10% of plates manually following automated plate counts to compare counts.\u2022 While performing manual counting, use magnifying lens or dissecting microscope to assist in countingsmall\/pinpoint colonies.\u2022 In manual counting, if counts are >300 CFU\/plate, estimate by counting 1\/2 to 1\/4 of the plate usingbuilt in gridlines, as long as the distribution of colonies is representative of the whole plate.\u2022 In manual counting, if there are >20 CFU\/square, estimate by counting a minimum of 4 squares(select squares that will give a good representation of the count of the whole plate). Divide this totalcount by 4 and then multiply by 20 to get the total plate count (total surface area of a Petrifilm plate is20 cm2).\u2022 If plate is too overgrown to get an estimate of the count, report as \u2019Too numerous to count\u2019 or TNTCin \u2018comments\u2019 section and report the count in results spreadsheet as \u2018300 CFU\/plate\u201918\u2022 With very high counts, the growth area of plate may look pink, and it may have very small coloniesaround the outer edge of growth area. In this case, report plate count as Too Numerous to Count(TNTC) and report count in results spreadsheet as \u2018300 CFU\/plate\u2019.\u2022 If RA is not available to enumerate Petrifilm plates after incubation period, plates may be sealed inbiohazardous bags and stored in freezer for up to one week. (*Do not refrigerate.)When ready to count the plates, remove from freezer and place on bench to warm up to room tem-perature for minimum of 30 minutes prior to counting in PPRA.8.18 Export of Plate Count Results8.18.1 Following completion of plate counting by PPRA, review the results on PPRA screen display andensure they are complete and in order.8.18.2 Select the results to export by date or by sample number.8.18.3 Export as an excel document and save to computer files. Upload results to lab culture spreadsheetsas well as send to Westech for upload to master Transit database.9 Laboratory - Organism IdentificationColonies growing on Petrifilm plates will be identified for potential disease causing bacteria. One third of allPetrifilm plates, both from control and copper stanchions, will be randomly selected and saved for colonyidentification - a randomization app is used to prepare a unique randomization table for each Audit, listingthe plates to save for organism identification.Petrifilm plates for ID will be imprinted onto Blood agar plates (BAP) and incubated for growth. Colonygrowth will be identified using MALDI (Matrix-assisted laser desorption\/ionization) methodology and Gramstain.9.1 Petrifilm Plate Randomization and Coding System9.1.1 A randomization app is used to prepare new randomization tables for each audit cycle to allow usto randomly select one out of our 3 triplicate culture plates to save for ID of colonies. (See sampleRandomization Table in Appendix 3).199.1.2 After enumeration of Petrifilm plates using PPRA, refer to randomization table and set aside the platesfor identification of colonies.9.1.3 For each vehicle, we will have a maximum of 21 plates saved for ID.9.1.4 Label one BAP for each Petrifilm plate using the following labelling system:For example, for Petrifilm plate with the following QR code:CM02 \u2013 18 \u2013 T102Label BAP: B2 18 02(Where: B2 = bus 2 = CM02 = CM 2124; 18 = stanchion 18; 02 = culture 2)* Shortened code is used on the BAP to simplify and save time as it will be entered into lab systemfor MALDI testing, etc.Vancouver vehicle codes for ID:Buses:B1 = CM01 = CM 2108B2 = CM02 = CM 2124B3 = CM03 = CM 2156Skytrains:S1 = ST01 = ST 301S2 = ST02 = ST 302S3 = ST03 = ST 303S4 = ST04 = ST 304Toronto vehicle codes for ID:Buses:B1 = TB01 = TTC Bus 8310B2 = TB02 = TTC Bus 8311B3 = TB03 = TTC Bus 8312Skytrains:S1 = TC01 = TTC Streetcar 4422S2 = TC02 = TTC Streetcar 4550S3 = TS01 = TTC Subway 527220S4 = TS02 = TTC Subway 52739.2 Imprinting Petrifilm to BAP9.2.1 Take first Petrifilm plate and using one hand, lift up the top clear film containing the agar layer. Useother hand to help hold the 2 layer of the Petrifilm plate apart.9.2.2 Remove lid from the BAP and carefully place the top clear layer containing the central agar circle ofPetrifilm plate in the centre of BAP agar surface. Allow this clear film containing agar and colonies toadhere to the BAP surface and use fingers to gently rub the top film ensuring the whole circular agarzone of Petrifilm plate has made contact with the BAP agar. Be careful to prevent contaminationof BAP with other surfaces of Petrifilm plate or from hands.9.2.3 Replace lid of BAP. Continue to imprint the remaining plates for ID and incubate plates at 37\u00b0C for 24hours.9.2.4 Remove BAPs from incubator after 24 hours of incubation and examine each plate looking for all ofthe different types of colonies present.9.2.5 Mark one of each \u2018type\u2019 of colony present on plate with an \u2018X\u2019 on agar side of plate using a permanentmarker and attach a letter to the \u2018X\u2019 to identify colony starting from A, B, C, etc.For example, if there are 5 different types of colonies on one BAP, you will have 5 \u2018X\u2019smarked on the agar side of the plate to identify exact location of the colonies to ID. These 5colonies will be identified as colonies A, B, C, D and E.9.2.6 If there are large spreading colonies that look like Bacillus colonies, it is not necessary to ID thesecolonies. Make note of them on worksheet listing.9.2.7 List all colonies in order on MALDI worksheets for identification.For example, For example, for a BAP plate with code: B2 18 01, with 4 distinct types ofbacteria, the four MALDI codes for identification of the 4 colonies will be:B2 18 01 AB2 18 01 BB2 18 01 CB2 18 01 D21These will be the 4 codes used to identify the colonies in MALDI system.9.2.8 Include descriptions of each colony for identification on worksheet. For example: small creamy white,large shiny yellow, small grey with alpha hemolysis, etc.9.3 Identification of coloniesMALDI mass spectrometry methodology will be used to attempt to identify each colony type on BAPs.If an identification by MALDI is not possible, Gram staining will be used to classify bacterial colonies asGram positive or Gram negative.9.3.1 After identifying all distinct types of bacterial colonies on BAP, carefully labelling each colony on platewith sharpie and listing each colony with its specific code on MALDI transit worksheet, use MALDI toidentify the colonies.9.3.2 Transfer the listing of colonies for MALDI ID from the worksheet onto the MALDI Table worksheet.9.3.3 Proceed to use MALDI methodology to identify colonies.9.3.4 Save BAPs on laboratory bench until all identification procedures are complete for Audit Cycle.9.3.5 Print out MALDI identification results and save in Transit Study binder under specific Audit Cyclesection.9.3.6 Enter colony identifications into Transit Study Identification Spreadsheets.9.3.7 In the event that MALDI ID comes back as: \u2018no peaks found\u2019, repeat MALDI procedure on that specificlabelled colony. If the result is the same after a 2nd MALDI, perform Gram stain.9.3.8 In the event that MALDI ID comes back as: \u2018No identification possible\u2019, perform Gram stain on colony.9.3.9 Enter MALDI ID results or Gram stain results into Transit Identification Spreadsheet.9.4 Transit Identification Spreadsheets9.4.1 Transit Identification Spreadsheets contain the following columns for completion:\u2022 Stanchion surface type\u2022 Number of colonies\/Petrifilm plate\u2022 MALDI ID \u2013 enter species only (unless it is a potential pathogen of concern)\u2022 Gram reaction22\u2022 Gram Negative Organism?\u2022 Freezer library ID (if colony saved)\u2022 Potential pathogens of concern\u2013 Staphylococcus aureus\u2013 Escherichia coli\u2013 other Coliforms, including ID\u2013 Pseudomonas aeruginosa\u2013 \u03b2-hemolytic Streptococcus spp.9.4.2 Attach MALDI identification sheets to MALDI worksheets and tables, and save all in Transit binderunder Audit Cycle.10 Sample Storage\u2022 Petrifilm Culture FilmsPetrifilm plates will be frozen and saved in microbiology laboratory following testing until organismidentification is complete for each audit.\u2022 Colonies from BAPEach laboratory will use Microbank\u2122 system to freeze organisms of interest from Blood Agar Platesin a Freezer Library:\u2013 Gram negative organisms and any potentially pathogenic organisms are saved using this system,including: Staphylococcus aureus, Escherichia coli, other Coliforms, Pseudomonas aeruginosa,\u03b2-hemolytic Streptococcus spp.Microbank\u2122 tubes are labelled with a number to identify the colony saved and tubes are saved in labfreezers. Colony ID number recorded in Identification Spreadsheet.11 Results\u2022 Master Transit Database \u2013 Westech will maintain the master database for all culture and ATPBresults for both Vancouver and Toronto audits. Vancouver and Toronto laboratory staff will send cultureresults directly to Westech after reading.\u2022 Vancouver and Toronto labs will maintain their own spreadsheets with culture results for each auditcycle.23\u2022 Vancouver and Toronto labs will maintain their own Transit Organism Identification Spreadsheets.Organism Identification results will not be sent to Westech.\u2022 Binders will be maintained in labs containing original paper worksheets, QC logs, reagent logs andresults sheets for each audit cycle.12 AppendicesAppendix 1 \u2013 Following 12 month in-transit study - our experience with the use of 3M\u2122 Petrifilm aerobicplates (direct contact) as bacterial enumeration media for copper and non-copper surfacesAppendix 2 - Colony Counting Guidelines: Petrifilm Plate CountAppendix 2 - Instructions for Evening Lab Staff receiving Petrifilm culturesAppendix 3 \u2013 Randomization Table (sample table)Part B: In-Vitro TestingIn-Vitro Testing will be performed at the end of the twelve-month study. Refer to separate protocol forprocedures.24Appendix 1: Following 12 month in-transit study - our findings onuse of 3M\u2122 Petrifilm aerobic plates (direct contact) as bacterialrecovery media for copper and non-copper control surfaces.3M\u2122 Petrifilm aerobic plates:Petrifilm aerobic plates may be used directly on environmental surfaces or indirectly (via recovery of organ-isms from surfaces using swabs\/sponges with subsequent inoculation onto Petrifilm in laboratory). In ourin-transit study, we chose to use Petrifilm plates directly on transit surfaces to recover organisms.Main reasons for the choice of direct Petrifilm (PF) plates:\u2022 Using plate media directly on surfaces (such as Petrifilm, RODAC) to recover organisms is known tohave higher recovery of organisms in comparison to indirect recovery methods such as swabs andsponges.\u2022 The flexible, thin structure of PF plates allows for ease of testing of curved surfaces such as the curvedstanchion poles on transit vehicles.\u2022 Bacterial enumeration from PF plates can be automated with use of PPRA (3M\u2122 Petrifilm PlateReader Advanced) Lab Automation*Interesting findings using Petrifilm plates following 12-month result analysis:Overall, Petrifilm aerobic plates showed good antimicrobial efficacy of copper surfaces compared to con-trols, however, we noted some interesting characteristics in the growth of organisms on PF from antimicro-bial (copper) surfaces after in-lab experimentation:Following triplicate in-lab experiments evaluating the recovery of organisms (Pseudomonas aeruginosa andStaphylococcus aureus) from copper and control surfaces using direct PF and comparing this to indirect col-lection using 3M\u2122 Quick Swabs and Romer cellulose sponges (with inoculation onto both Blood agar platesand PF (indirect) for bacterial enumeration), the following results were noted:\u2022 All three methods were proficient at recovering bacteria\u2022 Direct PF contact with copper surfaces consistently exhibited 2-3 log higher CFU\/20cm2 than indirectmethods\u2022 No significant difference in bacterial counts was observed comparing swabs to sponges applied tocopper surfaces\u2022 No significant difference in counts observed between direct PF, swabs and sponges when applied tonon-antimicrobial control surfaces.25\u2022 No difference was observed between PF and BAP counts when PF was used for recovery after indirectsample collection using Quick Swabs\u2022 ATP Bioluminescence (ATPB) and live-dead flow cytometry staining were performed in tandem tocorroborate bacterial recovery and antimicrobial findings: comparison of microbial counts with live-dead staining and ATPB results suggest that the PF direct collection method revived stressed anddying bacteria. This phenomenon was not observed with the indirect PF method.Conclusion:Based on our results, direct sampling of copper surfaces using Petrifilm confers a survival advantage tobacteria stressed by copper and may induce higher bacterial counts. Overall, Petrifilm aerobic plates (PF)still showed good reduction in bacterial growth and antimicrobial efficacy of copper surfaces compared tocontrols.26Appendix 2: Colony Counting Guidelines: Petrifilm Plate Counting\u2022 Enumerate plates after 48 hours of incubation at 30\u00b0C\u2022 Report colony counts as Colony Forming Units (CFU)\/plate - each Petrifilm plate has a surface areafor growth of 20 cm\u00b2\u2022 Plates are enumerated using 3M Petrifilm Plate Reader Advanced (PPRA) automated plate reader.\u2022 Preferable counting range of Petrifilm plate is \u2264 300 CFU\/plate, therefore, set countable range onPlate reader to 0-300 CFU\/plate.\u2022 If count is greater than 300, plate count is considered an \u2018estimate\u2019. In this case, the number recordedin the results spreadsheet is \u2018300 CFU\/plate\u2019, however the actual estimate count will be recordedin the \u2018Comments\u2019 section of spreadsheet with an \u2018E\u2019 following the count, representing an\u2018estimate\u2019 count.\u2022 In cases where the plate has a colony that releases an enzyme causing liquefaction of the agar, thePPRA will estimate the % of plate gel area that has been liquefied, and will give an estimate of thecount.\u2022 Un these case, recommend doing a manual count of the plate where no liquefaction of agar is presentand then calculate the Total count for entire plate using this calculation:Total plate count estimate = \u2018count on non-liquefied area of plate\u2019 \u00d7 (100 % \/ (100 % - % ofplate area liquefied)For example, if 28% of plate area is liquefied and count for remaining area of plate is 60 colonies,Total Estimated Plate Colony Count:60 CFU \u00d7 (100\/72) = 83.3 CFU\/plate\u2022 Count approximately 10% of plates manually following automated plate counts to compare counts.\u2022 While performing manual counting, use magnifying lens or dissecting microscope to assist in countingsmall\/pinpoint colonies.\u2022 In manual counting, if counts are >300 CFU\/plate, estimate by counting 1\/2 to 1\/4 of the plate usingbuilt in gridlines, as long as the distribution of colonies is representative of the whole plate.27\u2022 In manual counting, if there are >20 CFU\/square, estimate by counting a minimum of 4 squares (selectsquares that will give a good representation of the count of the whole plate). Divide this total count by4 and then multiply by 20 to get the total plate count (total surface area of a Petrifilm plate is 20 cm2).\u2022 If plate is too overgrown to get an estimate of the count, report as \u2019Too numerous to count\u2019 or TNTCin \u2018comments\u2019 section and report the count in results spreadsheet as \u2018300 CFU\/plate\u2019.\u2022 With very high counts, the growth area of plate may look pink, and it may have very small coloniesaround the outer edge of growth area. In this case, report plate count as Too Numerous to Count(TNTC) and report count in results spreadsheet as \u2018300 CFU\/plate\u2019.\u2022 If RA not available to enumerate Petrifilm plates after incubation period, plates may be sealed inbiohazardous bags and stored in freezer for up to one week. (*Do not refrigerate.) When readyto count the plates, remove from freezer and place on bench to warm up to room temperature forminimum of 30 minutes prior to counting in PPRA.28Appendix 3 \u2013 Instructions for Evening Laboratory Staff on Receipt ofPetrifilm culturesVancouver: Instructions for Evening Lab Staff for Petrifilm cultures1. Starting on Tuesday, October 5th, Transit Study samples will be delivered to the Medical MicrobiologyLab by Westech auditors as part of the Transit study of antimicrobial surfaces.2. Samples will be delivered every two months during one week on Tuesday, Wednesday, Thursday andFriday evenings. See audit schedule on lab scheduling board for dates.3. October approximate times of deliveries (these may vary if vehicles arrive at depot early): AuditorsArrive at Lab with samples:\u2022 Tuesday Oct 5: 3:00pm to 4:00 pm (2 bags)\u2022 Wednesday Oct 6: 3:00pm to 4:00 pm (1 bag)\u2022 Thursday Oct 7: 1:15pm to 2:15 pm (2 bags)\u2022 Friday Oct 8: 1:15pm to 2:15 pm (2 bags)4. Deliveries will consist of brown paper bags containing sets of Petrifilm culture film plates (approx. 60plates\/bag). (One to two bags will be delivered each of the evenings.)5. EVENING LAB STAFF INSTRUCTIONS:Wash hands and don gloves. Very carefully remove the sets of culture films from bags and placethem (label-side up) into small incubator (near the large walk-in incubator\/warm room) - QUINCY,INCUBATOR #42Incubator #4229Be very careful not to lift the top film off of the lower part of culture film.\u2022 Stack up to max. 20-high in incubator; leave space for airflow between stacks of films.\u2022 Record time and date of incubation of each set onto post-it note and place beside films onside of incubator.\u2022 Discard bags and information sheets.Call RA anytime if you have any questions. Thank you!Toronto: New Transit Study Specimen InstructionsAs of October 19th, 2021, Transit Study samples will be delivered to the UHN\/MSH Sinai Micro laboratoryas part of the TTC Transit study of antimicrobial surfaces.\u2022 Deliveries will consist of brown paper bags containing sets of Petrifilm culture film plates (approx. 60plates\/bag). One to two bags will be delivered each of the evenings.EVENING LAB STAFF:\u2022 Very carefully remove the sets of culture films from bags.\u2022 Place them label side up into the 30\u00b0C incubator MIIM16 (no order entry needed).DO NOT stack more than 20 films high.Leave space for airflow between stacks of film.30\u2022 Record time and date of each set onto transit study Log by the incubator. Document total number offilms received and any other comments (e.g. Received damaged, frozen.)\u2022 Discard bags and information sheets.For more information, questions, or concerns call RA.31Appendix 4: Randomization Table (Sample table from Audit Cycle 1)Randomization Table for selection of Petrifilm plates for Organism IDAudit 1 Cycle (October, 2021)StanchionNumber CM2108 CM2124 CM2156 ST301 ST302 ST303 ST3041 1 2 3 2 2 3 22 1 2 3 1 2 1 33 2 1 1 3 3 3 34 1 2 1 2 3 2 25 3 2 3 1 1 1 16 3 1 3 3 2 1 17 1 1 3 1 1 3 38 1 1 1 3 2 2 39 2 2 1 2 1 2 310 2 1 1 3 1 1 211 1 1 2 3 3 3 112 3 3 1 2 3 2 213 3 2 2 2 2 2 214 2 3 3 1 1 2 115 3 2 3 3 3 1 116 1 1 3 1 2 2 217 3 2 3 2 1 3 218 2 2 1 2 1 3 319 3 1 3 2 3 2 120 2 2 3 1 1 1 321 3 1 1 132","attrs":{"lang":"en","ns":"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note","classmap":"oc:AnnotationContainer"},"iri":"http:\/\/www.w3.org\/2009\/08\/skos-reference\/skos.html#note","explain":"Simple Knowledge Organisation System; Notes are used to provide information relating to SKOS concepts. 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