1. Part IGuidelines For Safe Working PracticesIn the GSBmE PC2 LaboratoryVersion 3, 2009 Lynn Ferris Mai Ly GSBmE University of NSW Office of the Gene Technology Regulator Part II

2. Introduction What is a GMO General practice Dry work Liquids Biological Safety Cabinets Spills Cultures Storage Transport Waste OHS Equipment Further information Part I: Safe Working Practices in the GSBmE PC2 LaboratoryIndex

3. Part I: Safe Working Practices in the GSBmE PC2 LaboratoryIntroduction • This training is directed at work only within the GSBME PC2 lab rooms 404 and 404a • You must be trained before you can work within the PC2 facility Part I: Guidelines for Safe Working Practices in the GSBME PC2 Laboratory Part II: Behavioural Requirements for working in the OGTR PC2 Laboratory at GSBME • GSBME PC2 labs are certified by the Office of Gene Technology Regulator (OGTR). In addition to this training, it is required by law that you must complete the training program “Behavioural Requirements for working in the OGTR PC2 Laboratory at GSBME” • A PC2 lab is the lowest level of containment required when working with genetic manipulation techniques or infectious agents. The aim of these work practices is to Physically Contain any genetically modified organisms (GMOs) within the PC2 facility and prevent their release into the outside environment

4. Part I: Safe Working Practices in the GSBmE PC2 LaboratoryIntroduction Cont • Except during entry and exit, doors must be closed at all times. • Windows must remain closed at all times. • Even if you are not working with genetic manipulation techniques or infectious agents, PC2 containment practices must still be used if you are working within the PC2 facility … Why? • Good microbiological technique • Containment of genetically manipulated organism • Prevention of cross-contamination of non-GMO with GMO work • Prevention of infection and damage to human health • Reduce the risk of accidents • It’s the law – to comply with the OGTR regulations, and our occupational health and safety obligations

5. Part I: Safe Working Practices in the GSBmE PC2 LaboratoryWhat is a GMO • Your cell culture is considered a GMO (Genetically Modified Organism) if its genetic material has been altered using genetic engineering techniques • All GMO work must be approved by the UNSW Institutional Biosafety Committee, unless your GMO is exempt. • Exempt dealings are a category of dealings with GMOs that have been assessed over time as posing a very low risk (i.e. contained research involving very well understood organisms and processes for creating and studying GMOs). • You must apply for an exempt dealing from the UNSW Insititutional Biosafety Committee if your GMO is exempt • Your GMO is exempt if one of the following vectors were used during gene transfection (as defined by the OGTR) • Non-conjugative plasmids • Non-viral vectors, or defective viral vectors unable to transduce human cells • Avipox vectors (attenuated vaccine strains) • Baculovirus (Autographa californica nuclear polyhedrosis virus), polyhedrin minus • None (non-vector systems)

6. Do not Eat, Drink or Smoke in the facility Apply cosmetics Mouth pipette Insert contact lenses Bring or store food Tongue moisten labels Contaminate materials (e.g. workbooks) that will be removed from the room without sterilization. Keep hands and pens away from your face. They may have been in contact with contaminated surfaces or aerosols Tie back long hair Windows must be kept shut. You must wear closed footwear Avoid using bleach or other chlorinated chemical disinfectants You must notify the Lab Manger of any spills or accidents immediately The lab doors should always be kept closed Take care that reading and writing materials do not become contaminated. Part I: Safe Working Practices in the GSBmE PC2 LaboratoryGeneral Practice

7. Always wear the PC2 lab gowns. They are kept within the PC2 room. Remove your external coat before entering the PC2 lab. Hooks are provided outside the room. Do not bring personal items such as mobile phones or backpacks into the PC2 lab Wear gloves All gloves should be disposed of in the biohazard waste Wipe down ALL work surfaces before and after use with the 80% (v/v) ethanol sprays provided Paper tissues are provided as wipes and should be disposed of in the biohazard waste Remove both gown and gloves just before leaving the room. Always wash your hands with the disinfectant provided before you exit the PC2 room Part I: Safe Working Practices in the GSBmE PC2 Laboratory Dry Work

8. Exposure to aerosols is the major cause of laboratory infections Aerosols can be produced during Vortexing Sonicating Centrifugation Pipetting Opening containers Minimise aerosol production on open benches. Use closed containers for shaking and mixing Use the biological safety cabinets where possible when you are opening, mixing, decanting or aliquoting fluids. Part I: Safe Working Practices in the GSBmE PC2 Laboratory Liquids

9. Wipe down the internal surfaces with 80% (v/v) ethanol Decontaminate the hood with UV light for a minimum of 20 minutes Organise all your materials for your session so that they are easily available. After use wipe down surfaces (80% (v/v) ethanol) and decontaminate with UV light for 20 minutes The biological safety cabinets are NOT fume hoods - They will not protect you from harmful chemical fumes. Fume hoods are located in rooms 429, 430 and 431 GSBME has safe-work instructions posted on each Cabinet. Part I: Safe Working Practices in the GSBmE PC2 LaboratoryBiological Safety Cabinets Prior to work in biological safety cabinets, you should:

10. Decontaminate work benches and equipment affected by spills and after work has been completed If infectious material is spilt, avoid breathing in any aerosol... wait 5-10 minutes until the particles have had a chance to settle Cover the spill gently with adsorbent towel, sand or vermiculite and dispose in the contaminated waste Wipe down the area and equipment with 80% (v/v) ethanol Report all spills of GMO’s to the Area Supervisor or the Lab Manager. Part I: Safe Working Practices in the GSBmE PC2 Laboratory Spills

11. Do not culture fungus within the GSBME PC2 laboratory Bacterial cultures should be cultured and incubated separately from the mammalian cell lines All cultures should be stored within the designated areas All cultures (including GMOs) must be labelled with:- date- identification- owner Any unlabelled viable materials must be treated as a GMO and handled in accordance these conditions Notify the Lab Manager immediately Part I: Safe Working Practices in the GSBmE PC2 Laboratory Cultures

12. Where possible all organisms or by-products should be stored within the GSBME PC2 laboratory All genetically modified organisms (GMO) will be stored in a designated biohazard containment area The register of GMOs will contain storage location details and other identifying information All work with GMOs must be approved by the UNSW Institutional Biosafety Committee. Part I: Safe Working Practices in the GSBmE PC2 Laboratory Storage

13. When material is moved outside the PC2 lab it must be confined in a primary sealed container, within a secondary airtight and robust container For GMOs, the secondary container must be unbreakable. The outside container must be labelled with a name and contact details in case the package is lost For GMOs, indicate that it is a GMO Both containers must be decontaminated or disposed as a biohazard waste after use to ensure no residue remains Organisms transported by road, rail or air must comply with transport regulations Any GMO samples or cultures taken from the PC2 laboratory shall go only to another PC2 containment facility Part I: Safe Working Practices in the GSBmE PC2 Laboratory Transport

14. All disposable goods used to work with biological materials e.g. plastics, towelling, gloves etc shall be placed in a metal drum lined with an autoclavable biohazard bag When the drum is full, the top of the bag is folded inside the drum and the lid placed on top The whole drum is then placed in the autoclave without the lid to allow steam penetration 500ml of water is poured down the side (inside the waste bag) to facilitate steam generation Heat sensitive tape must be applied to the biohazard bag to differentiate from unautoclaved waste. You must be trained before using the autoclave The autoclave waste is treated at 121°C for a minimum of 20 minutes (BE S006). Ensure that the entire contents are subjected to these minimum conditions. A full drum normally requires 60 minutes of treatment. An indicator is used once a month to validate successful sterilisation of waste After treatment, the bag is removed from the drum and placed inside a second biohazard bag, sealed with string and labelled with a red biowaste sticker Part I: Safe Working Practices in the GSBmE PC2 LaboratoryWaste Disposal

15. Sample label “Tissue culture waste” Name Weight “Biomedical Engineering, UNSW” Date The sterilised waste is then placed in the contaminated waste collection bin in the cool room (405) Liquid waste Liquid waste is treated with a chemical disinfectant eg 1% w/v available iodine solution Avoid using bleach or other chlorinated disinfectants as they may release harmful gases The treated liquid is then placed in the biological liquid waste container in the cool room (405) Part I: Safe Working Practices in the GSBmE PC2 LaboratoryWaste DisposalCont All waste is removed by the UNSW contractor

16. Ethanol is a flammable liquid - Do not spray it in the vicinity of an open flame or ignition source. When flame sterilising, have a cover ready to smother the flame Aliquot minimal amounts of ethanol Ensure that flame sterilised tools are long enough to hold safely Hold flame tools horizontally to prevent heat and flame transfer to your hands Part I: Safe Working Practices in the GSBmE PC2 Laboratory Occupational Health and Safety

17. Avoid using sodium hypochlorite (bleach) or other chlorine based chemicals to decontaminate materials Toxic chlorine gas can be released from chlorine when it is heated Iodine based disinfectants should be used instead at a 1%w/v available iodine minimum All injuries, spills or near misses should be immediately reported to the laboratory manager Immunisation eg hepatitis B may be recommended for particular workers. Part I: Safe Working Practices in the GSBmE PC2 Laboratory Occupational Health and Safety Cont

18. The use of sharps (needles, scalpel blades) is not permitted without proper training and authorisation The use of the autoclave is not permitted without proper training and authorisation For training see Veronika Tatarinoff room 434 ext 53923 ALL work must be covered by an authorised Risk Assessment. GMO work protocols must comply with statutory regulations All chemicals and reagents must be appropriately assessed and labelled according to the Hazardous Substances Regulations Use the GSBmE standard label on your preparations and quote your relevant Risk Assessment number Part I: Safe Working Practices in the GSBmE PC2 Laboratory Occupational Health and Safety Cont

19. Report any damaged or faulty equipment to the Area Supervisor or Lab Manager immediately No equipment is to be removed from the PC2 facility without the approval of the Area Supervisor or Lab Manager All equipment must be decontaminated before transfer outside the PC2 facility. All equipment must be decontaminated before repairs are made. All equipment must be cleaned and maintained. Records of maintenance must updated. Maintenance Contractors must report to the Area Supervisor or Lab Manager prior to starting work. Report any equipment or contractor issues to the Area Supervisor or Lab Manager Part I: Safe Working Practices in the GSBmE PC2 Laboratory Equipment

20. SOP BE L032-01Guidelines for Safe Working Practices in the PC2 Laboratory SOP BE L031-01Routine Cleaning of Equipment used in a PC2 Laboratory SOP BE S006-01 Overview of Sterilisation Techniques Autoclave Training Fundamental Autoclave Techniques https://www.gsbme.unsw.edu.au/ohs/autoclave.php Risk managementOHS Coordinator (Biosafety)Adam Janssen ext 52214 a.janssen@unsw.edu.au UNSW risk management http://www.riskman.unsw.edu.au Office of the gene technology regulatorhttp://www.ogtr.gov.au PC2 area supervisorMai Ly ext 53909 Laboratory Manager Lynn Ferris ext 53909 Part I: Safe Working Practices in the GSBmE PC2 Laboratory Further Information

21. Part IITraining Program“Behavioural Requirements”for working in the OGTR PC2 Laboratory at GSBmEVersion 1, 2009 Mai Ly GSBmE University of NSW Office of the Gene Technology Regulator Part I

22. Introduction Doors & windows Non-GMOs in the PC2 Lab Containment equipment Personal protective clothing Decontamination Labelling Removal & storage of GMOs Spills Further information Part II: Behavioural Requirements for GSBmE PC2 LaboratoryIndex

23. Part II: Behavioural Requirements for GSBmE PC2 LaboratoryIntroduction • This training is directed at work only within the GSBME PC2 laboratory rooms 404 and 404a. Some of the information may be repeated from Part I, it is important that do not miss any sections. • You must be trained before you can work within the PC2 lab Part I: Guidelines for Safe Working Practices in the GSBME PC2 Laboratory Part II: Behavioural Requirements for working in the OGTR PC2 Laboratory at GSBME • GSBME PC2 labs are certified by the Office of Gene Technology Regulator (OGTR). It is required by law that you must complete this training program “Behavioural Requirements for working in the OGTR PC2 Laboratory at GSBME” if you want to work in the GSBME PC2 lab • A PC2 lab is the lowest level of containment required when working with genetic manipulation techniques or infectious agents. The aim of these work practices is to Physically Contain any genetically modified organisms (GMOs) within the PC2 facility and prevent their release into the outside environment.

24. Part II: Behavioural Requirements for GSBmE PC2 LaboratoryDoors & windows • Except during entry and exit, doors must be closed at all times. • Windows must remain closed at all times.

25. Part II: Behavioural Requirements for GSBmE PC2 LaboratoryNon-GMOs in the PC2 Lab • Procedures must be implemented to ensure that non-GMO work is not cross-contaminated with GMO dealings • Physical separation of work; or • Working at different times and ensuring any contaminated surfaces are decontaminated prior to commencing work with non-GMOs • These procedures must be documented • The outermost container must be free of contamination with GMOs prior to being transported out of the facility

26. Part II: Behavioural Requirements for GSBmE PC2 LaboratoryContainment equipment • ALL work that produces aerosols containing GMOs must be performed in the biological safety cabinet

27. Part II: Behavioural Requirements for GSBmE PC2 Laboratory Personal protective clothing • The following personal protective clothing must be worn at all times when inside the PC2 laboratory • Lab gown (ensures protection to the front part of the body) • Gloves • Personal protective clothing must be removed before leaving the PC2 laboratory

28. All decontamination procedures must be carried out by trained personnel GMOs must be made non-viable prior to disposal Wastes containing GMOs must be decontaminated prior to disposal Work benches, surfaces and equipment must be decontaminated at the completion of procedures involving GMOs Personal protective clothing: Lab gowns must be decontaminated prior to resuse if it has been contaminated or suspected to be contaminated with GMOs Gloves are disposed of in the biohazard bin Decontamination can be: Autoclaving or other heat based treatment Incineration Chemical treatment Part II: Behavioural Requirements for GSBmE PC2 Laboratory Decontamination

29. Any heat-based treatment must be performed using a combination of temperature and time that has been validated as effective in rendering the GMOs non-viable Loads must be packed and loaded to allow adequate steam penetration The coldest part of the load must be exposed to a minimum of temperature of 121°C and 103 kPa for at least 20 minutes Loads that have been processed can be differentiated from unprocessed loads (e.g. autoclave tape) Refer to standard AS/NZS 2243.3:2002 Incineration must be performed in a high temperature, high efficiency incinerator Autoclaved waste at GSBME is collected and incinerated by a contractor Any chemical disinfectant treatment must be effective in rendering the GMO non-viable Refer to standard AS/NZS 2243.3:2002 Hands must be washed prior to leaving the laboratory Hand operated taps are not acceptable Part II: Behavioural Requirements for GSBmE PC2 Laboratory Decontamination Cont

30. Part II: Behavioural Requirements for GSBmE PC2 Laboratory Labelling • All GMO cultures must be clearly labelled - date - identification - owner • Any unlabelled viable material must be treated as a GMO and handled in accordance with these conditions

31. GMOs which require containment in a PC2 laboratory must not be removed from the lab unless: They are to be transported to another certified PC2 facility They are transported to another location for storage They are transported to another location to be decontaminated prior to disposal Written permission by the OGTR or permits, for transport to another destination within Australia or to the Australian border for export All GMOs transported out of the facility must be Confined in a primary sealed container, within a secondary airtight an unbreakable container The outside container must be labelled with a name and contact details, and indicate that it is a GMO Organisms transported by road, rail or air must comply with transport regulations Part II: Behavioural Requirements for GSBmE PC2 Laboratory Removal and storage of GMOs

32. All cultures of GMOs stored inside the PC2 laboratory must be sealed during storage Prevents dissemination of GMOs GMOs stored outside the PC2 laboratory (freezer, fridge, etc) must: Have a biohazard symbol posted on the storage unit Be locked when not in use, unless access is restricted to the room/area where the storage unit is located Prevents unintentional release of GMOs into the environment All GMOs stored outside of the facility must be Confined in a primary sealed container and labelled. The primary container must be stored in a secondary unbreakable container In the case of a small storage unit such as fridge, freezer or liquid nitrogen dewar, the secondary container may be the storage unit If the GMO is a notifiable low risk dealing (NLRD), you must obtain authorisation from the UNSW Biosafety Committee to store your GMOs outside the PC2 laboratory Visit the OGTR website for more information on the different classes of GMOs Part II: Behavioural Requirements for GSBmE PC2 Laboratory Removal and storage of GMOs Cont

33. Documented procedures to decontaminate spills involving GMOs inside or outside the PC2 laboratory must be in place and be made available upon request by the OGTR GMO spills inside the PC2 laboratory must be decontaminated as soon as reasonably possible in accordance with the spills procedure GMO spills outside the PC2 laboratory must be decontaminated and reported to the Lab Manager immediately. Any real or suspected unintentional release of GMOs outside the PC2 laboratory (including spills) must be reported to the OGTR as soon as reasonably possible Part II: Behavioural Requirements for GSBmE PC2 Laboratory Spills GMO spills are reported to: Lab Manager UNSW Institutional Biosafety Committee OGTR

34. SOP BE L032-01Guidelines for Safe Working Practices in the PC2 Laboratory SOP BE L031-01Routine Cleaning of Equipment used in a PC2 Laboratory SOP BE S006-01 Overview of Sterilisation Techniques Autoclave Training Fundamental Autoclave Techniques https://www.gsbme.unsw.edu.au/ohs/autoclave.php SWP Safe Blood Work Practices Blood Work Training Guidelines for Safe Blood Work Practices Risk managementOHS Coordinator (Biosafety)Adam Janssen ext 52214 a.janssen@unsw.edu.au UNSW risk management http://www.riskman.unsw.edu.au Office of the gene technology regulatorhttp://www.ogtr.gov.au Australian standards AS/NZS 2243.3:2002 PC2 area supervisorMai Ly ext 53909 Lab Manager Lynn Ferris ext 53909 Part II: Behavioural Requirements for GSBmE PC2 Laboratory Further Information Part I Part II