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Part I Guidelines For Safe Working Practices In the GSBmE PC2 Laboratory Version 3, 2009 Lynn Ferris Mai Ly GSBmE University of NSW Office of the Gene Technology Regulator Part II Introduction What is a GMO General practice Dry work Liquids Biological Safety Cabinets Spills Cultures

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part i guidelines for safe working practices in the gsbme pc2 laboratory version 3 2009

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

part i safe working practices in the gsbme pc2 laboratory index
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
part i safe working practices in the gsbme pc2 laboratory introduction
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
part i safe working practices in the gsbme pc2 laboratory introduction cont
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
part i safe working practices in the gsbme pc2 laboratory what is a gmo
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)
part i safe working practices in the gsbme pc2 laboratory general practice
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
part i safe working practices in the gsbme pc2 laboratory dry work
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
part i safe working practices in the gsbme pc2 laboratory liquids
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
part i safe working practices in the gsbme pc2 laboratory biological safety cabinets
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:

part i safe working practices in the gsbme pc2 laboratory spills
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
part i safe working practices in the gsbme pc2 laboratory cultures
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
part i safe working practices in the gsbme pc2 laboratory storage
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
part i safe working practices in the gsbme pc2 laboratory transport
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
part i safe working practices in the gsbme pc2 laboratory waste disposal
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
part i safe working practices in the gsbme pc2 laboratory waste disposal cont
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

part i safe working practices in the gsbme pc2 laboratory occupational health and safety
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
part i safe working practices in the gsbme pc2 laboratory occupational health and safety cont
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
part i safe working practices in the gsbme pc2 laboratory occupational health and safety cont18
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
part i safe working practices in the gsbme pc2 laboratory equipment
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
part i safe working practices in the gsbme pc2 laboratory further information
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 [email protected]

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
slide21

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

part ii behavioural requirements for gsbme pc2 laboratory index
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
part ii behavioural requirements for gsbme pc2 laboratory introduction
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.
part ii behavioural requirements for gsbme pc2 laboratory doors windows
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.
part ii behavioural requirements for gsbme pc2 laboratory non gmos in the pc2 lab
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
part ii behavioural requirements for gsbme pc2 laboratory containment equipment
Part II: Behavioural Requirements for GSBmE PC2 LaboratoryContainment equipment
  • ALL work that produces aerosols containing GMOs must be performed in the biological safety cabinet
part ii behavioural requirements for gsbme pc2 laboratory personal protective clothing
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
part ii behavioural requirements for gsbme pc2 laboratory decontamination
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
part ii behavioural requirements for gsbme pc2 laboratory decontamination cont
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
part ii behavioural requirements for gsbme pc2 laboratory labelling
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
part ii behavioural requirements for gsbme pc2 laboratory removal and storage of gmos
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
part ii behavioural requirements for gsbme pc2 laboratory removal and storage of gmos cont
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
part ii behavioural requirements for gsbme pc2 laboratory spills
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

part ii behavioural requirements for gsbme pc2 laboratory further information
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 [email protected]

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

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