Guidelines for

Safe Working Practices

in the GSBmE PC2 Laboratory Version 4, 2012

Lynn Ferris

Mai Ly

Sarah Walsh

GSBmE University of NSW

Introduction

This training is required for work within the GSBME PC2 laboratory, room 404. It outlines general principals and the specific work practices of the Graduate School of Biomedical Engineering

You must also complete the UNSW Biosafety for PC2 laboratories training. Register through MYUNSW.

A Physical Containment (PC)2 laboratory is suitable for work with material that may contain microorganisms classified under Risk Group 2. See UNSW Guideline for Risk Group Determination of Cell Lines (OHS651)

The GSBME labs are not registered with the Office of the Gene Technology Register (OGTR).

Genetically Modified Organisms (GMO’S) must not be brought into this facility without an exempt dealing certificate from the UNSW Internal Biosafety Committee (IBC) UNSW IBC

Why use PC 2 work practices?

If you are not working with potentially infectious

material, PC2 containment practices must still be use

… Why?

Good microbiological technique

Prevent cross contamination

Prevention of infection and damage to human health

Reduce the risk of accidents

It is the law – to comply with Work Health and Safety requirements

Doors & windows

Except during entry and exit, doors must be closed at

all times.

Windows must remain closed at all times.

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

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.

Safe Working Practices in the GSBmE PC2 Laboratory

Personal Protective Clothing

When entering the PC2 laboratory white lab coats must be removed and left on

the hanger outside the door.

The below personal protective clothing must be worn at all times when inside the

PC2 laboratory.

Blue gown (ensures protection to the front part of the body)

Gloves

Blue gown are kept on hangers within the PC2 laboratory.

Personal protective clothing must be removed before leaving the PC2 laboratory.

Never leave the PC2 laboratory wearing a blue gown.

Note Green Gowns are used when dealing with primary HUMAN tissue

Work with any unscreened human tissue requires additional

training

Dry Work Liquids

Always wear the blue PC2 laboratory gowns provided.

Do not bring personal items such as mobile phones or backpacks into the PC2 lab.

Wipe down ALL work surfaces before and after use with the 80% (v/v) ethanol squirt bottles or the isopropanol wipes provided

Remove both gown and gloves before leaving the room.

Always wash your hands with the disinfectant provided before you exit the PC2 room.

Exposure to aerosols is a major

cause of laboratory infections.

Aerosols can be produced during

Vortexing

Sonicating

Centrifugation

Pipetting

Opening containers

Minimise aerosol production on open benches by using closed containers for shaking and mixing.

Use the biological safety cabinets where possible when you are opening, mixing, decanting or aliquoting fluids.

Containment equipment

ALL work that produces aerosols which may cause a significant

risk to humans or the environment from the production of

infectious aerosols must be performed in the biological safety

cabinet

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.

Prior to work in biological safety cabinets, you

should:

Cultures

Do not culture bacteria or fungus in the GSBME PC2 laboratory.

All cultures should be stored within designated areas, see PC2 supervisor or lab

manager for details.

All cultures must be labelled with:

- date

- identification

- owner

Any unlabelled materials must be treated as potentially infectious and the Lab

Manager must be notified immediately.

All harvested cells or cells being introduced into GSBmE from an external

source must be mycoplasma tested. Cells must never ever be stocked in liquid

nitrogen dewars until confirmed mycoplasma negative. Until tested, cells can be

held in the -80°C freezer. See PC2 supervisor or lab manager for in-house

testing details.

Importing or Creating New Cell Line into GSBmE

IMPORTING OR CREATING NEW CELL LINE INTO GSBmE

Bringing Cells into GSBmE Sarah Walsh 08/03/2012

New cell line (created or external)

Mycoplasma Tested

Bank cells. See

procedure for details.

Not mycoplasma tested

Revive 1 vial of cells under Quarantine Conditions for at least 1 week.

Collect media (in contact with cells at least 3 days) and ask lab staff to

test for mycoplasma. Other vials can be temporarily stored in the -

80°C freezer until a result has been obtained.

DO NOT ADD ANY VIALS OF CELLS TO DEWARS UNLESS TESTED

NEGATIVE FOR MYCOPLASMA

Mycoplasma Positive

Mycoplasma Negative

Dispose of all cells from this batch

(revived and frozen) immediately. Alert

lab staff and external source contact.

Update OHS075 - MICROORGANISM

& BIOHAZARD REGISTER *

*REGISTER IS IN J:\Lab

Management\laboratory\PC2\PC2 (ask staff)

Cell Banking at GSBmE CELL BANKING

Sarah Walsh 8/03/2012

New cell line; tested negative for

mycoplasma and free from bacterial,

yeast or fungal infections

Bank vials of cells (~5 vials) except one, which

is to be resuscitated and cultured to expand.

Once the cultured cells have stabilised,

analyse cell count, viability and check cells

are free from bacterial, yeast or fungal

infections. Bank healthy cells(~5 vials).

Master Bank

Resuscitate and culture to expand. Once the

cultured cells have stabilised, analyse cell

count, viability and check cells are free from

bacterial, yeast or fungal infections. Bank

healthy cells(~5 vials).

Working Bank

Resuscitate and culture to expand. Once the

cultured cells have stabilised, analyse cell

count, viability and check cells are free from

bacterial, yeast or fungal infections. Bank

healthy cells(~5). General Use

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 small spills gently with 1% Virkon and large spills with Virkon powder,

dispose in the contaminated waste bin.

Wipe down the area and equipment with 80% (v/v) ethanol

Report all spills to the Area Supervisor or the Lab Manager.

Decontamination

All decontamination procedures

must be carried out by trained

personnel.

Viable materials must be made non-

viable by decontamination prior to

disposal.

Work benches, surfaces and

equipment must be decontaminated

at the beginning and on completion

of procedures.

Personal protective clothing:

Lab gowns must be

decontaminated prior to reuse if

it has been contaminated or

suspected to be contaminated

Gloves are disposed of in the

biohazard bin only

Decontamination can be:

Autoclaving or other heat based

treatment

Incineration (waste at GSBmE

is collected and incinerated by

a contractor)

Chemical treatment(must

render infectious agents non-

viable

Hands must be washed prior to leaving the laboratory

Hand operated taps are not acceptable

Storage

Where possible all organisms or

by-products should be stored

within the GSBmE PC2

laboratory .

All biologicals must be added to

the GSBmE Biological Register

See Laboratory Manager for

details

The Biological Register will

contain identifying

information and storage

location

When viable material is moved

outside the GSBmE facility it

must be confined in a primary

sealed container, within a

secondary airtight and robust

container.

The outer container must be

labelled with a name and

contact details in case the

package is lost.

Both containers must be

decontaminated or disposed as

a biohazard waste after use to

ensure no residue remains.

Transport

Domestic waste

Bins that are emptied by the UNSW cleaners are bins

are labelled Domestic Waste Only.

The only thing that should go in the bin is

uncontaminated wrapping and items of a non lab

nature

Never put plastic tubes or lids, gloves or

contaminated wipes in the domestic waste even if

they are clean

All other cleaning is done by users or special request.

Waste Disposal

Non-Hazardous Biological Waste

Non-hazardous biological waste is waste

that is or has been in contact with non-hazardous biological material. It is waste that will not cause harm or the spread of disease to humans, animals or the environment.

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.

Used culture flasks should be rinsed with a chemical decontaminant such as Betadine or Virkon solution prior to disposal.

When the biohazard bag is full, tie it up with string, label with a biowaste sticker and put in the large yellow bins in the cold room.

This waste is collected by the licensed UNSW waste contractors.

Hazardous Biological Waste

Hazardous biological waste is waste that

may cause harm or the spread of disease in humans, animals or the environment.

It is any material containing or contaminated with infectious microorganisms, infectious material, sample remains, human/animal blood, tissue or bodily fluids.

All such waste must be tied with string, labeled, autoclave tape attached and autoclaved in the metal bin at 121°C for 1 hour. The autoclaved waste is placed inside a second biohazard bag, sealed with string and labelled with a biowaste sticker before adding to the yellow bins in the cold room.

Small amounts of hazardous biological waste may be autoclaved in paper autoclave bags and then placed in the biowaste bins as non hazardous waste.

Liquid Waste Disposal

Collect liquid waste in the 500ml plastic beakers provided. Before adding waste, place 10% of final waste volume of disinfectant (Betadine, Virkon) in the container

NEVER put solid material in the liquid waste. No plastic tips of any kind

Avoid using bleach or other chlorinated disinfectants as they may release harmful gases when autoclaved.

The treated liquid is placed into the biological liquid waste cube under the sink in PC2. Once full, the drum is placed in the cold room (rm 405) awaiting collection by a UNSW contractor. Do not overfill.

A replacement waste cubes are found in the store room. Label it with a Non-

hazardous, iodine-treated tissue-culture waste sticker found in the Label Folder

in laboratory 429 and add ~50ml iodine to the bottom of the new drum.

Work Health and Safety

Ethanol is a flammable liquid - Do not

spray it in the vicinity of an open flame

or ignition source. Do not use a fine

mist spray.

Instead of flame sterilising, get training

in the use of the Bacti-steriliser, found

in PC1 (429a) or autoclave instrument

sets.

All injuries, spills or near misses

should be immediately reported to the

laboratory manager.

ALL work must be covered by an

authorised Risk Assessment or SWP.

Work protocols must comply with

statutory regulations.

All chemicals and reagents must be

appropriately assessed and labelled

according to Hazardous Substances

Regulations.

Use the GSBmE standard label on

your preparations and quote your

relevant Risk Assessment or SWP

number.

Immunisation eg hepatitis B may be

recommended for particular workers.

Ergonomics

Cell culture work can be physically stressful and

repetitive. Overuse injuries can result and be very

debilitation, if untreated.

Ensure that you have completed the UNSW

ergonomics training and can set up your work

stations correctly

Plan your work with breaks and minimise the number

of repetitive actions

Seek medical advice if pain persists and advise your

sueprvisor

Specific Training

Training information can be found on the GSBME OHS Database under Training

Register

Human blood work is not permitted without in-house training. Lynn Ferris

(laboratory manager) must be notified of all proposed research involving human

blood work. See the database or training techniques on the GSBME OHS

website Guidelines for Human Blood Work Practices at GSBmE:

Contact Lynn Ferris for details x53909 room 436.

The use of sharps (needles, scalpel blades) is not permitted without attending

Sharps Training. For training see Veronika Tatarinoff ext 53923 room 434.

Use of the autoclave is not permitted without Autoclave Training. For autoclave

training contact Lynn Ferris for details x53909 room 436. Prior to training one

must read the ‘Fundamental Autoclave Techniques’ PowerPoint on the GSBME

OHS website and complete the accompanying quiz:

Equipment

Report any damaged or faulty equipment to the Area Supervisor or Laboratory Manager immediately.

No equipment is to be removed from the PC2 facility without the approval of the Area Supervisor or Laboratory 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 be updated.

Maintenance Contractors must

report to the Area Supervisor or

Laboratory Manager prior to

starting work.

Tissue culture pipettes, marked

with a red label must not leave the

tissue culture laboratories.

Hazard Information Task/

Scenario

Hazard Associated harm Existing controls

Basic Cell culture

work with PC2

organisms

infection illness Work in a PC2 rated facility

Use PC2 work practices

Use a biological safety cabinet

Train all personnel in Biosafety for PC2 facilities

Immunised personnel against Hepatitis A & B and tetanus

Manual work Repetitive and

awkward

physical

movement

Ergonomic injury Complete the UNSW ergonomics training

Always adjust your workstation /microscope where possible

Plan your work to avoid long stretches of repetitive actions

Be aware of overuse symptoms and seek medical attention immediately

Biohazard hoods UV light

Biohazard

exposure

Burns

Illness

Never look directly at the UV light, never have the light on without the cover, never work in the hood with the

UV light on

Use the biohazard hood correctly by ensuring the air curtain is unimpeded and the hood is within its

calibration date.

See ergonomic injury

Never use hazardous fumes in the cabinet –it does not protect, use a fume hood.

Centrifuge crush

electrical

moving parts

Physical injury Ensure body parts are clear when closing lid

Always balance the load buckets with weight and placement symmetry.

Never leave the centrifuge unattended until it reached its run speed.

Pipettors Electrical

Manual

handling

biohazard

See electrical

shock and illness

illness

Always ensure there is a nose cone filter in the pipettor to block liquid entry in the tool

Ensure the pipettes fit tightly and do not leak.

Contain aerosols generated by pipetting

Always check the correct charger is used

CO2 Incubators Biohazard

Compressed

gases CO2

See illness

Asphyxiation

CO2 is an asphyxiant.

Leave the room immediately if the gas alarm sounds or the incubation indicates a high co2 level, Do not enter

unless the gas has been turned off and the sensor indicates normal levels.

If personnel are at risk call 56666 immediately

Always check gas connections for leaks when lines have been adjusted

Monitor the level in the gas bottles, report unusual usage levels.

Never shut the door in a small room with a CO2 incubator in operation with our a monitor.

Store supply bottles in large areas where possible.

Using equipment Electrical

equipment

fire

Electric shock

Burns , smoke

inhalation

Ensure all electrical equipment has a valid tag and test date

Inspect leads and connections to ensure they are undamaged and intact, especially if equipment is portable

Always use the correct charger for the battery operated pipetters

If equipment is damaged tag out of use immediately and report to management.

PC2 Roster

The PC2 Manager will hold a PC2 meeting every 3 months. PC2 meetings are

compulsory for all persons using PC2.

All persons using PC2 will be added to the PC2 roster. Each person is

responsible for their job on the roster until the next meeting.

PC2 Roster You are responsible for your job until the next meeting

You must organise to be trained- see Sarah/Jane

2012 March Please organise a replacement if you are away

Job Responsible Frequency date/s completed

Trash PC2 Denis Chang As needed

Trash PC1 Chris Kyung As needed

Benches/Restock Teddy Weekly

Bottles Steve/Bill/James CornwellAs needed

Gowns Khoon Fortnightly

DPBS* Yogi As needed

FBS* Josef/Romana As needed

Hoods* James/Eman Monthly

Incubators* Cameron Leo/Pallavi Monthly

Trash Tony As needed

Trypsin/Pen-Strep* Sally/Ryan As needed

Waterbath* Staffe /Anna C Once Weekly

Liquid Nitrogen Jane/Liyuan Once Weekly

Consumable Sheet

All persons using tissue culture consumables must fill in a Consumable Sheet

and give it to the PC2 Manager at each PC2 Meeting.

Tissue Culture consumables list Name

Supervisor Date total

Consumables Unit Week 1&2 Week 3&4 Week 5&6 Week 7&8 Extra

T25cm flask ea

T75cm flask ea

5/10/15ml tubes ea

50ml tubes ea

1 or 2ml pipettes ea

5 or 10ml pipettes ea

25ml pipettes ea

Syringe (1,3,5,10ml) ea

Syringe (20,60ml) ea

Trypsin mL

FBS mL

Pen/ Strep mL

Bottle of media 200mL

Water for irrigation 1L

well plate (any size) ea

Cryovials ea

Large filters ea

0.2um syringe filters ea

70ml jars ea

250ml Jars ea

Vi CELL vials ea

Petri Dish (any size) ea

Hood time Hours

Further Information

UNSW WHS Contact

Kate Noble Biosafety & Gene Technology Coordinator phone 93852911

k.noble@unsw.edu.au

UNSW resources

UNSW WHS: http://www.ohs.unsw.edu.au/

Biological forms and checklists

HS323 Biosafety Procedure

HS430 Register of Biosafety Legislation, Standards and Related Codes of Practice

Australian standards AS/NZS 2243.3:2002

Office of the Gene Technology Regulator http://www.ogtr.gov.au/

Lab Manager

Lynn Ferris ext 53909 room 436