Basic Biosafety: Safety in Experiments

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Basic BiosafetySafety in Experiments

• Environmental Health and Safety
• November, 2004

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Environmental Health and SafetyEHS

• EHS has been charged with ensuring the safety of
  this campus. We work to
• Ensure that biological research is conducted in a
  safe fashion
• Ensure that biological research meets regulatory
  requirements
• Support the Institutional Biological Safety
  Committee (IBSC)

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Biosafety means Practicing Safe Science

• Ask Questions BEFORE you start
• Plan safety into your experiment
• Biosafety manuals and UA manual each lab should
  have these and provide training on their contents
• Good lab practices
• Hazard Communication you must notify anyone who
  enters your area of risks
• Reduce risks to acceptable levels
• Concern for others and the environment we have
  an obligation to protect the public and the
  environment

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Biohazard Symbol

• Universal Symbol
• Communicates potential exposure
• Typically red or orange
• Symbol should be defaced when hazard is no longer
  present
• Use sparingly, explicitly
• Use for cultures of pathogens
• Human blood, tissue
• Equipment used with above
• Storage areas of above
• Cages of infected animals
• Door into laboratory

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Risk Assessment

• Risk assessments on lab procedures should be done
  before initiating them. They should answer the
  following
• What are the hazards?
• What might happen?
• How likely is it to happen?
• How serious are the consequences if it happens?
• What are the possible exposures?
• How can I mitigate exposure?
• What is the WORST that can happen?
• Remember! Familiarity affects your perception of
  risk!

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Tools for Risk Assessment

• Risk Groups
• Biosafety Levels
• Guidance Documents

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Risk Groups

• Based on transmissibility, invasiveness,
  virulence and lethality of the specific pathogen
• RG1 not associated with disease
• RG2 associated with disease that is rarely
  serious or for which there is treatment
• RG3 serious or lethal human disease with
  treatments
• RG4 serious or lethal human disease with no
  treatment options

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Biosafety Levels

• Correlates to Risk Groups
• More commonly used on campus
• Describes containment practices, equipment, and
  facility design features recommended for safe
  handling of these organisms

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Biosafety Level 1

• Class 1 agents are not associated with disease in
  healthy adult humans
• However, increased precautions may be appropriate
  when using these agents for transfection since
  foreign genes can be delivered even though
  disease is not caused
• Some examples include
• E. coli nonpathogenic laboratory strains
• Saccharomyces cerevisiae
• Agrobacterium tumefaciens
• Baculovirus
• Duck hepatitis B virus

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Biosafety Level 1 Good Microbiological
Techniques

• No eating, drinking, applying makeup, etc.
• No mouth pipetting
• Safety glasses worn
• Lab coats stay in lab
• Wash you hands
• Safe handling of sharps
• Decontaminate cultures and waste
• Laboratory access limited when work is in progress

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Biosafety Level 2

• Class 2 agents are associated with human disease
  which is rarely serious and for which
  preventative or therapeutic interventions are
  often available.
• Some examples would include
• E. coli, pathogenic strains
• Adenovirus
• Herpes simplex virus
• Chicken pox
• Moloney murine retrovirus, amphotropic

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Biosafety Level 2

• BSL 1 practices in effect
• Biohazard or restricted access sign on door
• Door closed negative air pressure
• Limit/restrict access to laboratory
• Minimize aerosols
• Biosafety cabinet for aerosol control
• PPE required gloves, lab coats, respirators in
  some cases
• High degree of precaution with sharps
• Decontaminate surfaces and equipment

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Biosafety Level 3

• Class 3 agents are associated with serious or
  lethal human diseases for which preventative or
  therapeutic interventions may be available (high
  individual risk but low community risk)
• Francisella tularensis
• Human immunodeficiency virus
• Histoplasma capsulatum
• Mycobacterium tuberculosis
• Aerosol is a common route of transmission, and
  increases the risk potential for these agents

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Biosafety Level 4

• Class 4 agents are likely to cause serious or
  lethal human disease for which preventive or
  therapeutic interventions are not usually
  available (high individual risk and high
  community risk)
• Ebola virus
• Herpes B virus (Cercopithecine)
• Lassa fever virus

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Relationships
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Recombinant DNA

• As an institution receiving research funds from
  the National Institutes of Health, we are subject
  to the NIH Guidelines for Research Involving
  Recombinant DNA Molecules
• All recombinant DNA experiments (rDNA) at the
  University of Alabama must be reviewed by the
  Institutional Biological Safety Committee
• All recombinant DNA work must be approved before
  work begins
• Protocols must be reviewed periodically

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Applicable Sections of the NIH Guidelines

• III-D, IBSC review prior to initiation
• BSL2 or higher microbes as vectors
• Introduction of rDNA into BSL 2 or higher
  microbes
• Large scale rDNA production (gt10L)
• III-E, IBSC notification
• Most BSL1 research
• Many plant experiments
• Many transgenic rodent experiments

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Exempt from NIH Guidelines Section III-F

• A common misconception is that low risk research
  is exempt from the Guidelines in fact,
  exemptions are limited and specific.
• S. cerevisiae and some E. coli K-12 hosts
• Viral genome (lt1/2) in tissue culture
• Sequencing and PCR
• DNA propagated solely in the species
• Gene transfer between species known to exchange
  DNA by physiological means
• Does not present risk, as determined by the NIH
  Director

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NIH Guidelines - Compliance

• As a condition for NIH funding of rDNA research,
  institutions shall ensure that such research
  conducted at or sponsored by the institution,
  irrespective of the source of funding, shall
  comply with the NIH guidelines.
• UA receives funding from NIH, so all research
  here must comply with the NIH Guidelines.
• Noncompliance may result in suspension,
  limitation, or termination of NIH funds at the
  institution.

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Some Available References for Risk Assessment and
Lab Safety
Laboratory Biosafety Manual, 3rd edition
World Health Organizatio Geneva 2004
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Pathogen Characteristics

• We must think of the characteristics of a
  pathogen in order to determine how best to
  contain it.
• Ecotropic pathogens are less risk than
  amphotropic pathogens because amphotropic can
  infect humans.
• Some pathogens are carried by vectors (e.g.,
  mosquitoes and malaria).
• Survivability in the environment varies greatly
  among pathogens, from minutes to years.
• Virulence can also vary greatly, even among
  different strains of the same microbe.
• Some pathogens produce toxins, many do not.
• Whether a pathogen stays local in the host or
  goes systemic (spreads to other organs) greatly
  affects virulence and host survival.

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Routes of Transmission

• Fecal - oral
• Vector e.g., mosquito
• Mucosal (splash)
• Cuts, scratches, bites
• Aerosol, inhalation
• Aerosol transmission tends to be the most
  hazardous and hardest to contain. We will give
  these some additional consideration

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Some Common Activities That Generate Aerosols

• Vortexing
• Pipetting
• Sonication
• Electroporation
• Popping tube caps
• Flame sterilizing tools
• Flow cytometry
• Centrifugation
• Infected animals

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Examples of Infectious Doses

• It is the low infectious doses that make some
  diseases so dangerous to human life.
• M. Tuberculosis 10 cells
• Salmonella typhi 100,000 cells
• Listeria lt 1,000 cells
• Cryptosporidium lt10 cells
• Rotavirus 10-100 i.u.
• Vibrio cholera 1,000,000 cells

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Host Susceptibility

• Age
• Immune Competence
• Medication
• Nutritional Status
• Pregnancy
• Metabolic Disorders
• Malignancy

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Some Other Hazards

• Bloodborne Pathogens for those working with
  human blood or tissue
• Mixed Hazards for those who use chemical and
  biological materials combined
• Physical Sharps Hazard for needles and broken
  glass

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Bloodborne Pathogens

• Universal Precautions handle human blood, body
  fluids and tissues as if they harbor pathogens
  like HIV, Hep B, etc
• Use BSL 2 containment
• Protect against cuts, needle stick injuries
• Use a BSC for aerosol control
• Hepatitis B vaccine recommended
• Wear gloves, lab coat, safety glasses, etc
• Decontaminate spills with approved disinfectant

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Mixed Hazards Chemical and Biological Some
Lab Practices

• Door closed negative air pressure
• Limit/Restrict access to lab
• Use of signs/labels
• Minimize aerosol production
• Containment fume hoods for chemicals
• PPE required gloves, lab coats, safety glasses,
  respirators in some cases
• High degree of caution with sharps
• Decontaminate surfaces and equipment

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What are considered Sharps?

• Hypodermic needles and syringes, IV needles and
  tubing, blades, etc., are regulated as medical
  waste
• Glassware exposed to an infectious agent must be
  managed as a sharp until it has been autoclaved.
  
• Sharps containers must be red in color and
  display the International Biohazard Symbol, say
  Medical Waste, or be labeled as Infectious Waste
• Sharps containers should be puncture proof.
  Sharps may not be disposed of in red bags.

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Some Protective Equipment

• Respiratory Protection
• Ventilation Devices
• Fume Hoods
• Laminar Flow Hoods
• Biological Safety Cabinets

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Respiratory Protection

• Surgical or dust mask for large particles
• Chemical masks for vapors, acids
• Filtering facepiece, air purifying respirator for
  microbes
• Must be FIT TESTED by EHS for respirator use!

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Ventilation Devices

• Fume Hood
• Laminar Flow Hoods
• Biological Safety Cabinet

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Fume Hoods

• Fume hoods protect workers from chemical vapors.
• Handle hazardous chemicals in fume hoods whenever
  possible.

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Laminar Flow Hoods

• These hoods are only appropriate for very few
  purposes and they are often misused.
• Dont work with any hazardous in a laminar flow
  hood.
• They protect the working surface only, NOT the
  worker!

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Biological Safety Cabinets (BSC)

• BSCs work to protect workers and material from
  microbes.
• Avoid working with volatile chemicals in a BSC.

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Proper Use of a BSC

• Turn it on!
• Maintain constant air curtain minimize
  movements in/out, traffic
• No Bunsen Burners
• Avoid clutter, keep grille clean
• Disinfect working surface and interior
• Certify performance annually (EHS does this!)
• Avoid extended use of UV lamps use them with
  care.

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Disposal of Potentially Hazardous Waste

• Chemical Disinfection
• Autoclave

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Disinfection

• Aim for total destruction of target organism
• Autoclave sterilization
• Bleach - effective against microbial agents of
  diseases at a concentration of 0.1 percent
• Detergents
• Alcohol (70)

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Factors Affecting Bleach Efficacy

• Amount of organic matter
• Exposure to light
• Age of bleach solution
• Concentration
• We recommend that you make a fresh solution of
  bleach at least monthly and store it in a opaque
  container that has a good seal. Date it so you
  know when to toss it. It is common for bleach to
  50 or more of its efficacy when in storage for
  just one month.
• Bleach itself is a hazard it burns the skin and
  it is corrosive.
• Do not autoclave a solution that has been treated
  with bleach. It is hard on the autoclave and can
  cause chlorine gas to be released.

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Can I autoclave something then put it into
regular trash?

• The short answer is YES on some things
• So what can I autoclave and dispose?
• Medical Waste
• Class 1 Agents
• Class 2 Agents that cant be aerosolized to class
  3

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Autoclavable Waste

• ADEM regulations have specific prohibitions on
  the regular trash disposal of all items bearing
  either an international symbol or any wording
  indicating that the items contain infectious
  waste, biohazardous waste, or medical waste.
• In order to dispose of treated medical waste as
  regular trash the autoclaved bag must not be red
  or orange nor contain any wording or symbols
  indicating that it contained medical waste.
• The state prohibits using an orange/red bag for
  autoclaving, then placing it into a black trash
  bag for disposal.

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How do I handle the collection of material?

• Many times the materials are biohazardous/medical
  waste until they are autoclaved then they can
  be regular trash. If I cant trash the red bags,
  how do I handle these?
• You should acquire outer secondary containers
  (ex trash receptacle) and affix a biohazard
  symbol to the exterior surface. Use a black
  autoclave bag inside the secondary container.
  This allows the material to be clearly identified
  in the lab and still allows disposal of bagged
  material in regular trash stream.

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Autoclave Guidelines

• Equipment should continuously monitor and record
  temperature and pressure during the entire length
  of each cycle.
• If not so equipped, temp sensitive tape should be
  affixed to each bag or container.
• Equivalent tests can be approved by ADEM
• Effectiveness must be evaluated under a full load
  at least once every 40 hours of operation
• In any routine monitoring of autoclave
  performance, biological indicators or
  thermocouples should be places at the center of
  each load.
• Sterilizers used for waste treatment shall not be
  used for sterilization of equipment, food, or
  other related items.
• Each bag must be exposed to a minimum of
• 250 degrees Fahrenheit
• 15 pounds of pressure
• At least 30 minutes time AT THESE CONDITIONS

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Autoclave Recordkeeping

• A written log or other means of documentation as
  approved by ADEM shall be maintained for each
  unit and shall contain the following
• Date, time, duration, and operator of each cycle
• Approximate weight/volume of medical waste
  treated during each cycle
• Temperature and pressure maintained during each
  cycle
• Method utilized for confirmation of temperature
  and pressure
• Dates and results of calibration and maintenance
• Written log must be maintained for three years

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Biosecurity

• Purpose of biosecurity measures is to protect the
  community, prevent theft, and comply with federal
  and state regulations
• CDC guidelines for even BSL 1 labs
• Access to the laboratory is limited or
  restricted at the discretion of the laboratory
  director when experiments or work with cultures
  or specimens is in progress.
• This means that everyone entering your lab should
  have approval to be there!

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Some things you can do to secure your lab

• Know the people in your area - question
  unfamiliar people/activities
• Maintain inventory of materials and equipment
• Safeguard hazardous materials unauthorized
  persons should not be able to access your
  inventory
• Limit access - everyone entering lab has approval
  to be there
• Lock lab whenever it is unattended do not prop
  lab doors open

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Summary

• It is up to you to protect yourself, your
  co-workers, the community, and the environment!
• Good lab practices are fundamental
• Communicate about hazardous materials
• PLAN PLAN PLAN
• When in doubt, ASK!!!!

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How to Reach Us

• Environmental Health and Safety
• 15 Research Drive
• Box 870178
• Tuscaloosa, AL 35487
• 205-348-5905
• Marcy Whitney mwhitney_at_bama.ua.edu

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Last Step!

• The final step is to take a brief quiz. This
  will test your knowledge and understanding of
  this material.
• Print the quiz, complete it and the information
  sheet, and send them (fax or campus mail)
• Marcy Whitney
• Box 870178
• Fax [phone]
• We will send you a certificate for training upon
  grading. You must get a 90 for a passing grade.

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Quiz page 1

• Name 3 things you would want to know about a
  pathogen that would help you determine an
  appropriate means of containment
• Name 3 things that could make you more
  susceptible to a pathogen
• Name three routes of transmission for an
  infectious agent
• List three references to aid you in Risk
  Assessment and Lab Safety
• Name two common laboratory activities that could
  generate an aerosol

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Quiz page 2

• If you handle human blood or body fluids/tissues,
  what containment level should you use?
• 7. List the minimum conditions a bag must be
  exposed to in an autoclave in meet ADEM
  requirements for Medical Waste treatment
• The efficacy of bleach for disinfection depends
  on (Mark all that apply)
• a. Concentration d. The amount of organic
  material present
• b. The brand e. The container in which it
  was stored
• c. The age of the solution
• Which of the following can go into regular trash
  streams?
• a. Class 4 biological agents c. Class 2
  agents that can aerosolize to Class 3
• b. Select Agents regulated by the CDC d. Class
  2 agents after being autoclaved according to ADEM
  regs
• Name three things you can do to protect your lab
  area

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Information Sheet

• Name ____________________________________________
  ____________
• Date_____________________________________________
  _____________
• Department ______________________________________
  _____________
• Supervisor ______________________________________
  ______________
• Building/Room ___________________________________
  ______________
• Phone ___________________________________________
  _____________
• Campus Mailing Address __________________________
  _______________
• Email ___________________________________________
  _____________
• Certification Statement I hereby certify that I
  have completed the training for Basic Biosafety
  and that I am submitting my quiz answers for
  review.
• Signature ______________________________________
  _
• Print __________________________________________
  _
• Date ___________________________________________
  
• Fax this form and quiz to Marcy Whitney at
  348-7773 or send through campus mail to Box
  870178. You should receive your certificate
  within 2 weeks.