Basic Course:
How to play it Safe in a Biosafety Lab
10.05.2017
Dr. Ursula Jenal, Jenal&Partner
Dr. Ines Raabe, SSHE, Laboratory and Occupational Safety
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Agenda
 Biosafety at the ETH Zürich: organization, responsibilities
 Basics in biosafety
 History of biosafety
 Why biosafety at all?
 Legal aspects
 Hand hygiene (incl. “Glo-Germ” experiment)
Coffee Break
 Overview: good microbiological practice, lab rules
 Personal protective equipment
 Which PPE should I use?
 When?
 When NOT?
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Biosafety at the ETH Zürich: organisation
and responsibilities
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Biosafety: Who is involved at the ETH Zürich?
head SSHE
CABS
other SSHE
units
„SSHE-BSO“
group leader
BSO (group,
institute)
cleaning and
maintainance
lab responsible
coworkers
staff
Legally, each group / institute is considered as an independent „company“ !
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Duties and responsibilities of the BSO (I)
 regular further training (e.g. seminars for BSO)
 create and update of a biosafety concept, incl.:






project list
list of organisms used in the group / institute
list of employees / students (provided by PL)
list of incidents / accidents
hygiene plan
etc.
 organize access rules for BSL2 labs: access only for authorized
people
 prepare an emergency plan (emergency measures, spill kit)
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Duties and responsibilities of the BSO (II)
 correct labelling of the labs (warning signs for BSL2, etc.)
 communicate safety measures and rules, e.g. concerning disposal of
hazardous waste, transport, SOPs...
 monitor that lab staff complies with safety measures and rules
 support / advice the PL
 coordination with other safety responsibles (radioprotection, etc.)
 self-inspection of the group / institute (checklist can be obtained from
ETH SSHE)
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Duties and responsibilities of Project Leaders
(PL) (I)
 ensure that all employees and students comply with regulations and
safety measures (CO, SAMV, SOPs, GMP, internal lab rules, etc.)
 ensure that all employees and students comply with regulations on inhouse transport of biological material
 provide personal protective equipment (PPE) for the employees and
students
 ensure that PPE is used (remember: labcoats mandatory even in
BSL1 labs)
 notification of activities, risk assessment ( inform the BSO of any
new activity)
 responsible for correct disposal of hazardous waste
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Duties and responsibilities of Project Leaders
(PL) (II)
 regular biosafety training and instruction for employees and
students (at least once per year)
 organization and check of health monitoring
 list of employees and students working in the different projects
 list of all incidents / accidents ( hands this list out to the BSO)
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Duties and responsibilities of the coworkers
(=YOU)
 Comply with safety regulations and SOP’s
 Use safe working techniques
 Comply with good microbiological technique
 Keep lab / workplace tidy
 Disinfection of lab / workspace
 Correct disposal of special waste
 Proper use of personal protective equipment (PPE)
 Damaged equipment, unsafe situations  report to BSO / PL
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Where to find all these information?
 In the biosafety concept of your group / institute


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SOPs
Lab rules
Hygiene plan
Maintenance plans
Disposal of special waste
Etc.
 Ask your BSO for any group-specific information!
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Where to find all these information?
 SSHE team: [email protected]
 SSHE homepage : http://www.sicherheit.ethz.ch
 General biosafety concept of the ETH
 factsheets, guidelines (not only for biosafety, but also for chemical safety, radiation
protection, lasers, maternity protection, etc.)
 Courses / seminars
 All general (and ETH specific) information
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Basics in biosafety
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ABC Biosafety I
-
Definition
Hazardous biological material
Origin
Principles of containment
Ursula Jenal
Baldwin, C.L. & Runkle, R.S.
Science 158: 264-265, 1967
ETH, 10.05.2017
[email protected]
Biosafety is controlling risks that could result
from exposure to hazardous biological material
Exposure of the public
Epidemiology
-
– Public areas, medical
practices, hospitals….
– Endemic areas
Human pathogens
• Hygiene
• Vaccination programs
Gene and cell therapy products
• Antibiotics
Live recombinant vaccines
– Clinical trials
Exposure of the environment, – Import control
– Endemic areas
field trials, marketing
• Pest control
- Plant and animal pests
- GMO plants
- Invasive species
Use inside facilities
- Life science industry
• Quarantine measures
• Control of dissemination and gene transfer
– Biosafety of field experiments
- Academia
- Microbiological diagnostics
– Biosafety
Misuse of biological agents,
biocrimes, bioterrorism
– Biosecurity
ETH, 10.05.2017
[email protected]
For which biological material is containment
necessary?
positive >/= negative effects ?
negative > positive effects ?
> risk
assessment
ECOSYSTEM
commensal
ammensal
destruent
pathogen
pest
symbiont
parasite
organism
predator
pray
invasive species
host
biogeochemical
circles
species alteration
through gene
transfer
ETH, 10.05.2017
[email protected]
Examples of research activities
involving GMOs, pathogens, invasive species and/or
biologically active genetic material
•
Research on disease prevention with human-, animal- and plant
pathogens.
•
•
Biological production of vaccines, antibiotics, food additives.
•
Establishment of new animal disease models, i.e. virus-producing
transgenic mice.
•
•
Activities with xenonucleic acids or xenoamino acids.
Gene therapy and stem cell research using viral vectors, also
CRISPR-Cas technology.
Pharmaceutical-producing transgenic plants, nutritionallyenhanced plants, pest-resistant plants.
•
Invasive animals, plants and microorganisms harbouring traits or
genetically engineered to contain inserts that lead to spread and
persistence in the environment, gene drives
•
•
•
Xenotransplantation, chimeric organs through genome editing
Biological control of pests through exotic species
Dual-use and gain of function research
ETH, 10.05.2017
[email protected]
Responsible life science research:
Should all research doable be done?
Reconstruction of 1918
Spanish flu ?
Biosecurity
Biosafety
Establish
transmissibility
of A/H5N1
influenza via air
by ferrets?
Human germline genome
editing ?
Bioethics
ETH, 10.05.2017
[email protected]
Biosafety - Biosecurity – Ethical Research
…. the containment principles, technologies and practices
that are implemented to prevent the unintentional
exposure to pathogens and toxins, or their accidental
release.
WHO Laboratory Biosafety
Manual, 3rd ed., 2004
…. to prevent their unauthorized access, loss,
theft, misuse, diversion or intentional
release.
Biorisk management
Laboratory biosecurity guidance
WHO/CDS/EPR/2006.6
…. the ethical acceptability of the research,
including its social value and scientific validity,
an acceptable ratio of potential benefits to risks
of harm…
Standards and operational guidance for ethics
review of health-related research with human
participants
ISBN 978 92 4 150295 5 6
ETH, 10.05.2017
[email protected]
Aspects of laboratory biosecurity and
interrelationship with biosafety
Public health
Biosafety
Biosecurity
http://www.bbic-network.org/
‘The protection, control and accountability for biological agents and toxins within
laboratories, in order to prevent their loss, theft, misuse, diversion of, unauthorized
access or intentional unauthorized release’
CWA15793 Biorisk Management
ETH, 10.05.2017
[email protected]
Biosafety is controlling biological risks
through team work
Protection of
people and the
environment…
…and yourself
and your
colleagues
Biosafety
Implement
best practices
and safety
measures
according
to risks
Comply to regulatory
requirements
Contained use ordinance, https://www.admin.ch/ch/d/sr/c814_912.html
Worker protection ordinance, https://www.admin.ch/ch/d/sr/c832_321.html
ETH, 10.05.2017
[email protected]
CEN Workshop Agreement 15793:2011 Biorisk
Management

Internationally applicable expert consensus document on biosafety &
biosecurity, http://www.uab.cat/doc/CWA15793_2011

Application of quality management to biosafety & biosecurity

To be transferred into an ISO standard 35001
Policy / philosophy
‘high standard in occupational and public
health as well as environmental safety’
Goals
‘apply best practice and
Evaluate
reports,
reviews and
initiate revisions
techniques for the safety of
people and the environment’
Report
Define & implement
biosafety concept
on problems
and successes
in implementation
Review & revise
processes periodically
for their adequacy
ETH, 10.05.2017
[email protected]
Biosafety yesterday - today
Alexander Yersin's lab
Karolinska Institut, Schweden,
in Kennedy Town, Hong-Kong,1894,
Stufe 3 Labor, seit 1994
for the analysis of Yersinia pestis
• USA, 1955: first safety conference related to bioweapons.
•
USA, 1964: first biosafety conference related to pathogens in general.
•
USA, 1975: Assilomar conference
•
WHO,1983: Basic concepts in biosafety, ‚Laboratory biosafety manual‘.
•
CH,
1986: Ordinance on major accidents.
•
EU,
1990: Directives on worker protection and contained use of organisms.
•
CH,
1999: Ordinances on contained use and deliberate release of organisms.
ETH, 10.05.2017
[email protected]
Laboratory acquired infections: yesterday
• Conte
nt
Needle sticks
10%
Spills
4%
25%
Broken glass
27%
Mouth
pipetting
18%
Infections
Accidents
18%
16%
accidents
No
identified
cause
82%
Bites /
scratches
Others
In 82% of the cases, no information on
the cause of infection
Belgian Biosafety Server, Laboratory-acquired infections, http://www.biosafety.be/CU/LAI/Intro_LAI.html
ETH, 10.05.2017
[email protected]
Laboratory acquired infections: today
1988: 82.7 cases/100’000 laboratory workers/year
1995: 16.2 cases/100’000 laboratory workers/year
Today: ~ 4 cases/100’000 laboratory workers/year
in USA (CDC)
• Decrease in LAI due to:
– increased awareness
– safety training
– safe work practices (personal protective equipment)
• Most LAI linked to an identified incident
– 65 % due to human error
– 20 % problems with instruments
– 15 % unsafe practices
• Main routes of infection
– Needle stick injuries
– Aerosol-producing procedures
– Bites and scratches
ETH, 10.05.2017
[email protected]
Behaviour
People at risk
• ignorance, rejection
• misinformation / insufficient information
• not supported by piers / management
• immune-suppressed
• pregnant
• lack of vaccination
ETH, 10.05.2017
[email protected]
Examples of laboratory acquired infections
• Exposure to aerosols with TB/ Neisseria meningitidis from using defective
biosafety cabinet and commonly used laboratory equipment
• Cracking of centrifuge bottle during run (Sabia Virus)
• Handling nude mice infected by an inadvertently contaminated cell line
(lymphocytic choriomeningitis virus)
• Handling broken blood tubes (HIV), droplet in eye lead to seroconversion
• Gloved hand contaminated with virus supernatant touched non-intact skin
(HBV, HIV), put finger in mouth (ulcer due to Helicobacter pyroli)
• Employee contaminated pastries in cafeteria (Shigella)
• Employee prepared dinner for family (Salmonella)
• Work on open bench with E. coli O157 (salad spinner)
• Cross-contamination: SARS found in West Nile Virus samples
• Virulent Francisella tularensis found in Tularemia vaccine strains
• Vibrio cholerae, spill clean-up in a shaker
Contained Use of GMOs and pathogens: Laboratory-acquired Infections:
References http://www.biosafety.be/CU/LAI/Recent_LAI.html#2016
American Biosafety Association LAI database http://www.uab.cat/doc/CWA15793_2011
ETH, 10.05.2017
[email protected]
Containment principles for laboratories to
avoid exposure to or environmental release of
biological material according to its risk
Level 1
- GMO
- no pathogens
Level 2
- pathogen (GMO)
ETH
Primary barriers
- exposure of personnel
- opportunistic
- self-curing
Level 3
- pathogen (GMO)
Secondary barriers
- release into the
environment
- potentially lethal
- treatment available
Tertiary barriers
- organisation
Level 4
- pathogen (GMO)
- potentially lethal
- no treatment
ETH, 10.05.2017
[email protected]
Avoid exposure to hazardous biological material
Bites
- when working with animals or arthropod vectors
- parasites, Dengue, Herpes B
Inhalation
Contact with
mucous
membrane
- aerosols
- splashes
- TB, influenza, SARS
- hands
- aerosols
- splashes
- Herpes Simplex Virus
Ingestions
- splashes, contact
- E. coli, Salmonella
Injury
Contact with skin
- broken glass
- needle stick
- Staphylococcus aureus
- Clostridium tetani
- splashes
- contaminated
material
ETH, 10.05.2017
[email protected]
Environmental protection from hazardous
biological material
Air
- close windows
- close doors
- filtration of exhaust air
- negative pressure
Vector
- disinfect hands
- remove lab coat
Water
- inactivate
Transport
- packaging
- decontamination
Waste
- inactivate
ETH, 10.05.2017
[email protected]
Exposure though contact with
contaminated material
• Outside surface of primary
containment
• Centrifuge
• Waste containers
• Transport containers
• Door handle, key boards
• Sink
• Lab coat
• Work bench outside BSC
ETH, 10.05.2017
[email protected]
Avoid contact exposure by wearing gloves
and personal protective equipment
https://www.youtube.com/watch?v=S4gyNAsPCbU
https://www.youtube.com/watch?v=TwJfNfCEnKU
and many more YouTube movies to check out….
https://www.yourglovesource.com/blogs/glove-knowledgebase/43943233-how-toput-on-nitrile-and-latex-gloves
ETH, 10.05.2017
[email protected]
ABC Biosafety II
Safe practice in biology
laboratories
ETH, 10.05.2017
[email protected]
Protection of personnel: hand hygiene
Washing = water and detergents remove particles.
Disinfection = inactivates microorganisms.
• Finger tip: 20 – 100 microorganisms/cm2
• Hand: 103 – 104 microorganisms/cm2
• Wash hands
–
–
–
–
–
before and after work
before and after a break
after using the toilet
after blowing nose
after contamination
• Level 1: wash hands
• Level 2: wash and disinfect hands
• Reduce jewellery to a minimum
ETH, 10.05.2017
[email protected]
Protection of personnel: hand hygiene
ETH, 10.05.2017
[email protected]
Hand Disinfection
ETH, 10.05.2017
[email protected]
Basic rules of good microbiology practice
• Good laboratory and hygiene practices
–
–
–
–
–
–
no mouth pipetting
no eating, drinking, storing of food or drinks
no contact with eyes and mouth
no contact lenses, no application of cosmetics
wear lab coat and gloves
wash hands when leaving lab
• Work practices
– keep lab clean and in order
– doors and windows closed (Level 2)
– clean work surfaces - disinfect when GMOs or
pathogens are handled
– avoid using sharps, get training for animal handling
– pack and label material appropriately
ETH, 10.05.2017
[email protected]
Minimize/avoid producing aerosols!
• Minute respirable airborne particles
• Droplets < 5µm reach alveoli of the lung
• Particles > 5µm reach mucous membrane of airways
• Disposition of larger heavy droplets onto surfaces, equipment,
personnel
•
•
•
•
Loop
Pipetting
Blenders, homogenizers, shakers, sonicators, mixers
Flow cytometry,
FACS fluorescent-activated cell sorter
ETH, 10.05.2017
[email protected]
How to avoid exposure to aerosols
• No blowing-out of pipettes.
• Use closed centrifuge tubes.
• Pay attention to liquid in lids when
opening the tubes.
• Transport cultures in closed containers.
• Outside of level 2 labs transport cultures
in closed secondary break-proof and
leak-proof containers to avoid spills.
• At level 2 use a biosafety cabinet at least
when liquid cultures of pathogens are
handled openly and whenever production
of aerosols cannot be avoided.
• (At level 1 for sterile practices with cell
lines).
ETH, 10.05.2017
[email protected]
Biosafety Guidelines
•
Biosafety in Microbiological and Biomedical Laboratories (BMBL)
CDC/NIH, 5th ed. 2009
http://www.cdc.gov/biosafety/publications/bmbl5/
•
Fleming & Hunt, Biological Safety: Principles and Practices
4th edition, 2006, ISBN-13: 9781555813390
•
Recommendations of the Swiss Federal Expert Committee
for Biosafety
www.efbs.ch
•
CEN Workshop Agreement CWA15973:2011 on
Biorisk Management
http://www.uab.cat/doc/CWA15793_2011
•
UK Health and Safety Executive
http://www.hse.gov.uk/biosafety/
The Genetically Modified Organisms (Contained Use) Regulations 2014
http://www.hse.gov.uk/pubns/books/l29.htm
The management, design and operation of microbiological containment laboratories
http://www.hse.gov.uk/pubns/books/microbio-cont.htm
ETH, 10.05.2017
[email protected]
Biosafety Training
 Swiss Biosafety Curriculum by the Swiss authorities
Training on level 1 (1 day), level 2 (3 days) and level 3 (3 days)
www.curriculum-biosafety.ch
 SUVA Lehrgang
Arbeitssicherheit und Gesundheitsschutz
http://www.suva.ch/home/suvapro/weiter_fortbildung/modularer_aufbau.htm
 DECHEMA, Frankfurt
Gentechnikrecht, Gefährdungspotentiale, Sicherheitsmassnahmen
und Rechtsvorschriften
http://dechema-dfi.de/Gentechnikrecht.html
 European/American Biosafety Associations
Annual pre-conference courses on specific biosafety topics
http://www.ebsaweb.eu/, http://www.absa.org
ETH, 10.05.2017
[email protected]
The not trained,
the well trained,
and the overly
trained…
Keep asking
questions!
ETH, 10.05.2017
[email protected]
https://pcscaliforniainc.files.wordpress.com/2015/10/rts-pictures1.jpg
Personal Protective Equipment (PPE)
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PPE as “last step” in the STOP concept
Decreasing efficacy
Strategic
measures
Technical
measures
Organisational
measures
Personal
protective
equipment
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PPE in a (micro-) biological lab
Triple protection
 Protection of lab personnel (legal basis: SAMV)
 Labcoat, gloves, safety glasses, dust mask
 Protection of sample (e.g. cell cultures)
 Labcoat, gloves
 Protection of the environment ( contained use!, legal basis: CO)
 Labcoat, gloves
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Labcoats
 Protection of lab personnel, environment and samples
 Mandatory in all (micro-) biological labs (even BSL1!) and when
handling hazardous substances, e.g. solvents, acids, liquid nitrogen)
 Not allowed: at desk, in cafeteria, in offices, etc.
 to be washed regularly (ask BSO how laundry is organized in your
unit)
 BSL2 labcoats have to stay inside the lab
 Different labcoats for BSL1 and BSL2
 In BSL2 disinfect after contamination
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Safety goggles – not only for chemists…
 Protection of lab personnel
 Mandatory when
 Risk of splashes / aerosol production / dust production
 Spill cleanup
 Handling chemicals (including solvents) or cryogenic liquids
 Working with lasers (class 3B and 4)
 Cutting glass
 Don‘t wear contact lenses (not even in combination with safety
goggles)
 When 1 person is doing such work in the lab, all
others must wear safety goggles, too!
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Safety goggles
 Optically corrected safety glasses can be obtained via SSHE
 Normal glasses don’t replace safety goggles!
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I wear gloves all the
time - I know only too
well how my colleagues
work!
Gloves
 Protection of lab personnel, environment and samples
 Mandatory when
 To be removed before
 contact with pathogenic material
cannot be excluded (BSL2)
 handling hazardous substances
 handling cryogenic liquids (liquid N2
etc.) or dry ice
 Working at desk / office space
 Touching computer keyboards
 Touching door handles,
telephones, etc.
 Leaving the lab
 Chose the appropriate glove for your work (wrong gloves  you’re
not safe at all!), check compatibility
 Regularly check and replace re-usable gloves, never re-use singleuse gloves
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Gloves
 Symbols for gloves standard
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Protective gloves against chemical risks
 Glove shows good degradation and penetration behavior
and protects against permeation of chemical substances.
 Terms:
 Degradation
Resistance, moisture expansion
 Penetration
A chemicals passes through a glove via defects or
porosities in the material and via untighten seams
(macroscopic)
 Permeation
A chemicals passes on the molecular level through the
material of a glove
Permeation time (min – h)
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Attention!
Protective gloves against
chemical risk
Simple protection
[139]; [140]
Protective gloves against
chemical risk
Full protection  testsubstance
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Which glove is the best?
Bad news: the one and only perfect glove does not exist!!!
Good news: there are suitable gloves for almost every kind
of chemicals - It all depends on which substances you work
with…
Be careful with latex / natural rubber (NR) gloves: they can
cause severe allergies  not recommended!
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Certification of gloves
Test substance
Compound class
A
Methanol
Primary alcohols
B
Acetone
Ketones
C
Acetonitrile
Organic nitriles
D
Dichlormethane
Chlorinated paraffines
E
Carbon disulfide
Sulfur containing organic compounds
F
Toluene
Aromatic hydrocarbons
G
Diethylamine
Amines
H
Tetrahydrofurane
Heterocycles and ethers
I
Ethylacetate
Esters
J
n-Heptane
Aliphatic hydrocarbons
K
Sodium hydroxide, 40%
Inorganic bases
L
Sulfuric acid, 96 %
Mineral acids
source: www.dguv.de/ifa/de/pra/chemikalienschutzhandschuhe/kennzeichnung/erlenmeyer/index.jsp
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Chemical resistance of different
glove materials
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Chemical resistance
NitroChem Plus
Ethanol
Ethidiumbromide in H2O
Ethylacetate
Formaldehyde 35%
Gasoline
Glutardialdehyde
Heptane 98% +
1-Butanol 2%
Hexane
Hydrochloricacid
Permeation level
in minutes
Protection level
0-6
8
0
> 480
6
<1
0
> 480
6
84
3
> 480
6
9
0
> 480
6
126
4
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How to Remove
Single-Use
Gloves
Source: www.HSE.gov.uk
https://www.youtube.com/watch?v=dyLEd9cng5U&feature=share&list=PLNhVwA0XZE27h8uh8ug8kX3362W2CAvai
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Sources of Information
 Material safety data sheet (MSDS)
 GESTIS Substance Database
 http://gestisen.itrust.de/nxt/gateway.dll?f=templates$fn=default.htm$vid=gestiseng:sdbeng
 http://www.ansell.com
 http://kcl.de
 http://www.mapa-professionnel.com
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Acetone (MSDS) – protection of hands
Wear protective gloves
Material of gloves
Butylcaoutchouc, thickness: 0,7 mm
The selection of the suitable gloves does not only depend on the material, but also on
further marks of quality and varies from manufacturer to manufacturer.
Penetration time of glove material
Value for the permeation: Level ≥ 6
The exact breakthrough time has to be found out by the manufacturer of the protective
gloves and has to be observed.
As protection from splashes gloves made of the following materials are suitable: Natural
rubber
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Dust Masks
 Protection of lab personnel
 Mandatory when risk of hazardous dusts / aerosols
 All potentially exposed people have to wear one
 Doesn’t protect against chemical vapors or toxic gases
 Doesn’t protect against lack of oxygen
 Doesn’t replace a fume hood!
 Surgical masks ≠ dust masks!
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What we don’t want to see any more…
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Thank you for your attention!
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Name Vorname | 09.05.2017 | 33