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Biologische Sicherheit / Biosafety
Biosafety is the responsibility of all persons
who manipulate pathogenic microorganisms and
recombinant DNA molecules.

• Development and implementation of
• administrative policies
• work practices
• facility design
• safety equipment

Purpose of Biosafety -- to protect the
worker the product the co-workers those
who interface with the lab the environment
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Biologische Sicherheit / Biosafety
Biological agents are included within the
definitions of a "substance hazardous to health
Biological agent" means a micro-organism, cell
culture, or human endoparasite, whether or not
genetically modified, which may cause infection,
allergy, toxicity or otherwise create a hazard to
human health or environment
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Biologische Sicherheit / Biosafety

• Principles
• General Lab Requirements
• Knowledgeable supervisor
• Knowledgeable personnel
• Aware of potential hazards
• Proficient in practices techniques
• Lab specific biosafety manual (operating
  instructions)
• Biosafety Levels (BSLs)
• Laboratory Practice and Technique
• Safety Equipment (Primary Barriers)
• Facility Design and Construction (Secondary
  Barriers)
• Biosafety cabinets (BSCs) - BSL 2/3
• Personal protective clothing
• Pipetting Devices
• Safety centrifuge cups and rotors

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Biologische Sicherheit / Biosafety
Gezielte Tätigkeiten specific activities
Nicht gezielte Tätigkeiten non-specific activities
Gentechnisch veränderte Organismen
(GVO) Genetically modified organisms (GMO)
Humane Proben (nicht charakt. Infektionsstatus)
Non-inactivated materials of human origin
Tierseuchen- Erreger Pathogenic agents
Mikroorganismen/ Krankheitserreger Pathogenic
agents
Biologische Arbeitsstoffe Biologic agents
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Biologische Sicherheit / Biosafety
Labor der Biosicherheitsstufe 2 Laboratory
biosafety level 2
Gentechnische Anlage genetic engineering
installations (S1, S2, S3)
Laborräume Laboratories
Anlagen S1 Anzeige S2
Meldung S3 Genehmigung PL, BBS
Meldung Arbeiten S2 Anzeige, Meldung
S3 Genehmigung
Erlaubnisinhaber Genehmigung authorization Beginn
, Ende der Arbeiten Anzeige notification
Behördl. Anzeige, Meldung, Genehmigung Authorizati
on Notification
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Biologische Sicherheit / Biosafety
Gentechnisch veränderte Organismen
(GVO) Genetically modified organisms (GMO)
Humane Proben (nicht charakt. Infektionsstatus)
Non-inactivated materials of human origin
Tierseuchen- Erreger Pathogenic agents
Mikroorganismen/ Krankheitserreger Pathogenic
agents
Biologische Arbeitsstoffe Biologic agents
Projektleiter Project manager
Erlaubnisinhaber Officially authorized person
Arbeitsgruppenleiter Group leader
Gefährdungsbeurteilung Risk assessment
Biosafety Level, Containment and Work
Procedures Operating Instructions
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Risk assessment and safety classification
Criteria for Risk assessment

• Provides the information used to inform the
  selection of biosafety levels, microbiological
  practices, safety equipment, and facility
  safeguards
• Primary factors to consider
• Agent hazards
• Laboratory procedure hazards
• Ability of laboratory staff to control hazards

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Risk assessment and safety classification
Criteria for Risk assessment

• Identify agent hazards and perform initial
  assessment of risk
• Risk groups of biological agents (List of ZKBS)
• Basis for categorisation of pathogens
• is the organism
  pathogenic for humans
• is it a hazard for
  laboratory workers
• is it transmissible
  to the community
• is effective
  treatment or prophylaxis available

• Considerations for GMOs
• Characteristics of donor and recipient
• Characteristics of the vector
• Characteristics of the modified micro-organism
  and the gene products
• Health considerations
• Environmental considerations

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Risk assessment and safety classification
Criteria for Risk assessment

• Identify laboratory procedure hazards
• Concentration
• Suspension volume
• Generation of droplets and aerosols
• Sharps
• Bites and scratches when working with animals

• Determine appropriate BSL and select additional
  precautions
• Evaluate staff proficiency and equipment
  integrity

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Risk assessment and safety classification
Risk assessment of microorganism by
Robert-Koch-Institute http//www.rki.de/GENTEC/ZK
BS/ZKBS.HTM
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Good microbiological practice (GMP)
Routes of infection

• Ingestion
• by mouth
• Inhalation
• breathed in
• Percutaneous
• across damaged skin or mucous membranes
• Instillation
• in eye
• Direct or by transfer on hands etc

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Good microbiological practice (GMP)
Basis for control measures

• Block all routes of infection
• Do not put anything in mouth
• Do not create aerosols
• Cover damaged skin and minimise risk of puncture
  wounds
• Do not produce splashes and droplets
• Do not transfer to any route with hands or other
  contaminated
• objects

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Good microbiological practice (GMP)
The Ten Conmandments

• Keep doors and windows closed.

• In working area wear lab coat and/or protective
  clothing
• Protective clothing devices must not be
  worn outside lab
• Open-toed foot wear must not be worn in lab

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Good microbiological practice (GMP)
The Ten Conmandments

• NO mouth pipetting.
• Materials must not be placed in mouth

• Avoid formation of aerosol.

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Good microbiological practice (GMP)
The Ten Conmandments

• After you are done decontaminate all waste, work
  space and equipment (which had been in contact
  with the organisms).
• After you are done desinfect and wash hands.

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Good microbiological practice (GMP)
The Ten Conmandments

• Regularly, verify identity of the biological
  material used.

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BSL 1 Work Practices and Procedures

• Applications
• Non-infectious agent and tissue culture, media
  preparation
• Prevent Cross Contamination
• Keep cultures covered
• Flame instruments and containers
• Use sterile media and equipment
• Keep hands or face away from cultures

• Biosafety Procedures
• Work with agents may be conducted on open bench
• Wash hands often
• No mouth pipetting
• No eating or drinking in lab
• Minimize aerosol generation
• Decontaminate work surfaces
• Wear applicable PPE

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BSL 2 Work Practices and Procedures

• Increasing emphasis on safety procedures and
  practices
• Increasing need for staff training
• Increasing need for competent supervision
• Restricted access
• Biohazard sign posted at entry door
• Biohazard labels affixed on regulated waste
  containers
• Use of personal protective equipment as a barrier
  to exposure lab coat, gloves, eye and face
  protection
• Some work on open bench allowed

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BSL 2 Work Practices and Procedures

• special emphasis on
• Gloves
• Mechanical pipetting
• Attention to sharps

• Use biosafety cabinets (class II) for
• work with infectious agents involving
• Aerosols and splashes
• Large volumes
• High concentrations

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Operating Instructions L2/S2
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Operating Instructions L2/S2

• Responsible persons
• Risks bilogical agents
• Regulation of access
• Instructions and medical care
• Laboratory practice
• Waste disposal
• Accidents, emmergencies
• Hygienic measures
• Special regulations related to the individual
  project

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BSL 2 Work Practices and Procedures

• Acess to L2/S2 areas
• is restricted to people, which have obtained
  safety intructions in yearly intervals.
• Visitors may only enter the L2/S2 areas in the
  presence of instructed people. (No access for
  children up to the age of 13, children between 13
  and 15 only in the framework of education under
  constant supervision).
• Personell of cleaning and maintenance of machines
  is only allowed to work in the L2/S2 area after
  having obtained safety instructions.
• Admitted persons with known medical conditions,
  pregnant or stilling mothers are required to
  contact the DKFZ doctor to fix individual
  regulations for their work.

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BSL 2 Work Practices and Procedures

• Instructions and medical care
• People possessing a permission to access have to
  be instructed about the dangers and rules of
  conduct in the lab before begin of work and in
  regular intervals.
• Medical surveillance
• Examination by the DKFZ doctor
• before starting the work , then annually and
  upon ending employment
• before initial work with agents for which
  immunisation exists, and for agents which cause
  chronic illness
• upon request by the employee
• Blood samples are taken
• upon contamination with infectious material
  (as negative value)
• upon request before starting and after
  finishing work at the DKFZ
• Immunization
• upon request HBV (vaccinia, measels)

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BSL 2 Work Practices and Procedures

• Aerosol generating procedures performed in a
  biosafety cabinet
• Homogenizing
• Vortexing
• Vigorous mixing
• Pipetting infectious liquids
• Sonication
• Pouring

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Hygienic Measures
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Hygienic Measures
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Use of gloves
Use disposable gloves for handling of
infectious material. however gloves do not
give absolute protection against infection.
? Need to desinfect hands after glove use!!
Do not touch common laboratory equipment with
gloves. Never wear gloves outside the lab.
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Use of gloves
Proper Glove Removal

• With both hands gloved, peel one glove off from
  top to bottom and hold it in the gloved hand
• With the exposed hand, peel the second glove off
  from the inside, tucking the first glove inside
  the second
• Dispose of the gloves promptly
• Never touch the outside of the glove with bare
  skin
• Wash hands as soon as possible

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Correct Use of Biosafety Cabinets

• Start Up
• Turn off ultraviolet light (if so equipped) as
  soon as you enter the room.
• Turn on all blowers and BSC illumination lights.
• Allow five minutes of operation to purge system
  check flow alarm system audio and visual alarm
  function (if so equipped).
• Decontaminate readily accessible interior
  surfaces and items with a disinfectant
  (appropriate for the agents or suspected agents
  present) before loading and wait at least 10
  minutes prior to start of work.

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Correct Use of Biosafety Cabinets

• During Use
• Load supplies prior to work.
• Do not overload cabinet.
• Separate clean and dirty side.
• Work in center of work area.
• Do not block front or rear grills.
• Minimize disruption of airflow (turbulence).
• Clean up spill promptly.
• Discard waste within the cabinet.

• DO NOT use Bunsen burners
• or open flames
• Fire hazard
• Can damage HEPA filter
• Interferes with proper air flow
• Microincinerator preferred
• Burner with pilot light not a good alternative
• Open flames react with disinfectants (flammables)

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Correct Use of Biosafety Cabinets

• Shut Down
• Decontaminate and remove all items from interior
  work area.
• Decontaminate readily accessible interior
  surfaces with a disinfectant appropriate for the
  agents or suspected agents present.
• Turn on ultraviolet light (if so equipped).
• Allow five minutes of operation to purge
  system. Then wait at least 10 minutes.
• Turn off BSC blower.

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Correct Use of Biosafety Cabinets

• Moving/InstallationBSCs must be decontaminated
  prior to moving. In order to ensure filter
  integrity, the equipment must be recertified
  after the BSC is installed at its final new
  location. Arrangements need to be made well in
  advance in order for contractors to meet your
  schedule. The PI is responsible for contacting
  the contractor or to schedule this work.
• DecontaminationDecontamination is usually
  performed by certified professionals.
• CertificationAll BSCs that are used for handling
  biohazardous materials must be recertified
  annually.

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Safe Use of Centrifuge

• For handling infectious material of BSL2
• Use safety centrifuge cups and rotors

• Load/unload centrifuge tubes, rotors and
  accessories in BSC

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Safe Use of Centrifuge

• Use sealed tubes, rotors, and safety cups/buckets
  that are sealed with O-rings
• Inspect tubes, O-rings and rotors for wear, and
  buckets for cracks, chips, erosion, etc.
• Do not use aluminum foil to cap centrifuge tubes
• Clean and maintain gaskets and O-rings
• Change O-rings if compromised
• Small, low speed centrifuges may be placed in a
  BSC however, high speed centrifuges pose
  additional hazards
• Do not overfill tubes
• Balance buckets, tubes and rotors properly before
  centrifugation
• Wait 5 minutes (or 30 mins. for high speed
  centrifuge) after each run before opening
• Do not decant or pour off supernatant. Use a
  vacuum system with appropriate in-line reservoirs
  and filters

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Minimizing Aerosols

• Use careful pipetting practices
• Avoid drops onto hard surfaces
• Wipe up spills promptly with appropriate
  disinfectant
• For ejection of liquid from micropipette
• No blowout
• No pressure ejection
• Use wall contact
• Use capped tubes when mixing, blending, or
  vortexing
• Pour liquids carefully
• Avoid bubbles

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Careful Pipetting Techniques

• Never blow out last drop in pipette
• Use pipetting aids with filters
• Discharge liquid down side of container, using
  tip-to-wall contact
• Deliver as close as possible to contents
• Work over plastic-backed absorbent matting
  (ensure it doesnt slide forward or backward
  blocking air grill)

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Use Extreme Care with Sharps

• Use sharps if only absolutely required as part of
  a process
• Percutaneous exposure risk
• Employ safe work practices
• Utilize safe sharp devices
• Aerosol exposure risk
• Use biosafety cabinet for removal of air from
  needle
• Use mechanical methods for needle removal
• Never bend, recap or manipulate sharps by hand
• Keep hands away from needle
• Use plastic ware

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Emergency Response to Biological Incidents
Don panic, keep cool and act prudently. Call for
help PI, BSO, safety officer In case of
emmergency immediately inform safety
department phone 2888
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Exposure Management
Contaminated persons

• skin
• Thoroughly disinfect with Spitacid or Desderman.

• eyes
• Immediately rinse out under flowing water while
  opened (10 min). Thereafter consult immediately a
  eye specialist.

• mucous membranes
• Immediately rinse with water. Thereafter consult
  immediately a specialist.

• extensive personal contamination
• First remove contaminated clothes, then disinfect
  skin with Spitacid or Desdoderm.

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Exposure Management
Contaminated persons

• Slight skin wounds
• (needlesticks or cuts with human blood, fluids,
• infectious agents or antibiotic resistant
  organism)
• Let wound bleed for 1-2 min, then apply wound
  disinfectant (Neokodan).

• Serious wounds
• Conventional first aid measures before
  disinfection!
• Call for help 2888

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Spill Clean-Up

• You can clean-up a biological spill if
• You are aware of the hazards and clean-up
  procedures (training required)
• There is no potential for personal or
  environmental damage
• The appropriate spill clean-up equipment is
  available
• Note Spill incident still needs to be reported
  to safety departrment and BSO. If spill is in
  liters, call safety department/BSO.

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Biological Spill Clean-Up Kit- Basic

• Tyvek-overalls
• Nitrile gloves (double gloving), safety goggles,
  shoe covers
• Partial filter P3-hals mask
• Small disposable broom with dustpan, tongs or
  forceps (for picking up sharps)
• Paper towels or other absorbent in the lab
• Sharps container and/or biohazard waste bags
• Disinfectant agent suitable for the agents in the
  lab

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Spill Clean-Up for BSL-2

• If there is a spill inside the biosafety cabinet
• Keep the BSC running during spill and clean-up to
  contain aerosol.
• Place absorbent paper on spill and soak with
  disinfectant.
• Allow 20 minutes of contact time. Wipe up spill,
  working from the edges to the center. Clean
  spill areas with fresh paper towels soaked in
  disinfectant.
• Disinfect the BSC interior and any other
  equipment in the BSC with disinfectant.
• Discard contaminated disposable materials using
  appropriate biohazardous waste disposal
  procedures.
• Place contaminated reusable items in biohazard
  bags or autoclavable pans before autoclaving.
• Run BSC 10 minutes after clean-up before resuming
  work or turning BSC off.
• Note If you are working in a BSC and the power
  went off in the room or the BSC fan stops
  blowing, IMMEDIATELY LEAVE THE ROOM.

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Spill Clean-Up for BSL-2

• If the spill is in the laboratory but outside the
  biosafety cabinet
• Call the safety department/BSO if the material is
  RG 2 or greater.
• Clear area of all personnel. Wait at least 30
  minutes for aerosol to settle before entering
  spill area.
• Remove any contaminated clothing and place in
  biohazard bag to be autoclaved.
• Put on disposable gown, safety glasses and
  gloves.
• Initiate cleanup with disinfectant as follows
• Place dry paper towels on spill then layer a
  second set of disinfectant soaked paper towels
  over the spill.
• Encircle the spill with additional disinfectant
  being careful to minimize aerosolization while
  assuring adequate contact.
• Allow at least a minimum of 20 minutes contact
  time to ensure germicidal action of disinfectant.
  Wipe up spill, working from the edges to the
  center. Clean spill areas with fresh paper
  towels soaked in disinfectant.
• Decontaminate all items within the spill area.
• Discard contaminated disposable materials using
  appropriate biohazardous waste disposal
  procedures.