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General Introduction to Biosafety

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Title: General Introduction to Biosafety


1
General Introduction to Biosafety
Robert Heckert, DVM, PhD, CBSP Robert Heckert
Consulting, LLC www.safevet.com
2
COURSE OUTLINE
  • Introduction
  • Laboratory Associated Infections
  • Blood-borne Pathogens
  • Classification of Biohazards
  • Infection/Biohazard Control
  • Spill Response
  • Biomedical Waste
  • Regulations

BIOSAFETY
3
INTRODUCTION
4
WHAT IS A BIOHAZARD?
  • A potential hazard to humans, animals or the
    environment caused by a biological organism, or
    by material produced by such an organism
  • Examples
  • Viruses, bacteria, fungi, and parasites and their
    product.
  • Blood and body fluids, as well as tissues from
    humans and animals.
  • Transformed cell lines and certain types of
    nucleic acids .

5
WHAT IS BIOSAFETY?
  • Measures employed when handling biohazardous
    materials to avoid infecting oneself, others or
    the environment.
  • Achieved through
  • Administrative Controls
  • Engineering Controls
  • Personal Protective Equipment
  • Practices and Procedures

6
WHY ARE WE CONCERNED?
  • Potential for acquiring a laboratory-associated
    infection (LAI)
  • Contamination of the environment
  • Contamination of research
  • Public perception

7
LABORATORY ASSOCIATED INFECTIONS
8
LABORATORY ASSOCIATED INFECTIONS
Infection Source
  • Cultures and stocks
  • Research animals
  • Specimens
  • Items contaminated with above

Route of Transmission
Susceptible Host
  • Percutaneous inoculation
  • Inhalation of aerosols
  • Contact of mucous membranes
  • Ingestion
  • Immune system
  • Vaccination status
  • Age

9
LAIS
  • Only 20 causative or defined event
  • 80 of which are caused by human factors
  • 20 are caused by equipment failure
  • Top 4 accidents resulting in infection
  • Spillages splashes
  • Needle and syringe
  • Sharp object, broken glass
  • Bite or scratch from animals or ectoparasites

http//www.weizmann.ac.il/safety/bio2.html
10
LAIS
WHO WHAT WHERE WHEN HOW
Researcher SARS Taiwan December 2003
Microbiologist West Nile Virus United States August 2002 Laceration
Laboratory Worker Meningococcal Disease United States 2000 Aerosol?
Laboratory worker Vaccinia virus United States 2004 Many ways
11
BLOOD-BORNE PATHOGENS
12
BLOODBORNE PATHOGENS (BBP)
  • Sources
  • Blood
  • Semen
  • Vaginal Secretions
  • Other Bodily Fluids
  • Cerebrospinal
  • Amniotic
  • Synovial
  • Tissue Cultures
  • Organ Cultures
  • Infected Experimental Animals

13
RISK OF EXPOSURE
  • Pathogen involved
  • Type of body fluid
  • Route of exposure
  • Duration of exposure
  • Volume of blood involved in exposure
  • Concentration of virus at time of exposure
  • PPE worn

14
SPECIFIC EXAMPLES OF BBPS
  • Hepatitis B
  • Hepatitis C
  • HIV

15
ISSUES TO CONSIDER
  • Symptoms
  • Mode of transmission
  • Incubation period
  • Survival outside host
  • Communicability
  • Immunization
  • Prophylaxis / Treatment

16
IF AN EXPOSURE OCCURS
  • Initiate first aid
  • Notify your supervisor / designated person
  • Report to hospital emergency department or local
    Health Services
  • Report incident to regulators (if necessary)

17
UNIVERSAL PRECAUTIONS
  • Minimum standard of practice for preventing the
    transmission of BBP includes
  • Education
  • Hand washing
  • Wearing protective barriers
  • Use safe work practices

If samples cannot be guaranteed non-infective
treat as infectious!
18
BIOHAZARD CLASSIFICATION
19
BIOHAZARD CLASSIFICATION
  • Conventional Agents
  • Recombinant DNA
  • Tissue Culture
  • Animal Work
  • Anatomical Specimens
  • Unconventional Agents

20
BIOHAZARD CLASSIFICATION
  • Organisms are categorized into a group base on
    the particular characteristics of each organism,
    such as
  • Pathogenicity
  • Infectious dose
  • Mode of transmission
  • Host Range
  • Availability of effective preventive measures
  • Availability of effective treatment

21
BIOHAZARD CLASSIFICATION
  • Organisms are categorized based on the measures
    required for handling each organism safely in a
    laboratory setting, such as
  • Engineering Requirements
  • Operational Requirements
  • Technical Requirements
  • Physical Requirements

22
CONVENTIONAL AGENTS
Risk Group Individual Risk Community Risk Containment Level Examples
1 Low Low Level 1 E.coli, B. subtilis, S. aureus, Trichoderma reesei
2 Moderate Limited Level 2 Streptococcus Salmonella spp, Measles, Adenoviruses, Hepatitis A, B C, Influenza
3 High Low Level 3 Bacillus anthracis, Mycobacterium tuberculosis, HIV, Yellow fever virus
4 High High Level 4 Lassa virus, Ebola virus, Marburg virus
Unlikely to cause disease in healthy workers or
animals
Rarely cause serious human or animal disease
May cause serious disease
Likely to cause very serious disease
23
RECOMBINANT DNA
  • Genetic Engineering in vitro incorporation of
    genetic material from one cell into another
  • The level of risk depends on source of DNA,
    vector and host.
  • The biosafety community may be able to assist the
    investigator in this determination.

24
TISSUE CULTURE
  • Have the potential to contain pathogenic
    organisms
  • In general
  • Human non-human primate, and mycoplasma-containi
    ng cell lines Level 2
  • Others Level 1

A detailed risk assessment should be undertaken
when using a new cell line.
25
ANIMAL WORK
  • Animals can harbour infectious organisms
    (naturally or introduced)
  • Level dependent on type of work being conducted.
  • Special Animal Care training is required for all
    personnel working with animals.
  • All work involving animal use must receive prior
    approval from the Animal Care Committee

26
ANATOMICAL SPECIMENS
  • All specimens should be considered infectious due
    to potential presence of infectious agents
  • Important to consider the type of specimen
  • blood, organs, tissues
  • Spinal sample, brain tissue
  • From infectious patient
  • In general Level 2 but it depends on the nature
    of the work.

27
UNCONVENTIONAL PATHOGENS
  • TSE prion diseases lethal transmissible
    neurodegenerative conditions
  • Creutzfeld-Jakob disease, Variant C-J Disease,
    Mad Cow Disease, Scrapie, Chronic Wasting Disease
  • Resistant to destruction by procedures that
    normally inactivate viruses.
  • Contact regulatory authorities to assess
    requirements (containment, procedures, waste
    disposal, etc.)

28
CONTAINMENT LEVEL 2
  • Clinical, diagnostic, research and teaching
    facilities with level 2 agents.
  • Requires a class I or class II biological safety
    cabinet if any potential for aerosol or splash
    exists.
  • An emergency plan for handling spills must be
    developed.
  • Access should be controlled.

29
CONTAINMENT LEVEL 3
  • Specialized design and construction
  • primary barriers to protect the individual
  • secondary barriers to protect the environment
  • All staff must undergo special training on the
    agents being used, PPE, equipment, waste
    management as well as practices and procedures
    above and beyond the scope of this course.

30
CONTAINMENT LEVEL 4
  • Only few level 4 facilities in the world
  • Design specifications are extremely stringent,
    worker is completely isolated from infectious
    material.

31
BIOLOGICAL SAFETY CABINETS
  • Effective means of primary physical containment
    for biological agents, especially when aerosols
    are generated.
  • HEPA filters remove particles (min 0.3 microns)
    with 99.97 efficiency.
  • There are 3 main classes of cabinets (I, II,
    III) which provide various levels of protection.

32
BIOLOGICAL SAFETY CABINETS
  • Laminar flow hoods
  • Vertical or horizontal laminar flow
  • HEPA filtered air (intake only)
  • Product protection only
  • Biological Safety Cabinet
  • HEPA filtered laminar air flow
  • Exhaust
  • Personnel, environment product protection

VS
33
WORKING SAFELY IN A BSC
  • Before using the cabinet
  • Ensure BSC is certified
  • Turn off UV lamp turn on fluorescent lamp
  • Disinfect work surfaces with appropriate
    disinfectant
  • Place essential items inside cabinet
  • Allow the blower to run for 5-10 min before work

34
WORKING SAFELY IN A BSC
  • While using the cabinet
  • Ensure material and equipment is placed near the
    back of the hood, especially aerosol-generating
    equipment. Do not block any vents
  • Use techniques that reduce splatter and aerosols.
  • General work flow should be from clean to
    contaminated areas
  • Minimize movement so as not to impede air flow
  • Open flame in BSCs is controversial

35
WORKING SAFELY IN A BSC
36
WORKING SAFELY IN A BSC
  • After using the cabinet
  • Leave blower on at least 5 minutes to purge
    cabinet
  • Remove and decontaminate equipment and materials
  • Disinfect cabinet surfaces
  • Turn off blower and fluorescent lamp, turn on UV
    lamp

37
WORKING SAFELY IN A BSC
  • Maintenance
  • Before and after each use - Work surfaces wiped
    down
  • Weekly - UV lamp should be wiped clean
  • Monthly - All vertical surfaces wiped down
  • Annually - UV lamp intensity verified
  • - Decontamination with formaldehyde gas
  • - Certification

38
PERSONAL PROTECTIVE EQUIPMENT (PPE)
  • PPE can become an important line of defence (last
    line of defense)
  • Responsibility of both the user and the
    supervisor to ensure that PPE is worn

39
PPE
  • Criteria for consideration
  • Routes of exposure that need to be blocked
  • Degree of protection offered
  • Ease of use
  • Only effective if correctly selected, fitted,
    used and cared for, and the individual is trained
  • Ensure PPE is removed before leaving the lab

40
PPE
  • Footwear
  • Closed toed shoes should always be worn. Booties
    are worn in some higher containment labs and
    animal facilities.
  • Lab Coats/Gowns
  • Long-sleeved, knee length with snaps
  • Elastic cuffs
  • Back-closing gowns
  • Periodic cleaning required

41
PPE
  • Gloves
  • Latex, nitrile vinyl for work with biological
    agents.
  • Exam gloves should not be reused, change
    frequently. Utility gloves can be disinfected and
    reused if they show no sign of degradation.
  • Consider tensile characteristics, length of cuff.
  • Double gloving.
  • BSO can provide assistance finding an alternative
    for people with allergies.

42
PPE
  • Eye Face Protection
  • Goggles, safety glasses to protect the eyes
  • Full face shield to protect facial skin.
  • Respirators
  • Only personnel who have been fit-tested and
    trained should wear respirators.

43
PRACTICES AND PROCEDURES
  • General Safety Guidelines
  • Good Microbiological Practice
  • Handwashing
  • Suspicious Packages
  • Specific Procedures
  • Centrifuges
  • Needles Syringes and other sharps
  • Pipettes
  • Blenders, Grinders, Sonicators Lyophilizers
  • Inoculation Loops
  • Cryostats

44
GENERAL LABORATORY SAFETY GUIDELINES
  • Mostly common sense, but you must understand the
    hazards you face in the laboratory and be
    adequately trained to deal with them.
  • Basic must be known for all labs.

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45
GOOD MICROBIOLOGICAL PRACTICE (GMP)
  • Basic code of practice that should be applied to
    all types of work involving microorganisms.
  • Objectives
  • prevent contamination of laboratory workers and
    the environment
  • prevent contamination of the experiment/samples
  • Application of aseptic technique, minimization of
    aerosols, contamination control, personal
    protection, emergency response

46
HANDWASHING
  • One of the single effective means of preventing
    infections if done properly and frequently
  • When to wash?
  • Before starting any manipulations
  • Before leaving the lab
  • When hands are obviously soiled
  • Before and after completing any task in a BSC
  • Every time gloves are removed
  • Before contact with ones face or mouth
  • At the end of the day

47
SAFE USE OF CENTRIFUGES
  • Before use
  • Stress lines? Overfilled? Balanced?
  • Caps or stoppers properly in place?
  • Run conditions achieved?
  • Use sealable buckets (safety cups) or sealed
    rotors
  • After run
  • Centrifuge completely stopped?
  • Spills or leaks?
  • Allow aerosols to settle (30 min) or open in a
    BSC.

48
NEEDLES AND SYRINGES
  • Avoid use whenever possible
  • Use a BSC for all operations with infectious
    material
  • Fill syringes carefully
  • Shield needles when withdrawing from stoppers
  • Do not bend, shear or recap needles.
  • Dispose of all used needles/syringes in yellow
    sharps containers

49
PIPETTES
  • Mouth pipetting is prohibited.
  • Never force fluids out.
  • To avoid splashes, allow discharge to run down
    dispense the receiving container wall.
  • Never mix material by suction and expulsion.
  • Reusable pipettes should be placed horizontally
    in a disinfectant filled pan.

50
BLENDERS, GRINDERS, SONICATORS, AND LYOPHILIZERS
  • Operate in a BSC whenever possible. Allow
    aerosols to settle for 5 minutes before opening.
  • Safety Blender
  • Do not use glass blender jars
  • Decontaminate immediately after use
  • Lyophilizers
  • Use glassware designed for vacuum work, ensure
    there is no damage before using
  • All surfaces should be disinfected after use
  • Use vapour traps whenever possible

51
INOCULATION LOOPS
  • Sterilization in an open flame may create
    aerosols which may contain viable microorganisms.
  • Use a shielded electric incinerator
  • Shorter handles minimize vibrations
  • Disposable plastic loops are good alternatives

52
CRYOSTATS
  • Wear gloves during preparation of frozen sections
    and heavy gloves when accessing the cryostat.
  • Decontaminate frequently (100 or 70 Ethanol)

53
SPILL RESPONSE
54
SPILLS
  • Spill response will vary depending on
  • What was spilled?
  • How much was spilled?
  • Where was the spill?
  • What is the potential for release to the
    environment?
  • Spills should be cleaned up immediately (unless
    an aerosol was generated), to ensure proper
    decontamination.
  • Ensure appropriate PPE is worn and clean-up
    equipment is readily available.

55
SPILLS-GENERAL CLEAN-UP
  • Cover spill area with absorbent material
  • Soak the spill area with an appropriate
    disinfectant (i.e. 10 bleach)
  • Pour disinfectant from the outside of the
    absorbent material towards the inside
  • Ensure any broken glass is picked up (with
    forceps!) and placed in a sharps container
  • Leave on for 20 to 30 minutes
  • Wipe up with absorbent material
  • Waste should be disposed in appropriate biohazard
    bags and where possible autoclaved

56
SPILLS-SPECIAL CASES
  • Within a Centrifuge
  • Within a BSC
  • Open Areas (lab, during transport)
  • A spill response plan should be prepared BEFORE
    the spill occurs

57
SPILLS
  • All users of biological materials should be
    familiar with the spill clean-up procedures.
  • All spills are to be reported ASAP to the lab
    supervisor and the BSO.
  • Additional assistance is available from
  • Your departmental safety officer
  • Local community services (if available)

58
BIOMEDICAL WASTE
59
DECONTAMINATION, DISINFECTION, AND STERILIZATION
  • Decontamination Free of contamination, the
    destruction of microorganisms to a lower level
    such that it removes danger of infection to
    individuals.
  • Sterilization The complete destruction of all
    viable microorganisms.
  • Disinfection Use of agents (physical or
    chemical) to destroy harmful organisms on
    inanimate objects (not necessarily all organisms)

60
DECONTAMINATION PHYSICAL
  • Heat
  • Autoclaving (most practical and recommended)
  • Incineration (for disposal of sharps and tissues)
  • Irradiation
  • UV light (wavelength of 253 nm is germicidal)
  • Gamma (disrupts DNA and RNA)
  • Filtration
  • HEPA (biological safety cabinets, ventilation)

61
AUTOCLAVES
  • Items that CAN be autoclaved
  • Cultures and stocks of infectious material
  • Culture dishes and related devices
  • Discarded live and attenuated vaccines
  • Contaminated solid items (petrie dishes,
    eppendorf tips, pipettes, gloves, paper towels)

62
AUTOCLAVES
  • Items that CAN NOT be autoclaved
  • chemicals (flammables, oxidizers, phenols, acids,
    alkalides)
  • chemotherapeutic or radioactive waste
  • Bleach (or other chlorinated products)
  • certain kinds of plastics

63
AUTOCLAVES
  • Preparation of waste
  • Use only approved autoclave bags
  • Do not overfill autoclave bags
  • Separate material for re-use from that which will
    be disposed and dry from liquid material
  • If outside of bag is contaminated, double bag
  • All flasks containing biological material should
    be capped with aluminum foil
  • Ensure items are labeled with contact information

64
SAFE USE OF AUTOCLAVES
  • Many autoclaves are now run by dedicated staff,
    however, if you are operating an autoclave
  • Learn how to use it!
  • Ensure PPE is worn
  • Recognize acceptable material and packaging
  • Proper loading and unloading
  • All users/operators must take the autoclave
    training

65
DECONTAMINATION CHEMICAL
  • Generally for disinfection rather than
    sterilization
  • Choice depends on
  • Type of material to be disinfected
  • Organic load
  • Chemical characteristics
  • Most common are chlorine compounds and alcohols
    (broad range)

66
WHAT TO USE FOR MY AGENT?
  • Vegetative bacteria (E.coli, Staph)
  • 2 domestic bleach
  • 75 Ethanol
  • Quaternary ammonia
  • 6 formulated Hydrogen peroxide
  • Mycobacteria and fungi
  • 10 domestic bleach
  • 75 Ethanol
  • Phenolic compounds
  • 6 formulated Hydrogen peroxide
  • Spore forming bacteria (Bacillus)
  • 10 domestic bleach
  • Gluteraldehyde
  • Formaldehyde
  • 6 formulated Hydrogen peroxide
  • Viruses
  • Enveloped (HIV, Herpes)
  • 2 domestic bleach
  • 75 Ethanol
  • Quaternary ammonia
  • 6 formulated Hydrogen peroxide
  • Non enveloped (Hepatitis, Adenovirus)
  • 10 domestic bleach
  • 6 formulated Hydrogen peroxide
  • Gluteraldehyde
  • Formaldehyde

67
WASTE MANAGEMENT
  • Discarded biological material from teaching,
    clinical and research laboratories and operations
    is biomedical waste. Biomedical waste includes
    but is not limited to
  • Animal waste
  • Biological laboratory waste
  • Human anatomical waste
  • Human blood and body fluid waste
  • Sharps

68
WASTE MANAGEMENT
  • All biological waste should be decontaminated
    prior to disposal (including level 1 agents).
  • Treated waste is no longer considered
    biomedical (i.e. microbiological waste, blood
    and bodily fluid waste) and can be disposed in
    the regular waste stream.
  • Any waste that cannot be treated (i.e. sharps,
    carcasses, tissues and body parts) remains
    biomedical waste and must be incinerated off site.

69
WASTE DISPOSAL
Biomedical Waste (untreated)
70
WASTE DISPOSAL
Biomedical Waste (treated)

In compliance with Sewer use by-laws
With H2O 110
71
SPECIAL WASTE
  • EtBr
  • Toxins
  • Recombinant DNA
  • Contact local authorities

72
REGULATIONS
73
KEY REGULATED ACTIVITIES
  • May be restrictions on pathogen use
  • Waste disposal (air, land, sewer)
  • Transportation
  • Storage
  • Information

74
INVENTORY
  • What material is presently being used and/or
    stored
  • Location
  • Expiry date
  • Use log book for remaining amount
  • MSDSs
  • Mandatory

75
SHIPPING AND RECEIVING
  • Transportation of Dangerous Goods Act Class 6.2
    of (Infectious Substances)
  • Local country restrictions
  • Ensure
  • Proper classification
  • Proper packaging
  • Proper labeling
  • Proper documentation
  • Import/Export Permits

76
TRANSPORTATION OF DANGEROUS GOODS
  • Pre-approved
  • Authorized Individuals
  • Lead time (International Regulations.)
  • Appropriate Scheduling (Holidays, Weekends)
  • Transportation within the building
  • Between lab to lab
  • Colleague to Colleague
  • Between Institutions

77
TRANSPORTATION
  • Important Considerations
  • does material need to be transported at all
  • packaging requirements
  • means and route of transportation
  • regulatory requirements
  • Between lab transfers - 4 sided cart, sealed
    primary container, secondary container, low
    traffic route.
  • Off Campus transfers consult your BSO

78
THE BOTTOM LINE
  • If you are not careful and diligent with
    biological agents you risk
  • Infecting yourself, others or the environment
  • Contaminating your research
  • Being in violation of local/national/international
    regulations

79
Thank you!
  • Tina Preseau
  • Rita Toussaint-Archambault
  • University of Ottawa
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