Biosafety
Training of Interns and National Service
Noguchi Memorial Institute for Medical Research
Shirley V. Simpson
8-Aug-16
Centers for Disease Control and Prevention
Office of the Director
1
Biosafety
Presentation Order:
• Risk Assessment
• Laboratory Biosafety Levels/ Biosafety cabinets
• Basic Principles of Good laboratory Practice (GLP)
• Waste Management; disinfection and autoclaving
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2
Introduction
• Biosafety
– Preventive measures with the objective to reduce or eliminate accidental
exposure to or release of potentially hazardous agents
• Biosecurity
– Preventive measures aimed at protecting biological agents against theft by
those who intend to cause harm
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3
Why Biosafety?
Protection:
• Workers
• “Products”
• Co-workers
• Lab support personnel
• Environment
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4
What is Risk Assessment?
 Risk: the probability that harm, injury, or disease will
occur; the probability of an adverse (health) effect
 Assessment: The process of gathering and judging
evidence in order to decide whether a person has achieved
a standard or objective
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Risk Assessment
Reduce the worker’s and the
environment’s risk of a laboratory
acquired infection (LAI)


Identify control measures to reduce risk
 Risk
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is never ZERO
6
Risk Assessment
“Biosafety is an inexact science, and the interacting
system of agents and activities and the people
performing them are constantly changing.”
• Every etiologic agent is different
• Every laboratory is different
• Every person is different
Biological Safety: Principles and Practices, 4th Ed. Fleming DO, Hunt
eds., p. 81. Washington, DC. American Society for Microbiology, 2006
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7
Ways to mitigate RISK in the lab

Anticipate the issues

Remove or reduce the hazard

Increase familiarity with the hazard

Train to deal with hazard

Increase protection from the hazard

Have a back up plan (in case all of the
previous attempts fail.
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8
CHAIN OF INFECTION
Pathogen/
Infectious
organism
x
Exposure/ Incident
x
Route of
transmission
x
Infectious dose
x
Host Susceptibility
x
INFECTION
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proper training, use proper PPE
Improper practices, training or equipment allows organism to escape
from vial
resistance in environment, improper disinfection
effective disinfection, proper ventilation, PPE,
reduce aerosols
route of entry provided: scratch, blood,
ingestion, mucous membrane, or
respiratory exposure
vaccination
available treatment
Compromised immune
status
surveillance
9
Laboratory Biosafety Levels/Biosafety cabinets
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10
What is Biocontainment?
• Describes safe methods for managing
infectious agents in the laboratory
• Purpose is to reduce or eliminate
exposure of laboratory workers and
outside environment to potentially
hazardous agents
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11
Biocontainment Barriers
Primary barriers (safety equipment)
• GLP
• Biological Safety Cabinets (BSC)
• Personal Protective Equipment (PPE) Full-body,
air-supplied, positive pressure personal suit
Secondary barriers (facility design)
Design parameters to protect people surrounding the
laboratory and the community
• Separate building or isolated zone
• Special ventilation systems and
controls
• Directional airflow
• Operational practices
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Elements of Biocontainment
The proper mix of
the three elements
is assessed by the Practices and Procedures
needs and specific
hazards of the
facility
Safety Equipment
Facility Design
Determine the Appropriate Mix
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13
Practices and Procedures
are a combination of
appropriate:
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Standard Microbiological
Practices
+
Special Practices
14
Practices
and
Procedures
Standard Microbiological
Practices
• Hand washing
• Strict adherence to aseptic
techniques
• No eating or drinking in the
lab
• Use of mechanical devises
like pipettes
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Practices
and
Procedures
•
•
•
•
•
Special Practices
Waste handling
Decontamination
Immunization
Use of Safety Manual and SOPs
– Program, operations, manuals, spill response,
emergency procedures etc
Training
•
Specific practices depends upon the
Risk Assessment
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Safety
Equipment
•
•
•
•
•
•
•
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Laboratory Equipment
Biological Safety Cabinet (BSC)
Autoclaves
Centrifuges
Freezers
Refrigerators
Pippettors
Vortexes
17
Safety
Equipment
Safety Equipment
Types of Biological Safety Cabinets
Class I BSC
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Class II BSC
Class III BSC
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Safety
Equipment
Safety Equipment
Personal Protective
Equipment
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Safety
Equipme
nt
•
•
•
•
•
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Powered Air-Purifying
Respirator (PAPR)
Disposable hood
Breathing tube
Motor/blower unit
Cartridges
Rechargeable battery
pack
21
20
Facility
Design
Facility Design
• Segregation from public
access
• Eyewash station
• Sink for handwashing
• Ventilation systems
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Facility
Design
Facility Design
• Slip-resistant floors
• Bench tops should be
impervious to water and
resistant to disinfectants,
acids, alkalis, organic
solvents and moderate
heat
• Walls, ceilings, floors and
furniture should be easy to
clean
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Laboratory Biosafety Levels 1-4
(BSL)and Animal Biosafety
Levels 1-4 (ABSL)
• BSLs are guidelines for
working safely in research
and clinical laboratories.
• Increasing levels of personnel
and environmental protection
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POTENTIAL HAZARD
LOWEST
Basic Laboratories
Containment Laboratories
23
Biosafety Level 1
Agents not known to cause disease in
healthy humans and of minimal potential hazard
 e.g., Bacillus subtilis, Bordetella pertussis, Clostridium
 botulinum
• Utilize Standard Microbiological
Procedures
• No Special Practices required
• Areas are not separated from other areas
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Biosafety Level 1 Laboratory
 Hand washing sink
 Windows with fly
screens
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Biosafety Level 2
Agents associated with human disease
 Hepatitis B virus, Salmonella spp.
• Work involving agents that pose moderate
hazards to personnel and the environment
• Limited risk for the spread of infection
• Personnel are trained to handle pathogens
• Restricted access while in operation
• Aerosol or splash-producing procedures are
conducted in containment equipment
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Biosafety Level 2
Facility Design
• Work surfaces and bench
tops are easily cleaned
and decontaminated
• Slip-resistant floor
• Emergency showers and
eyewash
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Biosafety Level 2
Facility Design
Autoclave available
in the facility
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Handwashing sink
28
Biosafety Level 2
Operational
Practices
• PPE
• Lab coat
• Gloves
• Use mechanical pipette a ids
• Transport of materials in
containers
• Decontaminate spills with
appropriate disinfectant
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Biosafety Level 2
Operational
Practices
Use BSCs when
performing work
that could create
an aerosol
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Biosafety Level 3 Laboratories
Agents associated with serious or lethal
human disease
 e.g., Yellow fever virus, Avian influenza Virus,
Hantaan virus, M. tuberculosis
• High individual risk, low community risk
• Effective treatment and preventive measures
available
• Typically for airborne transmitted organisms
• Often have a low infectious dose to produce
effects
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Biosafety Level 3 Laboratory
•Physically
separate from
access corridors
• Two sets
of
entry doors
•Exhausted air is
not re-circulated
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Biosafety Level 3
In Addition to BSL-2
requirements:
• “Hands-free sink”
• Restrict the use of “sharps” and glass
• Wrap-around/solid-front gown, head
covering and possibly shoe covers
• Eye protection and possibly
respiratory protection is worn
• Decontaminate solid materials/waste
prior to removal from the lab
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Possible
“Enhancements ”
Physical Laboratory Design
• Autoclave in lab
“Barrier” design
• Pass Through Box
• HEPA filtered Exhaust
Air
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Biosafety Level 3
Physical Laboratory Design
Directional inward
airflow
Pressure monitoring
devices at entry
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Biosafety Level 4 Laboratories
Agents are dangerous/exotic with a high risk to
the individual and the community, transmitted by
aerosol or transmission unknown
 e.g., Lassa fever virus, Ebola virus, Marburg virus,
Rift Valley Fever
• All work done in a Class III BSC or a Class II
BSC and positive pressure personnel suit
• Facility is separated from other work areas
• Access is strictly control (e.g., 24-hour guard
and check in / out logbooks).
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Biosafety Level 4
In Addition to BSL-3 requirements:
• Sign-in procedures to monitor
personnel
• Clothing change before
entering
• Chemical Shower required at
exit
• Isolated from other facilities
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37
Basic Principles of
Good Laboratory Practice (GLP)
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38
OECD Definition of GLP
OECD defines principles of GLP as “quality system concerned
with the organizational process and the conditions under
which nonclinical health and environmental safety studies
are planned, performed, monitored, recorded, archived and
reported”.
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OECD - Organization for Economic Cooperation and Development
39
Purpose of Principles
The purpose of the principles of GLP are to promote the
development of quality test data to ensure that studies are
reliable and can be trusted, conclusions reported are
verifiable and data can be traced
GLP makes sure that the data submitted are a true reflection
of the results that are obtained during the study.
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Fundamentals of GLP
•
•
•
•
•
•
•
•
Test systems
Apparatus/Equipment, material and reagent facilities.
Performance of the study.
Standard operating procedures (SOP)
Archiving of records and materials
Personnel and test facility organization
Reporting of study results.
Quality assurance programs.
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Why Was GLP Created?
GLP principles were conceived as a result of
irregularities and sometimes fraud committed in
research and even industry.
• Examples of some of these poor lab practices found
were
1. Equipment not been calibrated to standard form ,
therefore giving wrong measurements.
2. Incorrect/inaccurate accounts of the actual lab
study
3. Inadequate test systems
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GLP Principles
• Limits waste of resources
– Ensures high quality of results
– Ensures comparability of results
– Promotes mutual recognition of results
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GLP Principles: 5 Basic Points
• RESOURCES: Personnel, Facilities & Equipment
• RULES: Guidelines, Procedures Protocols/Study Plans
• CHARACTERIZATION: Test Article, Identification, Quality control system
• DOCUMENTATION: Raw data, Final Report, Archives
• QUALITY ASSURANCE: Audit/Inspection – Training - Advice
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To maintain GLP Principles
• Consensus Documents Required:
– Quality Assurance
– Laboratory Supplies
– Sample Collection/Storage
– Study/Lab. Director responsibilities
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RESOURCES
• Management is responsible for providing resources fit for the
task
• Personnel
– Laboratory Director
– Quality Assurance officer
– Laboratory staff
– Archivist
– Facilities/Equipment
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GOOD ORGANIZATION
 Adequate physical facilities, qualified
staff
 Planning of work and resource
allocation
 Definition of responsibilities & training
of staff
 Good record keeping and organized
archives
 Implementation of verification
processes
 Compliance with GLP
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PERSONNEL
• Organization shown in standard documents
– Organization charts
– Job descriptions
– Curriculum vitae
– Training records
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JOB DESCRIPTIONS
 Clearly define day-to-day responsibilities and tasks
 Make it clear who reports to whom
 Describe delegation of tasks
 Must be up-to-date
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Factors to consider
• Routine work
– Test systems
– Equipment
– Staff
• Safety & comfort of staff
• Possible impact on work
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Factors to consider
• Routine operational issues
–Access
–Security
–Cleaning
–Storage
–Utilities & maintenance
–Waste disposal
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EQUIPMENT
• Suitability
– Calibration
– Maintenance
– Documentation
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EQUIPMENT : Calibration
 Need proof of standard working conditions
 Calibration usually requires use of
standards




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“Secondary – working” standards
“Primary” standards
“National/ International” standards
Fix frequency of calibration
53
EQUIPMENT: Maintenance





Preventive maintenance
Curative maintenance
Back-up equipment/procedures
External service organizations
Alarms
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STANDARD OPERATING PROCEDURES
SYSTEM CHARACTERISTICS
 Part of lab master documentation system
 Cover all activities
 Administration/personnel management
 Safety/hygiene
 Technical
 Readable, clear, precise, practical
 Fully understood and followed
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DATA COLLECTION & RAW DATA
Remember lost/inaccurate data invalidate work
• Collect data on prepared forms so that it is clearly indicated:
– “WHAT” was done
– “HOW” it was done
– “WHEN” it was done
– “WHO” collected the data
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DATA COLLECTION AND RECORDS
• Data should be recorded:
– Directly/not transcribed from a rough copy
– Promptly
– Accurately
– Legibly
• Then finally:
• Signature & date
• Explain corrections
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Conclusion -GLP
Defines conditions under which all lab activity is:
– Planned
– Performed
– Recorded
– Reported
– Archived
– Monitored
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Waste Management; disinfection, autoclaves and
global harmonization
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Common Terminology
• Decontamination – the removal or inactivation of
biological agents by physical or chemical means
• Sterilizer – process or agent, physical or chemical, that
destroys or eliminates all forms of life, especially spores
• Disinfectant - an agent, usually chemical, that
inactivates viruses or kills vegetative bacteria but not
necessarily resistant forms such as spores
• Antiseptic - a substance that prevents or arrests the
growth or action of microbes, either by inhibiting their
activity or by destroying them (living tissue use)
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What to look for in disinfectant








Low cost
Effective (broad spectrum killing)
Environmentally friendly
Not otherwise hazardous (non-flammable, toxic,
corrosive)
Stock Strength / strength of active ingredient
Space filling and highly penetrating, but does not easily escape
room (for space decontamination)
Unaffected by room and contaminant conditions (temperature,
humidity ,pH, organic material presence, metals, etc.)
Formulae for calculating the % of bleach/hypochlorite use
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Key Disinfection Parameters





Time
Concentration of disinfectant (ethanol is
an exception)
Temperature
Disinfectant’s target material (lipid vs cell
envelope wall vs DNA)
Disinfectant specific issues
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Areas/items and sample of disinfectants and antiseptics that could be used
Area Or Item
Disinfectant
Dirty wound (wound dressing)
Surgical scrub, skin
disinfection
Cleaning
blood
utensils, equipment
Skin
Antiseptic
-
Normal saline, Povidone,
Potassium permanganate
Povidone, Chlorhexidine (Hibiscrub),
Chlorhexidine + Cetrimide (Savlon)
70% Alcohol rub(ethyl and isopropyl)
-
Diguanides, 70% Alcohol rub,
Chloroxylenol (dettol)
Non-alcohol based preparations e.g. steri -7
-
Detergents/soap
Antiseptic handwash e.g. hibiscrub
Quaternary ammonium compounds
spillage, Hypochlorite (bleach) 0.5%
Working surfaces
Floors
Chemical
sterilisation
endoscopes
Hand washing
Alcohol,
Hypochlorite
(bleach) + Detergent
Phenols
of Glutaraldehydes, peracetic
Serum, antibiotics preparation, Ultrafiltration
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culture media
63
Surface







Disinfectant
Classes
Quats
Alcholols
Phenolics
Chlorine-based
Peroxides
Peracetates
UV
Testing the potency of the
disinfectant after surface rub: inuse test
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Quaternary Ammonium Compounds (Quats)










Fairly odorless, and colorless compounds
Very stable in solution, but also significant
persistence in environment
Will leave residue which can become active when wetted
Although have detergent action, are inactivated by soaps and are
less effective with high organic loads
Effective against a variety of Gram positive and
negative bacteria, less effective against fungi.
Reported to be less effective against Hepatitis A Virus (HAV)
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Alcohols
Alcohols don’t leave residues (both good and
bad)
 For ethanol and isopropanol, optimal
concentration is ~70%



Target is protein denaturation, although also strips lipid envelopes
from enveloped viruses
Contact time is the major problem for alcohols
Good kill kinetics for enveloped viruses
and vegetative bacteria, but long
contact time (>10 min) for fungi and
mycobacteria. No effect on spores

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Phenolics


Residue can become active when wetted (can be tacky when dry)
Phenols are toxic, ineffective in hard water, and have a
noticeable “aroma”





But not affected by presence of organic material and are not
corrosive
Effective against enveloped viruses, most vegetative
bacteria, variable with fungi and limited activity on nonenveloped viruses
Common in hospitals and pharmaceutical manufacturing
areas
Trade names include: Vesphene, LpH, Amphyl
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Chlorine-based

Sodium Hypochlorite (bleach) and Chlorine Dioxide solutions (e.g., Clidox)

Bleach is 5.25% hypochlorite (or 6.25% for Clorox)

 10% bleach solution is 0.525% hypochlorite (or ~5,000 ppm)

Bleach
is a non-discriminatory oxidizer of organic
materials


But is also not considered stable in tap water- 24 h, and degrades presence of light
in
Use bleach in liquid waste traps, need to refresh daily Stabilized bleach solutions (e.g.,
Dispatch) are available Corrosive to many metals (limit to 10% bleach in BSC)
Effective against most organisms- need contact time and
concentration for spores (limited effectiveness)

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Ultraviolet Light

Special case- used in BSCs
 NSF 49 does NOT recommend (and cites CDC)
 But, research going back decades suggests it can be a useful
 adjunct to cleaning

• Effective against all agents except prions
• Use for 1h, with sash shut
• Must be >40 uW/cm
• (can’t tell without measuring it)
• Bulbs need to be cleaned
Won’t kill anything in shadows
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Waste Disinfection
Steam (autoclave)
 Ozone
 Incineration
 Gamma radiation (sample
disinfection)

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Steam (autoclave)
 Heat transfer during condensation of steam is key to killing



More efficient than dry heat (steam for 15 min at 1210C equal to
60 min at 1700C dry heat for surface sterilization)
But also means, no steam penetration, only inactivating by dry heat
• So, either bag must be opened to allow steam in, or water must be added (250-500 ml/bag)
to generate steam


Sufficiently common mode of disinfection
Two forms of autoclaves: gravity displacement and vacuum pre-treatment
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Incineration




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Outdated, but effective.
Can be used to disinfect air, liquid, or solid.
Destroys all known infectious agents, including prions.
Unlikely to be approved at new sites
72
AUTOCLAVES
Autoclaves use pressurized steam to destroy
microorganisms
Temperature = 121oC (250oF);
Pressure = 15 lbs/sq inch (psi) X 15 MIN (106 KPA).
If your autoclave is equipped to operate at 132oC
(270oF), you may be able to reduce processing
time if validated by Biological Indicators.
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AUTOCLAVE HAZARDS
• Explosive breakages of
glass vessels during opening
and unloading.
• Burns arising from
careless handling of
vessels containing
boiling liquids.
• Burns resulting from
physical contact with the
structure of the
autoclave.
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Safety Procedures
• Follow manufacturers’ guidelines
• Do not open when chamber is pressurized
• Avoid standing directly in front
of the autoclave door when opening it
• Place autoclave on preventive
maintenance schedule to ensure
autoclave is working according to
specifications of the manufacturer
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Autoclave Safety Procedures
•
Divide and Conquer
•
•
Do not place sealed containers into autoclave
Use shallow metal pans for best results and
heat transfer
Preventive Maintenance Program
•
– Divide large volumes into small volumes
– Autoclaving dense materials is not recommended
• Annual inspection by manufacturer’s technician
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Record Keeping
Record all information in a
Log book








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Name
Laboratory Location
Phone Number
Date
Dwell Time
Temperature
Type and amount of material
77
Biological Indicators
• When the bioburden is unknown, the most
appropriate method to validate sterilization is
the overkill method.
•
This method involves demonstrating that 106
l
spores (Geobacillus stearothermophilus) will be killed in a
half cycle.
• Thus a full cycle would result in a 12-log
•
reduction of spores and produce a Sterility
Assurance Level (SAL) of 10-6 or a one-in-a-million
chance of a non-sterile sample.
Monitoring of biohazard waste using
biological indicators should be performed
weekly.
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Additional Issues for
Autoclave Safety
• Transport of waste
– Distance travelled, container transport used
for
security of waste
– Where will autoclave bags/pans be kept
until they can be autoclaved?
PPE for autoclave
– Gloves for protection of heat
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Hazardous Identification
The hazard identification signal is a color-coded
array of four numbers or letters in
diamond shape.
Blue : health
Red : flammability
yellow : reactivity
White : variable
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Hazard Identification
 GHS
Classification (e.g.: Flammable,Carcinogen)
 Hazard Category – degree of severity
• Range from 1 – 5
• Not all classes have categories, but most do
• Not all categories have 5 levels; some have only two.
GHS Hazard Categories
Category
Category
Category
Category
Category
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1
2
3
4
5
~
~
~
~
~
‘Severe Hazard’
‘Serious Hazard’
‘Moderate Hazard’
‘Slight Hazard’
‘Minimal Hazard’
81
Safety Data Sheets (SDS)
• Required to be provided by
manufacturers or distributors
• Good general source of information on the
material
• No standard format, quality varies widely
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SDSs include :
• Name of chemical & supplier
• Physical & chemical properties
Hazards and toxicity data
• Storage and handling
• Emergency procedures
• Disposal and transportation
•
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Hazardous Materials Inventory
• What chemicals are at the
facility?
• Where/how are bulk chemicals stored?
How are they delivered to the lab?
• Who has authority to order specific
chemicals?
•
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Chemical
Storage
and Compatibility
• Classes of chemicals
– Organic acids (acetic, formic)
– Inorganic acids (phosphoric, hydrochloric,
sulfuric, nitric)
– Organic bases (amines)
– Inorganic bases (sodium
hydroxide, potassium hydroxide)
– Flammables (FP<100F) and combustibles
(100F<FP<200F)
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Classes
of chemicals
(cont)
• Oxidizers (peroxides, nitrates,
nitrites, permanganate, perchloric acid)
Peroxide formers (ethyl ether, tetrahydofuran,
• isopropanol)
Reactive chemicals (alkali metals) Toxics and
• carcinogens (formaldehyde, benzene,
• methylene
chloride)
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Standard Laboratory Practices
Separate
–
Flammables, oxidizers, acids, bases
Separate
– Organic & inorganic families
Separate
– Families into related compatible groups
and then store alphabetically
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Chemical
Proper
Storage
Storage
Segregation of chemicals
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Standard
Laboratory
Practices
• Store odorous
chemicals in
ventilated cabinets
•
Store highly toxic
chemicals in
approved
secondary
• containers
Secure highly toxic
chemicals
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Standard Laboratory Practices
Creative Waste Handling
• Use alternate (less hazardous) chemicals
• Minimize quantities used
• Recycle chemicals
• Reduce hazard before disposal
– Distillation
– Neutralization
– Evaporation
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