lab safety course biological safety 2013

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BIOLOGICAL SAFETY

Dr Mark Dixon Research Ethics and Biosafety Office, UWA

The research carried out in microbiological labs

can either investigate the characteristics of living cells

or use living cells as tools for other research.

Microorganisms:

• protozoa • fungi • free-living bacteria • cell-dependent bacteria • viruses

Cultures of cells or tissues from multicellular macroorganisms including humans, other animals and plants.

Hazardous proteins including prions and toxins.

Who?

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1) Microorganisms are too tiny to see.

2) Microorganisms replicate.

3) Microorganisms can infect people, causing disease.

1) The research can harm people.

2) The research can be harmed.

Safety in the lab is YOUR responsibility.

You are not just protecting yourself, but your colleagues, students, the public, and the environment.

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Know and love your Biosafety Office!

Australian/New Zealand StandardTM 2243.3:2010 Safety in laboratories Part 3: Microbiological safety and containment.

UWA staff and students have access to the Standard online through the library, by entering the search words ‘SAI Global’, entering the SAI Global website, and then searching for ‘2243.3:2010’.

Remember to log out of SAI Global as soon as you have the Standard you need, because UWA only has two licences to access the Standards at any time.

The electronic copy of the Standard will self-corrupt in a few days, so print it out if you need a long-term reference.

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AS/NZS 2243.3:2010

Who is responsible for microbiological lab safety?

At all times, all lab work must be carried out putting the safety of lab occupants, the wider community and the environment first.

Every single worker in the lab, from the newest Honours Student to the most Senior Postdoc, is individually responsible for ALL ASPECTS OF LAB SAFETY.

This means you!

AS/NZS 2243.3:2010 - Responsibility

Management responsibility : senior lab staff, Lab Managers, School Managers, Heads of Schools, Deans, DVCs and the VC.

1) providing and maintaining the lab structure and the protective equipment.

2) developing, providing and maintaining a policy relating to safe work practices for storing, handling and disposing of microorganisms.

3) ensuring that staff are trained in those practices and carry them out in the lab.


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The Deputy Vice Chancellor Research has an Institutional Biosafety Committee (IBC) to assist her to meet her responsibilities about microbiological safety.


AS/NZS 2243.3:2010

Risk Assessment

The Risk Assessment process is the single most important part of any research activity.

When you are planning to do some research or teaching using a microorganism, you must first do a written Risk Assessment:

• to determine the appropriate level of containment for the microorganism

• to demonstrate that any hazards have been identified and are going to be controlled

AS/NZS 2243.3:2010 – Risk Assessment

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The UWA Biological Risk Assessment

The UWA Biological Risk Assessment

AS/NZS 2243.3:2010

Laboratory Management

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Your lab is meant to have a Lab Manager to set-up and run the lab safely:

• ensuring that safe procedures are developed, documented and put

into practice (Risk Assessments, Emergency Plans)

• implementing initial (lab induction) and continuing training

programs (training records)

• ensuring staff are supervised

• ensuring building and equipment maintenance is carried out in

accordance with safe procedures (maintenance records)

• ensuring that casual visitors do not have unrestricted access to the

lab

At UWA this role can be shared between the Lab Head(s), Senior Researcher(s), School Manager, Safety Officers and Lab Technician(s).

AS/NZS 2243.3:2010 – Management

AS/NZS 2243.3:2010

Laboratory Acquired Infections

20% follow known accidents with infectious material.

Up to 80% are due to inhalation of aerosols.

- vortexing - dropping cultures of high-titre material

- sonicating - blowing out the last few drops in a pipette

- homogenizing - removing a needle from a rubber seal

- centrifuging - vigorous shaking or mixing

- grinding - intranasal inoculation of animals

- harvesting infected tissues from animals and eggs

- opening containers of infectious material whose internal pressure is different from

ambient pressure

AS/NZS 2243.3:2010 – Lab Acquired Infections

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Cultured microorganisms usually contain higher titres than occur naturally and can therefore:

• be more infectious

• sometimes be infectious via a different route


Get vaccinated.

Protect at-risk persons.

Avoid cross-contamination.

Use containment equipment and procedures.

Report incidents and near misses.


AS/NZS 2243.3:2010

Microbiological Risk Groups

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AS/NZS 2243.3:2010

Classifies microorganisms into Risk Groups

so that we know how dangerous they are

and what precautions to take when handling them.

AS/NZS 2243.3:2010 – Risk Groups

Classification

• Microorganisms are classified into fourrisk groups, based on several criteria(termed Risk Groups 1 - 4)• Laboratories are classified into fourcorresponding Physical Containmentlevels (termed PC1 – PC4 laboratories)


Risk group allocation:

based on the degree of hazard to the individual, the community and the environment

Degree of hazard determined by:

• infectivity

• ease of transmissibility

• resultant effect

• host range of agent

• availability of vaccines/effective treatment

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HUMANS AND ANIMALS - RISK GROUP 1

- poses low individual and community risk.

- is unlikely to cause human or animal disease.

- for example: bakers yeast that everyone eats every day.



- poses moderate individual risk, limited community risk.

- is a pathogen that can cause human or animal disease, but is unlikely to be a serious risk to laboratory workers, the community, livestock, or the environment.

- laboratory exposures may cause infection, but effective treatment and preventive measures are available, and the risk of spread is limited.

- Includes some viruses, parasites, fungi, bacteriaLegionella, Shigella, Hepatitis B



- poses high individual risk, limited to moderate community risk.

- is a pathogen that usually causes serious human or animal disease and may present a serious hazard to laboratory workers.

- it could present a limited to moderate risk if spread in the community or the environment, but there are usually effective preventive measures or treatment available.

- viruses, bacteria, fungiJEV, NDV (exotic), Lyssa virus, B. anthracis, Brucella, Y. pestis, MDRMycobacterium tuberculosis


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- poses high individual and community risk.

- is a pathogen that usually produces life-threatening human or animal disease, represents a significant risk to lab workers, and is readily transmissible from one individual to another.

- effective treatment and preventive measures are not usually available.

- for example: Ebola (highly infectious and deadly).


Risk group = Physical Containment level

Each risk group must be worked at corresponding laboratory containment level

eg Risk Group 2 organisms --> PC2 labs (Physical Containment Level 2)

If you want to work with a Risk Group 2, 3 or 4 microorganism at UWA you will first need to apply to the UWA IBC for permission to do the work.


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Some high-risk microorganisms, as well as some toxins, are classed as Security Sensitive Biological Agents (SSBAs).

UWA is not currently registered to work with SSBAs and so you must not store or work with them.


HUMAN AND ANIMAL CLINICAL AND DIAGNOSTIC SPECIMENS – (BLOOD)

Treat all human and animal clinical and diagnostic specimens,

and primary cell cultures, with care and as at least RG 2.


AS/NZS 2243.3:2010

Chemical disinfectants.

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Microorganisms vary in their susceptibility to chemical disinfectants.

• Lipid-containing viruses and the vegetative forms of most bacteria are relatively susceptible.

• Fungi, acid-fast bacteria (Mycobacterium spp.) and non-lipid-containing viruses are less susceptible.

• Bacterial spores are resistant to the action of many chemical disinfectants.

• The agents of scrapie, Creutzfeldt-Jakob disease and other prions are extremely resistant to chemical disinfection.

AS/NZS 2243.3:2010 – Chemical Disinfectants

Many chemical disinfectants are available under a variety of trade names.

Examples of chemical disinfectants with a broad spectrum of activity against a range of microorganisms, including some sporicidal activity, are:

• Halogens (e.g. chlorine, iodine)• Aldehydes (e.g. formaldehyde, glutaraldehyde)• Oxidizing agents (e.g. peracetic acid, peroxygen biocide, hydrogen peroxide, chlorine dioxide)

Chemical disinfectants with a more limited antimicrobial spectrum include:

• Alcohols (e.g. ethyl and isopropyl alcohols)• Phenolics• Quaternary ammonium compounds• Chlorhexidine• Acids and alkalis


Variables that may affect the action of chemical disinfectants include the:

• concentration and formulation of the disinfectant• effective period of contact time• temperature• pH• relative humidity• inactivation by organic matter or cellulosic and synthetic materials

Mechanical brushing and rubbing facilitate the action of chemical disinfectants and other forms of decontamination.


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AS/NZS 2243.3:2010

Spills

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SPILLS

Spills are inevitable and must be planned for.

Plan every step of your work to minimize the chance of a spill.

All researchers will learn the procedures for controlling spills.

Clean-up materials and equipment.

Label your samples so someone else could decontaminate them.

Spills must be cleaned up properly.

AS/NZS 2243.3:2010 – Spills

Spills of microorganisms are complex events.

They can involve:

• contaminated gases, liquids and solids• sharps• people

A spill can spread a very long way before being contained.


1. the bulk of the liquid that remains in an irregular puddle

2. the portion that separates as splashes and rivulets

3. the small portion that is separated as airborne particles


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SPILLS

Step 1 – get people to safety

Step 2 – prepare for cleanup

Step 3 – define the contaminated area

Step 4 – decontaminate the area

Step 5 – report the spill


Time for some practice.

PRINCIPLES OF CONTAINMENT

Contamination control = Hazard control

AS/NZS 2243.3:2010 – Physical Containment

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Contamination

-the presence of hazardous or unwantedparticles (microorganisms) in an areawhere they do not belong

Primary and Secondary Barriers

Primary:provides immediate containment at thesource eg BSC, waste box (autoclave),animal room

Secondary:usually the lab, to provide secondarycontainment if the primary barrier fails

Primary, secondary and tertiary containment measures.

Optimal microbiological containment is provided by the ‘box-within-a-box’ principle, where the highest hazards are enclosed by multiple containment measures.


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PHYSICAL CONTAINMENT LEVEL 1 (PC1)

A PC1 lab is suitable for work with microorganisms where the hazard levels are low, and where laboratory or facility personnel can be adequately protected by standard lab practice.

PC1 lab practices and equipment are usually suitable for student and undergraduate teaching labs.

The organisms used should generally be classified as RG1.

Specimens that have been inactivated or fixed may be handled in PC1 facilities.



A PC2 lab, with its practices and equipment, is applicable to research and diagnostic work with RG2 microorganisms, or material likely to contain RG2 microorganisms.

It has the ability to contain these microorganisms, and to work with them and dispose of them safely.

If working with specimens containing microorganisms transmissible by the respiratory route, or if the work produces a significant risk to humans or the environment from the production of infectious aerosols, you must use a Biological Safety Cabinet.



A PC3 lab, with its practices and equipment, is suitable to do research and diagnostic work with RG3 microorganisms, or material likely to contain RG3 microorganisms.

A PC3 lab provides additional building features and services to minimize the risk of infection to individuals, the community and the environment.

UWA has one PC3 facility, located at the Biomedical Research Facility on the Shenton Park Campus.


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A PC4 lab, with its practices and equipment, is suitable for work with RG4 microorganisms.

UWA has no PC4 facilities.


OTHER KINDS OF MICROBIOLOGICAL LABS

Australian Quarantine and Inspection Service (AQIS) - Quarantine Approved Premises (QAP)

Office of the Gene Technology Regulator (OGTR) - certified facility

Door signage


Laboratory Work Practices - PC1

(comprise 'good microbiological practices')

• Risk Assessment of work – prior to start and

approved

• all procedures to be carried out to minimise the formation of aerosols and droplets (NB sonication, homogenisation, mixing)

• no mouth pipetting

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• wash hands after handling infectious material and animals, after removing gloves and before leaving the lab

• chemicals stored re AS2243.10 (minimise)


• clean up spills immediately with appropriate disinfectant; work surfaces to be decontaminated at the end of each day

• segregate waste and treat appropriately (decontaminate)


• label and date samples; use self adhesive labels

• report all significant spills (records)

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Additional requirements for work atPhysical Containment Level 2 (PC2)

(ie with risk group 2 microorganisms)

ALL the requirements for PC1

Plus:

• biohazard sign with PC2

• OGTR PC2 sign for gene technology work

• instruction, training, supervision specific to

microorganisms used


• biosafety manual must be available and implemented

• limit access to labs (doors closed)

• BSCI or II to be used to contain aerosols (‘significant risk’ or ‘transmissible by respiratory route’)


• wear gloves in BSC and when handling human samples

• supply of fresh disinfectant available and containers for infectious materials

• provide separate area for reference documents and papers

• provide separate area outside lab for report writing/ reading etc

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• minimise use of needles and syringes

• needles not to be resheathed, bent, capped, sheared

• use sharps containers

• use leur-lock syringes


• transfer cultures to other areas in secondary sealed container; decontaminate outer surfaces

• incinerate or autoclave all infectious waste before disposal

• protective clothing to remain in lab


• medical surveillance as required

• baseline serum samples where appropriate

• immunisation where necessary

• train cleaners, maintenance staff in hazards

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AS/NZS 2243.3:2010

Personal Protective Clothing and Equipment (PPCE)

AS/NZS 2243.1

PPCE

You will wear some protective clothing at all times when in the lab – whether you are working or just passing through:

closed footwear, a lab coat, safety glasses

You will add extra PPCE when you are working in the lab, depending on the work you (and others) are doing:

gloves, respiratory protection, hearing protection

Before leaving the lab, remove your PPCE (except your shoes) and wash your hands.

AS/NZS 2243.3:2010 – PPCE

Eating and Drinking

• There should be NO eating or drinking in a lab EVER, no matter what the level of containment!

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AS/NZS 2243.3:2010

Transporting microorganisms

Every time you need to transport your experiments, or wastes containing microorganisms, between:

• two labs in the same building separated by non-lab corridors • two labs in different buildings • from the lab to the storage area• from one campus to another • by foot• by car, truck, bus or train• by post• by plane

you will need to package them to ensure that they will not:

• become contaminated by the environment• escape containment to infect people or the environment

no matter what happens to the package during the transport.

AS/NZS 2243.3:2010 – Transporting

Walking - double contain

Transport Box

• large plastic lunchbox• sealing lid• labelled with the biohazard symbol• labelled with the 24 hour contact details of a responsible person


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If you send microorganisms by post you need:

• to pack them correctly• written permission from the receiver• copies of any required quarantine permits

If you receive posted microorganisms you need:

• all appropriate AQIS, Quarantine WA and OGTR permits and certified facilities• facilities for after-hours delivery • suitable unpacking procedures


AS/NZS 2243.3:2010

Waste

WASTE

There will be several different kinds of clearly labelled bins in the lab,

so that you can segregate different kinds of wastes at the point of discarding them,

and dispose of them appropriately according to local regulations.

AS/NZS 2243.3:2010 – Waste

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NON-CONTAMINATED WASTE

Non-infectious, non-hazardous waste will be:

• collected in a bin lined with a plastic bag • usually located next to the hand basin by the entry door• emptied by the cleaners to go to landfill

AS/NZS 2243.3:2010 – Waste

SHARPS WASTE

Sharps (including pipette tips) will be:

• collected in sharps containers • yellow incineration wheely bins • high-temperature incineration

AS/NZS 2243.3:2010 – Waste

CONTAMINATED WASTE

Disposable infectious material containing live microorganisms:

Collected in a solid bin containing a robust plastic autoclave bag displaying the biohazard symbol.

Autoclaved - disposal with non-contaminated waste.

Commercially incinerated.

AS/NZS 2243.3:2010 – Waste

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CONTAMINATED WASTE

Re-useable material contaminated with live microorganisms:

Autoclaved and then washed.

Chemical disinfectant and then washed.

AS/NZS 2243.3:2010 – Waste

TOXIC WASTE

Commercially by incineration.

Ethidium Bromide

AS/NZS 2243.3:2010 – Waste

AS/NZS 2243.3:2010

Cleaning the lab

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CLEANING

It is your responsibility to tidy and clean your own:

• lab bench (every day)• shelves• cupboards

Your lab manager will give you specific instructions about what else you also need to clean and how to clean it (e.g. walls, equipment).

AS/NZS 2243.3:2010 – Cleaning

CLEANING

Set up your work bench organised so that it can be cleared completely every day in order to be cleaned.


PROFESSIONAL CLEANERS

Only the floor and the windows.

Remove clearly-marked uncontaminated waste.

Never leave any infectious materials for the cleaners to handle.


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BIOLOGICAL SAFETY CABINETS (BSC)

Filter infectious aerosols and particles out of the air using HEPA filters.

Class I and Class II.

Require annual maintenance.

AS/NZS 2243.3:2010 – Equipment

Class I BSCs protect the researcher from infection.


Class II BSCs protect both the researcher and the biological material from contamination.


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HEPA FILTERS

High Efficiency Particulate Air (HEPA) filters.

Found in BSCs and in the ventilation system of PC2 facilities.

Disposable and replaceable.

Decontaminated using formaldehyde gas.


LAMINAR FLOW CABINETS

Clean workstations (laminar flow clean benches) protect the research but not the researcher.


FUME CUPBOARDS

Fume cupboards protect the researcher from hazardous chemicals.


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Undertaking Gene Technology work atUWA

To undertake a dealing at UWA you will need approval

from the UWA IBC

What is a Dealing?

Conducting experiments with or, making or breeding any GMO. This includes the possession, use, transport or disposal of the GMO.

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Doing a GM dealing at UWA

First you will need to go to the Biosafety webpagehttp://www.research.uwa.edu.au/staff/biological/GMO

You will also need to take and pass the online Gene Technology Awareness Session (GTAS).

This will help you figure out what class of dealing you need and what the next steps are.

Or you can just call me for help!

lab safety course biological safety 2013

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