Bio and GM Safety v2 07/01/2014 · Bio and GM Safety v2 07/01/2014 5 Occupational Health & Safety Service 25 Workshop Exercise 2 Organism B: Organism B is a catalase-positive bacterium

Bio and GM Safety v2 07/01/2014

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Occupational Health & Safety Service

Biological Safety Course 2014

Dr Samantha Dainty– University Biological Safety Advisor


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Objectives for today

• Part 1: Why Bio-safety Matters What are the implications if (when?) things go wrong?

• Part 2: Risk Assessment (BioCOSHH)- Hazards and risks relating to biological agents. How will you identify the risks in your project?

• Part 3: How to contain biological agents and GM hazards.

How will you prevent yourself and co-workers becoming contaminated/infected?


Why Bio-safety matters


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Biological Safety Law


Some of the enforcing authorities

• Environment Agency – ~ everything that may affect the environment

• HSE – GMM’s, GMO’s, human pathogens

• Defra/Fera – plant pests, plant and animal pathogens

• Northumbrian Water – GMM’s, chemicals

• Local Council – waste disposal / land fill sites

• Human Tissue Authority – human materials

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Biological Safety Law

• IF you want a long career in science / research this becomes even more important the more senior you become

• Technicians – responsible for ensuring lab equipment is “safe”, appropriately tested and “legal”

• Post docs (supervising students) – responsible for their safety

• Group leader – responsible for entire groups safety

• Institute director – responsible for everyone on site

• Vice Chancellor – responsible for everyone in University

• As you become more senior - need to KNOW all regulations and laws!

• Postgraduate students are regarded as employees at Newcastle University


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YOU have a legal obligation to follow the biosafety (and other) regulations: • As implemented in the University • As directed by biosafety personnel and your

supervisor • Including ‘if in doubt – find out!’ • Undergo appropriate training

What this means Occupational Health & Safety Service

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Injury to staff, students and the public

through laboratory acquired infections

What are we trying to prevent?


Biosafety case study: Smallpox Exposure

• Janet Parker, medical photographer was the last recorded person to die from smallpox in 1978

• Worked in a dark room and offices located above the microbiology lab in the University of Birmingham Medical School

• Contracted smallpox through the ventilation system

• Had previously had variola (smallpox) vaccine!

• Lab inspected - Dangerous Pathogens Inspectors (HSE)

→ Lab was inadequately sealed and operated for this hazard group 4 agent

→ Lab dishonestly claimed smallpox use had decreased

→ A school leaver (<9 months experience) was found to be handling the virus!!

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Variola virus

WOULD YOU WANT TO WORK IN THIS LAB ???


The ten most frequently reported laboratory-associated infections worldwide

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Weinstein R A , Singh K Clin Infect Dis. 2009;49:142-147

© 2009 by the Infectious Diseases Society of America

Hazard group

3

3

3

2

3/2

3

2

3

3

3

(Brucella melitensis)

(Coxiella burnetti)

(Salmonella enterica)

(Francisella tularensis)

(Mycobacterium tuberculosis)

(Microsporum spp)

(Chlamydophila psittaci)

(Coccidioides immitis)

Agent type

bacterial

bacterial

viral

bacterial

bacterial

bacterial

fungal

viral

bacterial

fungal


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Consequences of “getting it wrong” Loss of institute reputation Occupational Health

& Safety Service

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Consequences of “getting it wrong” Serious financial penalties

£37,500

£42,159

£46,000


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What does an accident comprise of ?

• Time taken with actual injury – first-aid, hospital, clear up

• Medical follow up

• Reporting the accident

• Retrieval of details of experimental procedures and safety training; training records, CoP’s, RA’s

• Interviews with supervisor

• Interviews with safety staff, occupational health

• Interviews with HSE inspector

• Retraining courses, demonstrations, competence checks

• Follow up by safety staff, HSE


Consequences of “getting it wrong” Research costs/resources

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Risk Assessment of Biological Agents -

BIOCOSHH


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Who carries out the risk assessment? - a ‘competent person’

• A competent person under COSHH is somebody with the skill, knowledge, practical experience and training to enable him or her to assess the risks arising from work activities involving substances hazardous to health.

• A competent person should:

• Understand hazard and risk

• Know how the work can expose people to substances/agents hazardous to health

• Have the ability (and authority) to collect all the necessary information

• Have the knowledge, skills and experience to make the right decisions about how to control exposure.

• DOES NOT need to be an expert – but needs to know where to obtain biosafety advice and information


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• Unlikely that new postgraduate students are competent to carry out risk assessments on their own

• The process is a joint effort between the Principal Investigator, the student(s), any other researchers and biosafety personnel (BSO)

• Competent authority (HSE, Defra) involvement for certain projects and activities

What this means Occupational Health & Safety Service

Risk Assessment

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Exposure – if a biological agent is inhaled, ingested, enters through broken skin (cut, sharps injury)

Infection – when the microorganism establishes within organs/blood and produces colonies (infectious dose)

Virulence

Amount of organism entering system

Health status of exposed person

aim is to avoid exposure

to prevent infection ever happening


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• You are required to read and understand the BioCOSHH risk assessment form for your project

• example:

RA Comprises:

• Section 1: Project

• Section 2: Hazards

• Section 3: Risks

• Section 4: Controls

• Section 5: Emergency Procedures

you need to know what to do in an emergency

• Section 6: Approval

BioCOSHH Risk Assessment Form

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http://safety.ncl.ac.uk/biocoshhriskassessment.aspx


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Hazard, Risk and Risk Assessment

What is risk?

Risk is the chance or probability that a person

will be harmed or experience an adverse health

effect if exposed to a hazard.

What is a risk assessment?

Risk assessment is the process where you:

identify hazards, analyze or evaluate the risk

associated with that hazard, and

determine appropriate ways to eliminate or

control the hazard.

What is a hazard?

A hazard is any source of potential damage,

harm or adverse health effects on something or

someone under certain conditions at work.

Basically, a hazard can cause harm or adverse

effects to individuals as health effects.

control measures


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Hazard Group Classification of Biological Agents

• Devised by Advisory Committee on Dangerous Pathogens (ACDP) 2013

Classification based on:

• the likelihood that the agent will cause disease by infection or toxicity in humans

• how likely it is that the infection would spread to the community

• the availability of any prophylaxis* or treatment.

* Treatment which will prevent infection and/or may reduce the effect of an exposure or an infection. This includes vaccines.


Hazard Group Classification of Biological Agents

Group Description Examples

1 Unlikely to cause human disease B. subtilis, E. coli (K12 and BL21 strains)

2* Causes human disease

Unlikely to spread to community

Usually effective prophylaxis or

treatment

Influenza virus, Adenovirus, EBV, E. coli spp, S. aureus, primary cell lines, human/primate samples

3* Causes severe human disease

May spread to community

Often effective prophylaxis or treatment

Pandemic influenza virus, * HIV, * HBV, HCV,

HDV, E. coli 0157, M. tuberculosis,

4 Causes severe human disease

Likely to spread to community

Often no effective prophylaxis or treatment

Pandemic influenza virus, Variola virus, Ebola virus, Herpesvirus simiae

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HG2, HG3, HG4 are HSE notifiable

no HG4 work in Newcastle


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Workshop Exercises

From the following descriptions of

biological agents, allocate them to a

Hazard Group.

Hazard Group Classification of Biological Agents Occupational Health

& Safety Service

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Workshop Exercise 1

Organism A:

Organism A lives as a commensal organism* on the skin and in the nose of humans and other

animals, as well as in the environment. It can infect other tissues when normal barriers have

broken down (e.g. skin or mucosal lining). This leads to furuncles (boils) and carbuncles (a

collection of furuncles). In infants, infection can cause a severe disease. Infections can be spread

through contact with pus from an infected wound, skin to skin contact with an infected person,

and contact with objects such as towels, sheets, clothing, or athletic equipment used by an

infected person. Deep infections can be very severe. Prosthetic joints are particularly at risk, and

endocarditis (infection of the heart valves) and pneumonia may be rapidly fatal. Usually there is

effective prophylaxis and treatment.

*= Living on or within another organism and deriving benefit without injuring or benefiting the

other individual.

Organism A should be in Hazard Group HG2

Justify your answer: Although only in certain

circumstances can this organism can infect

tissues or cells of humans and although a

commensal it still can cause disease/infections

(harm to humans). Effective treatment is

available.

Organism A is Staphylococcus aureus


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Workshop Exercise 2

Organism B:

Organism B is a catalase-positive bacterium that is commonly found in soil. Like other such

species, it has the ability to form a tough, protective endospore, which allows it to tolerate

extreme environmental conditions. Organism B can contaminate food and rarely causes food

poisoning. The spores can survive the extreme heating that is often used to cook food, and it is

responsible for causing ropiness in spoiled bread. Nevertheless, a strain of Organism B is used

in the commercial production of the Japanese delicacy natto. Other strains have other

commercial applications. For instance, one strain has a natural fungicidal activity, and is

employed as a biological control agent.

Organism B should be in Hazard Group HG1.

Justify your answer: Widespread and only

causes problems in food contamination and

rarely – food poisoning (caused by bacterial

toxins rather than infection).

Organism B is Bacillus subtilis


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Organism C:

Organism C infection often leads to bloody diarrhoea, and occasionally to kidney failure. Most

illness has been associated with eating undercooked, contaminated beef. Person-to-person

contact in families and childcare centres is also an important mode of transmission. Infection can

also occur after drinking raw milk and after swimming in or drinking sewage-contaminated water.

Effective prophylaxis and treatment.

Workshop Exercise 3

Organism C should be in Hazard Group HG3.

Justify your answer: Causes severe human

disease and risk of spread but effective

prophylaxis.

Organism C is Escherichia coli O157:H7


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Workshop Exercise 4

Organism D:

Organism D is a non-segmented, negative stranded virus. Symptoms include uncontrolled

vomiting, severe diarrhea, headache, dizziness, and trouble breathing. Later bleeding from the

nose, mouth, and rectum occur and the disease is associated with up to a 90% mortality rate in

some epidemics. No effective prophylaxis and no treatment.

Organism D should be in Hazard

Group HG4.

Justify your answer: Severe

disease, high mortality and risk of

spread. No prophylaxis and no

treatment.

Organism D is Ebola virus


Biological Agents Guidance

3 key documents: -

• ACDP Approved list of biological agents

• ACDP Biological agents: Managing risks in laboratories and healthcare premises

• ACDP The management, design and operation of microbiological containment laboratories

• Links are available to these documents from the Safety Office website

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Example Hazards : Clinical/Human Samples – BBV*

• HIV and Hepatitis are major hazards • Amniotic fluid

• Blood

• Breast milk

• CSF

• Pericardial fluid

• Tissues with traces of infected blood..

• Unscreened clinical samples in the lab !

• Sharps increase risk.

• Hep B vaccinations ?

• Consider if Health Surveillance needed ?

• Peritoneal fluid

• Pleural fluid

• Semen

• Synovial fluid

• Vaginal secretions

* BBV = blood-borne viruses

not analysed for viruses

or other agents


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Example Hazards : Animals

• Laboratory or fieldwork

• Infection from animals, bites, scratches

− Laboratory animals often screened for common pathogens

− Introduced pathogens/GMO

• Serious injury

− Large animals = Large teeth (usually at one end!)

• Escape risk

− Extra concern if GM involved


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Example Hazards: Primary and Continuous Cell Cultures

• Human, animal or plant cell culture

• Adventitious biological agents in primary cells – patient has a virus/disease? HepB, HIV

• Cell culture - ATCC did NOT screen any mammalian cell lines for HIV/HepB pre-2010

• Unintentional culturing of an environmental pathogen

• Some cell lines already contain viral components: HPV-E6, SV40, adenoviruses


What Are The Hazards ? : Microbial and other Toxins

Toxin

Caffeine

Ammonium Dichromate

Nicotine

Osmium tetroxide

Hydrogen cyanide

Sodium cyanide

Tetrodotoxin

Aflatoxin B1

Clostridium tetani

Clostridium botulinum

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NOTE: Microorganisms do not need to be viable for toxins to be present and harmful – toxins may also be present in spent cell-free culture media

LD50 mg/kg ~ LD for 75kg person

192 14.4g

67.5 5.065g

50 3.75g

14 1.05g

10 750mg

6 450mg

0.3 22.5mg = 20 x

0.048 3.6mg = 125 x

0.000000025 185ng = 2472 x

0.000000010 75ng = 6000 x


What Are The Hazards ? : Allergens

Biological/chemical materials which cause hypersensitivity

• Animals, plants and microorganisms or their products can cause hypersensitivity

• Sensitization can occur by acute or chronic exposure

• Asthma – animal house bedding, fur, dander

• Dermatitis – many chemicals, animal furs/hair

• Anaphylaxis – natural products

• If you exhibit any allergic response contact school safety officer immediately

• Important to declare known allergies at commencement of employment

• Continued exposure can lead to severe anaphylaxis (<5 - 30min response) 33


Schedule 5 items

• Schedule 5 materials require secure storage under part 7 of the Anti Terrorism, Crime and Security Act 2001 (ATCSA)

• These organisms or toxins are generally capable of causing death in humans.

− Examples: B.anthracis, dengue fever virus, lassa fever virus, C. botulinum, ricin protein etc

• Local anti terrorism police (CTA), HSE and University staff will all inspect laboratories housing these agents.

• Lab requires “Bio-security” policy !!

• Involves an extensive project application procedure

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Pathogen and Toxin Registration

Certain biological agents and hazards must be registered using

Pathogen or Toxin Registration Forms before they are even

brought into University

• Hazard group 2 and 3

biological agents

• Any pathogen or toxin

listed in Schedule 5

Detailed guidance on what must be registered on USO

website


Risk Estimation

Risk is estimated by combining severity of harm if the hazard was to occur and the likelihood of occurrence in specific circumstances

• Severity of harm (severe, moderate, minor, negligible)

• Likelihood of harm (high, medium, low, negligible)

Risk = Likelihood x Severity

Risk = Effectively zero, Low, Low/Medium, Medium or High

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Risk Estimation Matrix*

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Likelihood of Harm (occurring)

High Medium Low Negligible

Severe High High Medium Effectively

zero

Moderate High Medium Medium/

Low Effectively

zero

Minor Medium/

Low Low Low

Effectively zero

Negligible Effectively

zero Effectively

zero Effectively

zero Effectively

zero

Severi

ty o

f H

arm

*A risk matrix is included at the end of every risk assessment form


Assessing Risk

Who is at risk in the lab?

• Workers – co-workers

• Visitors – public, school students

• Contractors - cleaners

• Immunocompromised status − infections, drug treatments,

cancer

• Pregnancy

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New and Expectant Mothers

If you become pregnant and may come into contact with the agents listed below -

• Visit Occupational Health Site…

• Avoid work with − Radiation sources

− Hazardous Chemicals

− Carcinogens/Mutagens

• Avoid work with some microorganisms − HG2

− HG3

− Animal infection

− Workplace exposure

− Blood screening?

• Avoid farm animals (pregnant ewes)

− esp. chlamydiosis, toxoplasmosis and listeriosis

• Inform SSO asap for assessment

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Assessing Risk: infectious dose

Agent Dose Route

Tuberculosis 10 Inhalation

Influenza A2 790 Inhalation

S.typhi 105 Ingestion

Vibrio cholerae Cholera 108 Ingestion

E.coli 108 Ingestion

E.coli 0157 10-100 Ingestion

Shigella 10 Ingestion

Polio virus 1 2 Ingestion

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Definition: The amount of pathogenic organisms that will cause infection in susceptible subjects. Dependant upon organism AND host


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Routes for transmission of infection

Mucous Membranes: Exposure of mucous membranes of the eyes, nose and mouth through splashes, splatter or aerosols.

Inhalation: Breathing in respirable sized aerosols (<5μm), centrifuge leaks, spills, pipetting, etc.

Ingestion: Swallowing through mouth pipetting, eating, drinking or smoking in the lab.

Percutaneous/injection: Through intact or non-intact skin via NEEDLESTICK, puncture with contaminated sharp object, animal scratch or bite, through wounds abrasions, or eczema.

Contact (indirect transmission): Via mucous membranes or non-intact skin from hands that have been in contact with a contaminated surface (i.e. benches, phones, computers, equipment handles) or by failure to wash hands after working


Scale: Quantity and Concentration

• Small amounts of dilute substances “generally” pose little hazard and risk

• Handling of large quantities of a substance increases risk – bioreactors (100L+)

• Centrifugation and filtration increases concentration of agents

• Highly concentrated substances, can make them:

− More infectious − Higher aerosol potential − Emergencies require specialist

spill clean up − Waste inactivation issues

− Higher risk

• Quantities and concentration requires outlining in risk assessment 42


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• Infection

• Communicable diseases

• Environmental release (GM)

• Occupational disease

− Asthma, dermatitis

• Organ damage

• Cancer – some years later

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Consequences of exposure to

hazardous agents and substances Occupational Health & Safety Service

Guidance and Information

Websites • University Safety Office - Biological Safety section

(http://www.safety.ncl.ac.uk/biologicalsafety.aspx)

• University Occupational Health Service

• Health and Safety Executive

• ACDP – Advisory Council for Dangerous Pathogens

• Department for Environment, Food and Rural Affairs (Defra)

• Health Protection Agency (HPA)

Publications • Microbiology and biology textbooks

• Scientific papers

• Detailed Internet searches 44


Containing Biological Agents and GMO’s


Risk Estimation Matrix*

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Likelihood of Harm (occurring)

High Medium Low Negligible

Severe High High Medium Effectively

zero

Moderate High Medium Medium/

Low Effectively

zero

Minor Medium/

Low Low Low

Effectively zero

Negligible Effectively

zero Effectively

zero Effectively

zero Effectively

zero

Severi

ty o

f H

arm

*A risk matrix is included at the end of every risk assessment form


Risk Estimation

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High hazard – low risk? Medium hazard – high risk?

which lab would you rather work in?


Biological Agent Containment

• BA containment is vital

• Minimise the risk to human health

• Containment is not just physical confinement (labs and equipment) but also operational activity or ‘Good Microbiological Practice’ as described by the labs ‘standard operating procedures’ and used on a day-to-day basis.

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http://www.safety.ncl.ac.uk/biologicalsafety.aspx




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Good Microbiological Practice Example items of Good Laboratory Practice (adapted from Sheffield University)

• Personal items irrelevant to the microbiological work (sports gear, items of shopping, etc) must not be taken into the laboratory.

• Protective clothing must be worn for all microbiological work. PPE also dependent on the RA.

• Scrupulous personal hygiene must be observed in the laboratory. Wash hands correctly after work and before leaving the laboratory.

• Eating, drinking, smoking and the application of cosmetics in the laboratory are forbidden.

• Mouth pipetting is forbidden in all microbiology laboratories.

• Minor cuts, scratches and abrasions on the hands must be sealed with waterproof dressings before entering the laboratory.

• The use of "sharps" (scalpel blades, syringes and needles, etc) in microbiological labs must be minimised and contaminated sharps disposed of in approved "sharps" containers for incineration. Containers must be exchanged regularly and not allowed to become over-full.

• Workplaces must be kept clean and free from clutter (lab-books, pens, equipment). After use benches must be decontaminated using the approved disinfectants.

• Every microbiological lab must have a disinfectant policy (SoP) describing which and how to use disinfectants effective against the specific agents used in the lab.


Containment Laboratories

Containment levels required for microbiological as well as animal and plant laboratories

• CL1 for low risk HG1 biological agents

• CL2 for medium risk HG2 biological agents

• CL3 for high risk HG3 biological agents

• CL4 for extremely high risk HG4 biological agents – not permitted in Newcastle


Lab Containment Levels for different hazard groups Occupational Health

& Safety Service

Containment Level 1 Laboratory

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CL1 Specifications:

• Sealed, coved floors

• Sealed bench surfaces

• Biohazard sign on door

• Specified disinfection procedures

• Dedicated handwashing facilities at exit



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CL2 Specifications

• as for CL1 plus:

• Negative pressure if ventilated

• Secure; restricted access

• Microbiological safety cabinet for aerosol containment

• Secure storage of biological agents

• Sealed, impervious bench surfaces

• Autoclave in same building



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CL3 Specifications

• as for CL1 and CL2 plus:

• Dedicated laboratory and airlock

• Negative air pressure

• HEPA filtered air extract system

• Observation window

• Sealed, impervious floor surfaces

• Sealable to permit disinfection - fumigation

• Dedicated autoclave


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• Very high security units – only ~6 in the UK – Pirbright, HPA (Porton Down), MOD etc.

• Completely sealed labs with airlocks; fumigation and effluent treatment

• Total suit containment – though depends on organism

• Individual filtered air supply

• Use of totally sealed microbiological safety cabinets (class 3); often connected

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Class 3 MSC


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Basic Controls for Animal Containment

• Hazards – Urine, blood, faeces, saliva, hair, sharps, bites, allergies, viral vectors

• Animal escape AND biosecurity issues

• Containment laboratory, dedicated equipment and PPE

• Access control and locked rooms

• Isolators and individually ventilated cages (IVC) (HEPA filtered)

• Home Office licences and Defra licenses for animal welfare

• Extensive training in animal handling required


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Control & Containment of Research Animals

• Animal containment level 1- 4

• Protective equipment and procedures

• Security and access

• Disinfection and disposal procedures

• Air handling

• Operating procedures

• Small or LARGE animals? Risks?

• Animal welfare and transport


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Biological Controls help contain Biological Agents

• Highly effective means of containing biological agents and GMM’s

• Substitution of wild type strains or genetic environments for less harmful ones

• Reduced replication capacity eg lentivirus, adenovirus vectors

• Inactivated bacterial strains (‘lab’ strains)

• Auxotrophs require nutrients from media

• Species that cannot survive outside of lab environment

• Most GM cloning hosts are genetically disabled in one way or another


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Microbiological Safety Cabinets (MSC)

Class 1 Class 3 Class 2

MSC’s are designed for containment of contaminated aerosols – airborne biological agents


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Which Protective Cabinet to use?

1. Microbiological safety cabinet (MSC)

2. Laminar flow hood

3. Chemical fume hood

4. PCR hood (UV light)

2-4 ARE NOT Microbiological Safety Cabinets and must not be used for microbiological work

1 2

3 4


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• Do not obstruct grill – leads to loss of sterile curtain

• Adverse air flow can affect sterile air curtain and compromise containment

• Bunsen burners, centrifuges and walk-past all disrupt the airflow in MSC

• Use of caustic materials that vaporise can attack aluminium separators in HEPA filter

• MSC does NOT sufficiently vent harmful chemicals – MSC’s are NOT fume hoods so avoid volatile chemicals – methanol, acetic acid, mercaptoethanol

Correct MSC Operation is essential for Containment Occupational Health

& Safety Service

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Containing Aerosols in Centrifuges

• Centrifuges are a major source of aerosols

• Use Biosafety Rotors for biological agents - sealed tubes, buckets and rotors

• Open rotor, tubes and buckets inside MSC

• Do not open centrifuge immediately after a breakage or spillage – let aerosols settle first

• Disinfect after spillages


Personal Protective Equipment

• Microbiological lab coats (Howie-style) should be worn for all microbiological work but certainly for CL2 and CL3

• Risk assessment will determine what PPE needed

• Suitable gloves must be worn for hazardous activities, nitrile gloves for most biological hazards

• Eye/face protection should be worn when necessary

• Overshoes for sterile areas, containment animal facilities or for emergency spillage cleanup

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Respiratory Protective Equipment

• RPE may be required for certain hazardous activities

• MUST be the appropriate type (many types)

• Types for allergens, dusts, microbes, volatile chemicals and microbiological hazards

• Paper filter types of limited use (not BA)

• RPE must be face-fit tested to each individual person

• Monitor usage of reusable RPE (logs)

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Immunisation* – if containment has failed

and exposure has occurred

Area Activity ** Required Recommend

Clinical Patients/clinical materials contact – exposure prone procedures Hep B*

Patients/clinical materials contact – non exposure prone procedures Hep B

Laboratory Working with unscreened human blood or tissue Hep B

Working with screened human blood or tissue Hep B

Working with novel human cell lines from uncontrolled sources Hep B

Working with established human cell lines from controlled sources

NB Has the cell line been screened? Pre 2010 ATCC?

Hep B

Working with non human material

Agricultural /

Horticultural

Handling soil or plant material Tetanus

Animal

Technicians

Working with colony bred animals Tetanus

Working with wild caught primates if not conditioned Rabies, Hep A Tetanus

* remember immunity takes several weeks to become fully effective Hep B = 12 weeks

** routine work with hazardous materials warrants immunisation (if available) as one prophylactic measure


Transport and Storage of Biological

Agents


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Transport of Biological Hazards

• Transport properly to prevent accidental exposure or release of biological hazards – overlooked area!

• Containers, suitable? Strength, leakproof?

• Hazards warning signs and correct labels

• What would happen to samples if anything happened to the carrier person? Who would be informed and how? Accompanying PAPERWORK!

• Samples leaving the University – speak to the school safety officer first, many legal issues!

• Use reputable couriers

• Pathogenic / GM samples arriving at University? Check before requesting!

• Consult guidance on Safety Office Biosafety web site http://safety.ncl.ac.uk/transportofbiologicalhazards.aspx


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Be careful when packaging with dry ice!!


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Storage of Biological Agents – Containment in the lab

• Store cultured biological agents correctly

• Discard contaminated and used plates

• Label plates with organism, name, date, antibiotic resistance

• Consider long-term, medium-term, short term storage

• Consider spillage and transport. Containment!

bad storage practice!


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Good storage practice

• sealable • clear plastic box • robust (at low temp?) • clear labelling • indelible marker • discard • update

Name: Date: Organism / Sample: Antibiotic: Contact: Plus any other relevant biosafety information

If in doubt - test it!


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Precautions

• Must wear PPE e.g. full face visor

• Use only in well ventilated area (extract fan) or with oxygen monitors

• Avoid liquid N2 - store samples in vapour phase N2

• Use cryotubes NOT eppendorf tubes

• Store cryotubes in secondary container

• Place vials into secondary containment immediately after removal from LN2

• Liquid nitrogen is hazardous: cold b.p. -196oc, and an asphyxiant – several deaths due to nitrogen asphyxiation (1L liquid → 700L gas)

• There is a risk that vials may crack or explode as liquid nitrogen exaporates

• Oxygen condenses in LN2 – enriched oxidant

Ultra-Low Temperature Storage LN2 Occupational Health

& Safety Service

Disposal and Inactivation of Biological Agents and GMO’s

http://safety.ncl.ac.uk/transportofbiologicalhazards.aspx




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Disposal of Biological Agents/GMOs

• Biological agents and GMO’s must be safely disposed of using correct containers and waste route

Incineration Autoclave Sharps


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Sharps Controls

Go

od

Pra

ctic

e

Don’t dispose of sharps in ordinary waste bins

Don’t dispose of sharps in clinical waste bags

Never resheath needles

Use gloves and never resheath needles

Don’t transfer used sharps to other workers

Dispose of sharps immediately after use in a sharps bin

Take disposal bin to sharps

Dispose of bins on reaching max level (marked)

Treat all contaminated sharps as hazardous

Bad

Pra

ctic

e


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Inactivation of BA/GMO

• Autoclaving is the most effective method for inactivating BA and GMO waste

• Standard 121°C or 134 °C for 15-30 minutes

• Validation of effectiveness using annual (12-point) thermocouple testing is required

• BUT Do Not autoclave GMO’s containing radioactive or hazardous chemicals

• Verification of autoclaving efficacy needed for pathogen inactivation


autoclave tape

20 minutes @ 121oC (115psi)

TST indicator strip

Thermalog™ indicator strip

Autoclave indicators

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results with indicators inside a sealed air-filled container

results with indicators

buried inside a

waste load

X

Autoclave indicators

do not tighten caps

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• autoclave cycle monitor

• measures the actual temperature within the autoclave or load

• monitors temperature continuously so can verify that a given load has achieved and maintained temperature for the required period.

• printout can be kept for waste disposal records (CL3 and CL4)

Autoclave thermal monitor

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“Effective” Disinfectants

Disinfectant Bacteria Bacterial

spores

Fungi Enveloped

viruses

Non-enveloped

viruses

Prions Comments

Phenolic YES NO YES YES Limited NO Toxic

Hypochlorites YES YES Limited YES YES YES Toxic/Corrosive

70% Alcohol YES NO NO YES Limited NO Flammable

Aldehydes* YES YES YES YES YES NO

Irritant/allergen,

glutaraldhyde resistance

bacteria

*Formaldehyde YES YES YES YES YES NO CL3 level - very toxic,

need to seal the lab!

Peroxygen

(Virkon) YES YES YES YES YES NO

Dust irritant, limited

solution life, corrosive to

metals

Is a quick spray of ethanol good enough to protect you or your work from biological agents? (in some cases YES!; in ALL cases – NO!)


• disinfection policy (code of practice) is a requirement for all microbiological labs

• important to match your target organism to an effective disinfectant

− viruses, bacteria, spores, fungi

• how do you know if disinfection has been successful?

− data from manufacturer – kills 100% of all known germs!

− data from other users / labs

− ‘in-use’ testing

• know the limitations of disinfectants

Effective Disinfectant Use


Effective Disinfectant Use

• some disinfectants can be ‘inactivated’ under different conditions

• avoid disinfectant combinations

• limit age of disinfectant solution (fresh)

• use at correct dilution (‘in-use’ concentration)

• presence of ‘organic material’ - compensate

• exposure time

• COSHH properties; compatibility with hardware

PERACETIC ACID DISINFECTANT – active life


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Typical Spill kit (DIY)

• Be prepared - Have an emergency spill clean up kit available (project specific)

• Learn the standard operating procedure for spillage clean up and decontamination

autoclave bags

gloves mask

lab coat

absorbent towels

disinfectant

goggles

absorbant

granules

SOP

gloves

(heavy duty)

sealable bin (autoclavable)

biohazard

tape

overshoes


Post lab Inactivation

• Wash hands before leaving the lab! – sounds simple but this is a KEY control measure!

• Treat area with respect as this is also an Emergency Station

− Skin Contamination

− Mouth contamination

− Eye wash

• Handwash sink must not be used for lab waste, reagents, solutions, washing up.

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Personal Contamination or Injury

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For medical emergency

• Know what to do

• Dial emergency 999

• Dial security 6666

• Occupational health?


15


Security and Bioterrorism

• Need to consider security arrangements if handling and storing hazardous pathogens and toxins

• the Anti-terrorism, Crime and Security Act 2001

• Under the legislation, the holding, storage or use, of any micro-organism or toxin or relevant genetic material on Schedule 5, is subject to notification to the Home Office

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Training, Instruction and Supervision

• It is critical that all staff / students have had appropriate training and instruction in all hazardous processes – including handling biological agents

• Class-room exercises and “on the job” training – theoretical and practical

• Staff / students should be supervised until deemed “competent” in all processes

• Training records must be kept as a means of evidence of training and competency

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• remember when it comes to the enforcing authorities, such as HSE, Defra/Fera ……….

“ If it’s not documented – it’s not done! ”

Documentation

• So if you have no evidence (usually a printed document) that your work has been properly and adequately risk assessed it is assumed (by HSE) that this has NOT been done

• this applies to risk assessments, standard operating procedures, testing of facilities/equipment and training records/schedules for staff and students

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Summary

• Know and respect different containment levels

• Follow all risk assessments, SOPs, guidelines

• Dispose of all materials in the correct manner

• Decontaminate all work areas after use

• Be aware of the emergency procedures – be prepared!

• Seek advice if you are unsure, never assume! – you are NEVER alone – senior lab staff, supervisor, PI, SBSO, UBSO

• Get trained in relevant procedures and for using equipment

• Document (sign and date) all your biosafety training

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Any questions about Biological Agents?

Please make sure that you sign the attendance sheets for this course

and the GM course to make sure that you are registered as having attended these courses and that you receive your GM certificate

Bio and GM Safety v2 07/01/2014 · Bio and GM Safety v2 07/01/2014 5 Occupational Health & Safety Service 25 Workshop Exercise 2 Organism B: Organism B is a catalase-positive bacterium

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