Transcript Principles and Practices of Biosafety

Principles and Practices of
Biosafety
Environmental Health and Safety
San Diego State University
Abbreviations












BSC – Biosafety Cabinet
BSL – Biosafety Level
ABSL – Animal Biosafety Level
BSO – Biosafety Officer
BUA – Biological Use Authorization
CA – California
CDC – Center for Disease Control and
Prevention
DGR – Dangerous Goods Regulations
DOT – Department of Transportation
EHS – Environmental Health and
Safety
EPA – Environmental Protection
Agency
HMR – Hazardous Materials
Regulations












IACUC – Institutional Animal Care
and Use Committee
IATA – International Air Transport
Association
IBC – Institutional Biosafety
Committee
IRB – Institutional Review Board
NIH – National Institute of Health
OSHA – Occupational Health and
Safety Administration
PI – Principal Investigator
PP – Physical Plant
PPE – Personal Protective Equipment
PS – Pubic Safety
SD – San Diego
USPS – U.S. Postal Service
Introduction
The management of biological hazards through the
proper application of engineered containment and
administrative controls is referred to as biosafety
or biohazard control.
Biosafety or biohazard control is a team effort
involving the PI, research lab personnel, BSO,
IBC, IRB, IACUC, EHS, PP and PS.
Regulatory Requirements and
Guidelines
NIH Guidelines for Research Involving
Recombinant DNA Molecules (April 2002)
 CDC/NIH Biosafety in Microbiological and
Biomedical Laboratories (May 1999)
 CAL/OSHA Bloodborne Pathogen Standard
 CA Medical Waste Management Act
 DOT Transportation of Hazardous Materials

SDSU Biosafety Requirements
and Guidelines


Policies established by the Institutional Biosafety
Committee (IBC) that meet or exceed applicable
guidelines and regulations for use of RG 2 and
RG 3 biohazardous materials or agents as well as
non-exempt NIH Recombinant DNA research.
Policies established by Environmental Health and
Safety (EHS) that meet or exceed applicable
regulations and guidelines for minimizing
bloodborne pathogen exposure and disposal of
biohazardous wastes.
Biohazardous Material




Infectious microorganisms
(bacteria, viruses, fungi,
parasites, prions,
rickettsiae, etc.) affecting
humans and animals
Diagnostic (clinical)
specimens
Recombinant DNA (viral
vectors, gene therapy,
cloning)
Genetically Modified
Microorganisms
(transgenic plants and
animals)



Human and non-human
primate cells, cell culture
(primary and
immortalized), tissues,
blood (whole blood or any
components) and body
fluids
Animal or plant cells, cell
cultures, fluids, tissues or
derived wastes which
may contain pathogens
Animals known to be
reservoirs of zoonotic
disease
Institutional Biosafety Committee
and
Biological Use Authorization
Biological Use Authorization
(BUA) Application

BSL 1, ABSL 1 and NIH-Exempt

Reviewed and approved by BSO

BSL 2 or 3, ABSL 2 or 3 and NIH- Non-exempt

Reviewed and approved by IBC
Currently, no facilities at SDSU meet the
minimum criteria for BSL 4; thus this type of
work is prohibited.
Submission of New BUA








New infectious agents
New cell lines
New vector system
Enhanced replication or infectivity
Expression of toxic products
Partial genomes increased to more than two-thirds
of whole genome
New or altered procedures that pose increased risk
(e.g., aerosol or other type of exposure)
Work with non-human systems being changed into
work with human system
SDSU Biosafety Approval
Institutional Biosafety Committee (IBC)
 Protocol Review
 Biological Use Authorization (BUA) with
respect to containment level (biosafety
level)
 Training
 Inspection
Principal Investigator





Develops specific protocols to ensure the safe use
of biohazardous materials.
Submits a BUA application and obtains approval
from IBC prior to commencement of work.
Complies with specific biosafety protocols,
practices and procedures described in the
biohazard control manual.
Ensures that all laboratory staff are appropriately
trained on biosafety.
Reports any significant problems, violations of
the policies, practices and procedures, or any
significant research-related accidents and/or
laboratory acquired infection to the BSO
immediately.
Laboratory Staff
Comply with the specific biosafety
protocols, practices and procedures
described in the biohazard control manual.
 Report to the PI or the lab manager all
problems, spills or violations in procedure
immediately.

Risk Group
and
Biosafety Level
Classification of Infective Agents
by Risk Group
Pathogenicity
 Infectious Dose
 Mode of Transmission
 Host Range
 Availability of Effective Preventive
Measures and Treatment

Classification of Infective Agents
by Risk Group
Risk Group 1
Risk Group 2
Risk Group 3
Risk Group 4
Severity of
Disease
unlikely to
cause human
or animal
disease
can cause disease,
unlikely to be
serious, effective
treatment and
preventive measures
available
can cause serious
disease, does not
ordinarily spread
from one person to
another, effective
treatment and
preventive measures
usually available,
exposure route:
inhalation (often)
likely to cause serious or
lethal disease, can be
readily transmitted from
one individual to another,
effective treatment and
preventive measures are
not usually available,
transmission: direct,
indirect, inhalation
Host Range
human
(healthy adult)
and animals
human (healthy
adult) and animals
human (healthy
adult) and animals
human (healthy adult) and
animals
Individual
Risk
low
moderate (potential
hazard)
high
high
Community
Risk
low
low
low
high
Classification of Containment by
Biosafety Levels
Practices and Procedures
 Containment Equipment (Primary Barriers)
 Containment Facility (Secondary Barriers)
Note: CDC/NIH has 4 biosafety level classifications
currently in use. Each level is appropriate for:
 Operations Performed
 Routes of Transmission (ingestion, inoculation,
inhalation, mucous membrane exposure)
 Laboratory Function

Risk Group and Biosafety Level
Classifications
Not appropriate to use risk group or
biosafety level when assessing toxins.
 Can be found in the American Biological
Safety Association website:
www.absa.org/resriskgroup
 Risk Groups and Biosafety Levels are not
always the same!

Bloodborne Pathogen
Standard
Introduction




California Code of Regulations, Title 8, Sec 5193
Applies to all employees who could “reasonably
anticipate” as a result of performing their job tasks
contact with blood and other potentially infectious
material (OPIM) i.e., body fluids, unfixed tissues
or organs
Limit occupational exposure to blood and OPIM
which could result in transmission of bloodborne
pathogens i.e., Hepatitis B virus (HBV), HIV
Requires a written Exposure Control Plan
Elements of an Exposure Control
Plan








Exposure Determination
Method of Compliance
HIV, HBV and HCV Research Laboratories
Hepatitis B Vaccination and Post-Exposure
Evaluation and Follow-Up
Hazard Communication
Record Keeping
Evaluation of Exposure Incidents
Sharps Injury Log
Exposure Determination
Exposure evaluation based upon the job
description.
 Exposure evaluation based upon reasonably
anticipated contact (skin, eye, mucous
membrane, parenteral contact, etc.) with
blood or other potentially infectious
materials resulting from performing the
assigned tasks.

Method of Compliance


Universal Precaution – treating all human blood and
certain human body fluids as if infectious for bloodborne
pathogens
Engineering and Work Practice Controls






Needleless system or needles/sharps with engineered sharps injury
protection
Needles and other sharps shall not be recapped, bent or broken
Needles and other sharps shall be disposed in rigid, punctureproof, leak resistant and properly labeled sharps container
Sharps container shall be closed immediately prior to removal or
replacement to prevent spillage or protrusion of contents during
handling or transport
Specimens of blood or OPIM shall be placed in a closable,
leakproof, properly labeled red bag prior to handling, collection or
transport
Personal Protective Clothing and Equipment
Hepatitis B Vaccination and PostExposure Follow-Ups
Hepatitis B vaccination shall be provided at
no cost to employee who has a potential for
becoming exposed to blood or OPIM.
 Post-exposure follow-ups shall be provided
if an occupational exposure occurs.

Hazard Communication
1.


2.
Signs and Labels
Signs shall be posted at the entrance to the work areas
which shall bear:

Name of infectious agent

International symbol for biohazard in fluorescent
orange-red

Special requirements for entering the area

Name and telephone number of lab director or other
responsible person

Warning labels shall be affixed to containers of
infectious wastes, refrigerators and freezers
containing blood or OPIM, or other containers used
to store or transport blood or OPIM.
Labels shall have the international symbol for biohazard
in fluorescent orange-red
Training
Pathogenic Microbiology
Bacterial Laboratory Acquired
Infections
76% of exposures occurred in clinical labs;
6% in vaccine manufacturing facilities; 8%
in research labs.
 Exposure modes: 60% inhalation, other
exposure modes included ingestion
(intentional, poor technique such as mouth
pipetting, smoking and eating in the lab)
and secondary transmission.

Viral Laboratory Acquired
Infections




>70% associated with research labs; 32% of all
viral LAIs associated with animals
18% of total were Hantavirus; of these, 8% were
working with known infectious material or rodents
- others thought they were working with
uninfected rodents
16% were in clinical labs; rest were in production
or field work
Major exposure modes: inhalation, percutaneous
(especially from animals)
Rickettsial Laboratory Acquired
Infections




All rickettsial LAIs were associated with research
laboratories
95% of overt infections were by Coxiella burnetii;
remainder were Murine typhus
All Q fever cases by inhalation; all infected staff
worked with or were in close proximity to sheep
Remaining cases were by percutaneous,
inhalation, mucous membrane or unknown
exposure modes
Molecular Biology
It’s a Matter of Perspective
The investigators who submit IBC protocols
want to perform their experiments safely.
 However, their perception of the risks
involved will not necessarily be the same as
that of a biosafety professional.

Risk Assessment
The following risk assessment will identify the
biological containment system to be used:
 Properties of the donor organism
 Nature of the DNA sequences that will be
transferred
 Properties of the recipient organism
 Properties of the environment
Biological Expression System
Most routine genetic engineering experiments
can be performed safely in E. coli
K12/pUC18 at BSL 1 provided the inserted
foreign DNA sequences do not require a
higher BSL.
Donor Organism and Cloned DNA
Insertion of well-characterized DNA sequences that
are unlikely to be involved in pathogenicity may
not require additional safety measures.
In cases where these sequences are not characterized,
a situation that is typically encountered when a
library of genomic DNA of an organism is being
established, a higher BSL will be required.
Cloning of genes coding for proteins that have
potential pharmacological activity such as toxins
may therefore require higher BSL.
Viral Vectors for Gene Transfer
Although viral vectors used in gene therapy or
gene transfer are replication-defective, they
should be handled at the same BSL as the
parent viral vector from which they are
derived since the virus stocks may be
contaminated with replication-competent
viruses, which are generated by rare
spontaneous recombination events in the
complementing cell line.
Transgenic and “Knock-Out”
Animals
Animals carrying foreign genetic information
(transgenic animals) should be handled in the
containment level appropriate to the characteristics
of the products of the foreign genes. For each new
line of transgenic animal, the routes by which the
animals can be infected, the inoculum size
required for infection, and the extent of the virus
shedding by the infected animal must be
determined.
Animals with targeted deletions of specific genes
(“knock-out” animals) do not generally present
particular biological hazards.
Human and Other Primate
Cells and Tissues
Human Source Material







Blood and blood products
Vaginal secretions
Semen
Amniotic fluid
Unfixed tissues
Cerebrospinal, synovial,
pleural, pericardial and
peritoneal fluids
Cell cultures







Saliva
Urine
Tears
Sputum
Feces
Vomit
Other excretions and
secretions
Second column not covered in Bloodborne Pathogen Standard, possibly not
occupationally related.
Human Source Material



May transmit infectious agents
Imperfect knowledge of infectious status
 Incubation period (asymptomatic)
 No test for every pathogen
Most tissues and body fluids
 Bloodborne Pathogens (HBV, HCV, HIV,
HTLV-1)
 Pathogens causing Malaria, Syphilis,
Babesiosis, Brucellosis, Leptospirosis,
Arboviral infections, Relapsing fever,
Creutzfeldt-Jacob disease and viral
hemorrhagic fever
Cell Culture Risks




Contaminating pathogenic agents
 natural (often zoonotic) or inadvertent
 ~20 LAIs from primary cultures in last 30 years
 e.g., Herpes B (CHV-1), prions
Oncogenic potential
 could be oncogene or oncogenic agent
 e.g., HPV-18, MPMV genomes in HeLa cells
Unexpected (adventitious) agents
 e.g., SIV, STLV, SV5 in primate cells, HHV-8
in BCBL-1 cells
Hazardous chemicals added to culture medium
Cell Culture under Bloodborne
Pathogen Standard


ABSA requested OSHA’s interpretation in 1994:
 Do human cell cultures fall under the
Bloodborne Pathogen (BBP) Standard?
Response:
 All primary human cell cultures (explants) and
subsequent in vitro passages fall under the
BBP Standard
 To be exempted from the BBP requirements,
cell strains and lines must undergo testing and
characterization (documented) for bloodborne
pathogens (not just HBV, HCV and HIV)
Cell Culture Safety

Extend Universal/Standard Precautions to all
human and animal cell cultures
 Consider working at BSL 2 (most work
there already to protect the cell cultures)
 Handle all cultures in a biosafety cabinet
 If human origin and not demonstrated to
be free of human bloodborne pathogens,
adhere to requirements of the BBP Standard
 Wear PPE appropriate to human source material
Summary
Human Source Materials





May be regulated
Can be biohazardous
Use Universal Precautions at
all times
Visible blood means
increased risk
Don’t consider “normal”
source
Human and Nonhuman Primate Cell
Cultures




Treat human cultures as
possible biohazards
Beware of non-human
primate cells
Beware of CNS, corneal,
pituitary cells
Some cells may be OK at
BSL 1
Animals and Allergens
Risk Assessment for Work with
Research Animals



Risks associated with the research agent used in
the animal
 chemical, physical, biological
Risks associated with the species of animal used
 zoonotic agents
Risks associated with animal maintenance
 ergonomic factors, bites, scratches, allergens
Risks Associated with the Agent
Used


Chemical agents
 carcinogens, mutagens
 toxic chemicals
 anesthetics
Physical agents
 radiation
 heat
 sound
Risks Associated with the Agent
Used

Potentially biohazardous agents
 deliberate use of an infectious agent in
animals for research purposes
 maintenance of infected animal for
duration of experiment
 sacrifice, necropsy and harvesting of
agent or infected tissue
Transmission of Biohazards
During Work with Animals
Airborne
 Release of infectious aerosols by animal by
sneezing, coughing
 Release during nasal infection or aerosol
challenge
 Aerosolization from bedding and excreta
 During surgical procedures
 During birthing
Transmission of Biohazards
During Work with Animals
Direct Inoculation
 Needlesticks during injection/inoculation
process
 Bites and scratches from infected animal
Transmission of Biohazards
During Work with Animals
Direct exposure of mucous membranes
(by splash or splatter)
 During surgical procedures
 During injection
 During necropsy
Transmission of Biohazards
During Work with Animals
Indirect transmission and ingestion
 From contaminated hands or gloves to mouth
 Facial contamination directly from animal
 Transfer of parasites by animal handling
Indirect transmission with eye or mucous
membrane exposure
 Dust from bedding
 Splash during cage washing
 “Dirty” environment
Risk Reduction: Containment of
Infectious Agent

Containment must include:
 Primary containment





Enclosed filtered caging system
Biosafety cabinets
Safety equipment
PPE
Secondary containment

The containment facility
• Negative pressurization
• Nonrecirculated air supply
• Ventilation must consider wellbeing of animal
Containment Caging Systems
No Containment
 Open (standard) cage
 Some Containment
 Filter top cage
(microisolator cage)
 Full Containment
 Fully enclosed in
ventilated rack

Containment Caging Systems

Microisolator Cage
works like a Petri dish
 open gaps around lid edge allow limited air
exchange
 may lead to more labor intensive husbandry due
to moisture and ammonia buildup

Containment Caging Systems
Individual cages sealed into rack with
supplied air under negative pressure
 Both supply and exhaust usually HEPA
filtered
 Ventilation must control humidity and
buildup of ammonia

Containment Caging Systems
Can install cages in class III biosafety
cabinet
 Cages are completely contained with glove
port access
 Very motion-limiting
 Transfer in and out may be an issue

Containment Caging Systems
BioBubble (Ft. Collins, CO) makes softwall ventilated enclosures
 Can be containment or barrier style
 Large equipment can be surface-mounted in
wall

Special Animal Housing Situations


Barrier colonies
 Special breeds - often immunocompromised,
“fragile”, expensive (SCID-Hu, nude athymics)
 Transgenics - often even more fragile and
expensive (knockouts, microinjected, combos)
 Specific pathogen-free (SPF) - bred and raised
to be missing certain specific microorganisms
Isolation colonies
 Extensive SPFs and defined flora animals
 Gnotobiotes (an entirely different animal!)
Zoonoses

Zoonotic disease: A disease of animals that
can be transmitted under natural conditions
and cause disease in humans
 Wild caught animals most hazardous
 Random source animals (e.g., from a
pound) are also a risk
 Purpose bred animals pose least risk
Some Animals and Their
Zoonoses

Animal
Macaque monkeys
Sheep
White mouse
Dogs, cats, skunks,
raccoons, bats
Cattle, NHP
Cats
Parrots, macaws
Chickens

Disease
Herpes B virus
Q fever
Hantavirus
Rabies
Tuberculosis
Toxoplasmosis
Psittacosis
Avian influenza
Rodent Zoonoses




Rat bite fever (Streptobacillus moniliformis,
Spirillum minus)
 transmission: direct contact (bites)
Lymphocytic choriomeningitis (LCM, a virus)
 transmission: inhalation
Leptospirosis (Leptospira spp.)
 transmission: inhalation
Others include ringworm (fungal), scabies (mites,
an ectoparasite)
Transmission of Zoonoses



Enteric route (fecal/oral)
 Salmonella, Shigella, Campylobacter,
Giardia, Toxoplasma, Cryptosporidium,
Entamoeba, Hepatitis A
Respiratory route
 Q fever, Chlamydia, Measles
Skin contact
 Ringworm (Tinea), Measles, Monkeypox
Control of Zoonoses



Get information on
species and agent
Quarantine animals prior
to use
Use Engineering controls







written SOPs and
manuals
Use PPE

facility construction and
secondary barriers
Consider the need for
containment caging
Use Administrative
controls
additional protection for
worker
Practice good facility and
personal hygiene
Provide staff training
Laboratory Acquired Allergies
(LAA)




Significant occupational disease
Affects >30% of all personnel working with
animals
No minimum safe exposure levels to allergens
have been established
Animal allergens found in hair, dander, urine,
saliva, serum
 fel-d-l cat allergen (in saliva and thus on skin)
is one of the strongest allergens known for
humans
Sources of Exposure to LAA




Hair and dander shed
from animal
Urine and feces dried
in bedding
Particulates shed from
bedding material
Animal saliva
Routes of Exposure to LAA
Inhalation of airborne allergens
 during cage changing
 during animal handling
 Skin or eye contact
 usually indirect by touching skin, eyes
 Percutaneous exposure
 animal bites (saliva)

Risk Factors for Development of
LAA



Exposure to allergens
 duration
 frequency
 intensity
Previous allergic conditions
Other predisposing conditions
 illness
 Immunocompromised
 pets
LAA: Exposure Control




Engineering Controls
 enclosure
 dilution ventilation
Administrative Controls
 reduce time with animals
 reduce density of animals
 housekeeping practices
Personal Protective Equipment
 respirators and clothing
Medical Surveillance
Disinfection
Why Disinfect?


To reduce or eliminate exposure risk
 Biohazard waste disposal
 Spill cleanup
 Routine surface decontamination
To eliminate contamination risk
 Preparation of microbiological media and
supplies
 Preparation of work area for cleanliness-critical
tasks
Resistance to Disinfectants









Prions
Bacterial spores
Coccidia
(Cryptosporidium)
Mycobacterium
Nonlipid viruses (Hep A,
Polio)
Fungi
Rickettsiae, Chlamydiae
Vegetative bacteria
Lipid-containing viruses
Classes of Disinfectants
Chlorine
 Iodine
 Alcohol
 Phenolics
 Quaternary Ammonium
 Glutaraldehyde
 Hydrogen peroxide

Factors Influencing Efficacy




Surface/Topography – uneven, cracked or pitted
surfaces especially wooden surfaces can hide
microorganisms and are difficult to disinfect
Temperature - elevated temperatures may
enhance germicidal action but also evaporation
rate
Relative Humidity – many disinfectants have
optimal relative humidity range for maximum
effectiveness
Water Hardness – some disinfectants may be less
effective when diluted in hard water

Organic Load
 Blood, sputum, milk, bedding, feed,
manure
 Proteins physically protect and stabilize
many microorganisms
 Adverse effect on action of many
disinfectants

Concentration
 In most cases, the higher the
concentration, the more rapid the kill
 Consider potential damage to surfaces or
tissues
 Reducing concentration to avoid
damage will require additional contact
time
 Ultimately, disinfectant will no longer
be active enough to be useful

Contact Time
 Disinfectants should be effective with a
short contact time
 Manufacturer’s recommended contact
time may be unrealistic under in-use
condition
 Contact time may depend on the method
of application
 For surface applications, loss by
evaporation may require frequent
applications to achieve contact time
Some Other Factors








Dirt, grease and oils – all can protect the organism
and will repel water based disinfectants
Types of microbes present – spores, vegetative
cells, viruses
Dried spills (from media, buffers) can protect
microorganisms from contact with the disinfectant
pH
Age of the product/solution
Method of application (spray vs. wipe)
Rate of application
Storage condition
Medical Waste Disposal
(Biohazardous and Sharps Wastes)
Who Regulates Medical Waste?
Federal
 EPA (40 CFR part 60.51c)
 DOT (49 CFR Part 173.134)
 OSHA (29 CFR Part 1920.1030b)
 USPS (39 CFR 111.1)
 State CA Health and Safety Code
 Local SD Code of Regulatory Ordinance

Medical Waste
…biohazardous waste and/or sharps waste
that is produced or generated as a result of
diagnosis, treatment or immunization of
human beings or animals; research
pertaining thereto; production or testing of
biologicals or removal of regulated waste
from a trauma scene.
Forms of Medical Waste



Solid
 Labware (flasks, tubes, plates, bottle, vials)
 Pipettes (could also be sharps)
 Lab waste (stocks, specimens, cultures, swabs,
vaccines)
 Gloves, apparel, wipes
Liquid
 Aspirates, culture fluids, rinses, washes
 Sera, body fluids
Sharps
 Anything with a point or edge capable of
piercing or cutting
Medical Waste Does Not Include:





Waste generated in food processing
Urine, feces, saliva, sputum, nasal secretions,
sweat, tears, or vomitus, unless it contains fluid
blood
Medical solid wastes i.e., paper towels or empty
specimen containers that are not biohazardous,
bandages/dressings containing dried blood
Hazardous waste, radioactive waste, household
waste
Waste generated on agricultural or livestock
practices on a farm or ranch
Biohazardous Waste



Laboratory Wastes
 Human or animal
specimen cultures
 Cultures and stocks of
infectious agents
 Wastes from production of
biologicals, live and
attenuated vaccines,
culture dishes and devices
Pharmaceuticals
Human surgery specimens or
tissues, including those fixed
in fixatives



Animal parts, tissues,
fluids, or carcasses
Wastes containing
recognizable fluid blood
or blood products,
containers with fluid
blood, blood from animals
Wastes contaminated or
containing
chemotherapeutic agents
Sharps Waste
… means any device having acute rigid corners,
edges, or protuberances capable of cutting or
piercing.


Needles, needles with syringes, contaminated
syringes, blades, needles with attached tubing
Broken glass items i.e., Pasteur pipettes, blood
vials contaminated with biohazardous waste
Mixed Waste
… means mixtures of medical and nonmedical
waste. Mixed waste is medical waste, except for
all of the following:
“Mixed Waste Hierarchy”
Biohazard Bag
…means a disposable red bag that is
impervious to moisture and has a strength
sufficient to preclude ripping, tearing, or
bursting under normal conditions of usage
and handling of the waste-filled bag.
Containment and Storage
Biohazardous Waste


Must be segregated from other types of wastes
Must be contained in “biohazard bags”
 Bags must be red.
 Bags must be labeled either with the word
“Biohazardous” or with the biohazard symbol
and the word “Biohazard”.
 Bags must also be labeled with the generator’s
name, address and phone number.
 Bags must be securely tied to prevent leakage
or expulsion of contents.
 Bags must be placed in a rigid container for
storage, handling and transport.



Containers
 Containers shall be leak resistant, have tight-fitting
covers, kept clean and in good repair.
 Containers may be of any color and shall be labeled
with the word “Biohazardous” or with the biohazard
symbol and the word “Biohazard” on the lid and on the
sides so as to be visible on any lateral direction.
Reusable containers shall be washed and decontaminated
unless protected from contamination by disposable liners
or bags.
Reusable containers shall be washed to remove visible soil
and decontaminated by:
 Exposure to hot water (180°F) for 15 secs.
 Exposure to the following sanitizer for 3 mins.
 Hypochlorite soln. (500 ppm avail. Chlorine)
 Phenolic soln. (500 ppm active agent)
 Iodoform soln. (100 ppm avail. Iodine)
 Quaternary ammonium soln. (400 ppm active agent)
Sharps Container
…means a rigid puncture resistant container
that, when sealed, is leak resistant and
cannot be reopened without great difficulty.
Containment and Storage
Sharps Waste
 Must be segregated from other types of wastes.
 Must be contained in “sharps container”.
 Tightly close or tape closed the lid of a full sharps
container ready for disposal.
 Store sharps container ready for disposal for not
more than 7 days.
 Label sharps container with the word “Sharps
Waste” or the biohazard symbol and the word
“Biohazard”.
 Must also be labeled with the generator’s name,
address and phone number that is legible and
easily visible on the outside of the container.
Disposal



Take biohazard bag and sharps container to
designated Accumulation Sites:
 Life Science, Room 14
 Student Health Service, outside shed
 Generators at other locations may call EHS at
(619) 594-6778 for biohazard waste pick-ups
A biowaste vendor will collect biohazard bag and
sharps container for disposal on a weekly basis
from Life Science and Student Health Services.
Biohazardous and sharps wastes will be
autoclaved while animal carcasses will be
incinerated.
Solid Medical Waste Collection
Must be rigid, puncture-proof, leak-proof
Not acceptable in CA
Labels have to be affixed on all 4 sides of the container.
Sharps Waste Collection
Sharps containers <7 gal. should not be on the floor. Lids have to be difficult to
open. Labels have to be affixed on all 4 sides of the container.
What’s Wrong with these
Pictures?
Left: Sharps sticking out of Sharps Waste container.
Right: Sharps Waste container past full line. No generator label.
What’s Wrong with these
Pictures?
Left: Bottle not labeled.
Right: Cardboard box is not allowed for liquid waste. No labels. No lid.
What’s Wrong with these
Pictures?
Left and Right: Cardboard box is not an appropriate Sharps Waste container.
No labels. No lids.
What’s Wrong with these
Pictures?
Left: Red bag should be inside the secondary container. Cardboard box is not an acceptable
secondary container.
Right: Bag must be red. Secondary container does not have to be red. No biohazard label.
Red bag on floor ready for disposal must be transported to the accumulation site immediately.
What’s Wrong with these
Pictures?
Left: Do not fill red bags completely. Replace more often.
Right: No biohazard label. Red bag on floor ready for disposal must be
transported to the accumulation site immediately.
What’s Wrong with these
Pictures?
Left: Do not deface container. Incorrect label placed on container (need generator label).
Right: Red bag must be transported in a secure secondary container to the accumulation
site. Red bag must have biohazard label and generator label.
What’s Wrong with these
Pictures?
Left: Proper Sharps Waste container not used. No generator label.
Right: Generator label should be on the outside of the red bag. Secondary container needs
biohazard label on all visible sides including top. Use appropriately sized red bag for
secondary container.
What’s Wrong with these
Pictures?
Left: Incorrect label placed on container (need generator label). Keep lid closed when not in
use.
Right: No lid. Use appropriately sized red bag for secondary container. Secondary container
needs biohazard label on all visible sides including top.
Containment Equipment and
Facilities
Biocontainment
The principle of holding or being capable of
holding or including within a fixed limit or
area
 Preventing the unintentional release of
biological agents through a combination of
laboratory practices, containment
equipment (primary barrier) and laboratory
facility design (secondary barrier)

Primary Barrier
Primary barriers contain the agent at the
source
 Equipment/Engineering Control
 Biological safety cabinet, fumehood,
glove box, animal housing, centrifuge,
fermenter

Secondary Barrier
Secondary barrier is the structure
surrounding the primary barrier
 Facility/Engineering Control
 Rooms, building
 Types of Facilities
 Basic laboratory
 Containment laboratory

Primary Barriers - Equipment



Personnel Protection
 Any aerosol generated within the cabinet is
contained and kept away from the researcher
Product Protection
 Air within the work space of the cabinet has
been filtered so that is is virtually free of
airborne particles and organisms; thus
protecting the work from outside contamination
Environmental Protection
 Aerosols generated within the unit are removed
from the air before the air is discharged
Ventilation Equipment
Classes and Types
Chemical Fume Hood






100 fpm face velocity
Offer only personnel protection
Always exhaust air to the outside
Do not offer protection to the product or the environment,
as there is no filtration of intake and exhaust air
(Sometimes air cleaning treatment is added to the exhaust.)
Do draw contaminants in the laboratory air directly over
the product being worked on
Used for work with chemical hazards
Any Comments?
Fumehood - keep hood clean, sash should be closed when hood is not in
use, equipment should be 9” from sash
Clean Bench / Laminar Flow Hoods





Provide product protection only
Product protection is provided by creating a unidirectional
airflow generated through a HEPA filter
Discharge air goes directly into workroom
Applications
– Any application where the product is not hazardous but
must be kept contaminant free
– Preparation of non-hazardous intravenous mixtures and
media
– Particulate free assembly of sterile equipment and
electronic devices
Eliminate Clean Bench in containment laboratory
Biological Safety Cabinets






Designed to contain biological hazards
Inward airflow for personnel protection
HEPA filtered exhaust air for environmental protection
Supply air HEPA filter for product protection (except Class
I)
Separated into Classes and Types
– Class I
– Class II
• Type A1, A2
• Type B1, B2
– Class III
Microbiological studies, cell cultures, pharmaceutical
research and procedures…
Class I Cabinet





75 fpm face velocity
Provides personnel and environmental protection
No product protection
Requires an exhaust blower to pull the air through
- usually to the outdoors
Applications
– Housing centrifuges, fermenters
– Cage dumping in an animal lab
– Aerating cultures
Class II Cabinets





Ventilated cabinet
Provides personnel, product, and environmental
protection
Open front with inward airflow for personnel
protection
Downward HEPA filtered laminar airflow for
product protection
HEPA filtered exhaust air for environmental
protection
Any Comments?
BSC - remove unnecessary objects, keep grill at front of cabinet unobstructed
Primary Barriers
Personnel
Chemical Fumehood
Product
Environment
x
Laminar Flowhood
x
Class I Biosafety Cabinet
x
x
Class II Biosafety Cabinet
x
x
x
Class III Biosafety Cabinet
x
x
x
Isolators
x
x
x
Types of Biosafety Cabinets
NSF/ANSI Standard 49 – 2002
Type
Face velocity
(lfpm)
Airflow Pattern
Radionuclides/
Toxic Chemicals
Biosafety
Level(s)
Class I
75
In at front; rear and top through
HEPA filter
No
2, 3
No
Class II
Type A1
75
70% recirculated through HEPA;
exhaust through HEPA
No
2, 3
Yes
Class II
Type A2
100
30% recirculated through HEPA;
exhaust via HEPA and hard ducted
Yes (Low
levels/volatility)
2, 3
Yes
Class II
Type B1
100
No recirculation; total exhaust via
HEPA and hard ducted
Yes
2, 3
Yes
Class II
Type B2
100
Yes
2, 3
Yes
Class III
NA
Yes
3, 4
Yes
Same as B1, but plena under
negative pressure to room and
exhaust air is ducted
Supply air inlets and exhaust
through 2 HEPA filters
Product
Protection
Biological Safety Cabinet
Certification





First Certification
Annually
When moved
When filter is changed
When repaired or modified
Note: Certification is paid by the researcher, not EHS
Other Primary BarriersEngineering Control







Gasketed blenders, homogenizers
Cotton plugs, filters for flasks in shakers
Filtered pipette tips
HEPA and hydrophobic vacuum line filters
Plasticware substituted for glassware
Gas burners with shield, microincinerator
Centrifuges
 Interlock, solid cover, safety buckets, O-rings
Secondary Barrier- Facilities
Laboratory Biosafety Level 2






Lockable doors (a must for restricted agents)
Sink
Bench tops impervious and easily cleaned
Biological safety cabinet (if applicable)
Eyewash
Inward airflow (desirable)
Biosafety Practices and
Procedures
Hierarchy of Controls
Administrative Control
 Engineering Control
 Work Practices
 Personal Protective Clothing or Equipment

Administrative Controls





Substitution
Authorization/Approval
Written biosafety procedures required for the
experimental procedures and equipment including
inventory of biological agents or materials
Laboratory personnel biosafety training
Medical Surveillance (BSL 2 and above)
 Health history
 Medical screening
 Immunization
 Serum storage
 Post-exposure prophylaxis
Engineering Controls
Biological safety cabinets, glove boxes
 Animal containment caging systems
 Safety equipment (filtered or sealed
equipment)
 Ventilation system
 Containment facilities

Personal Protective Clothing and
Equipment
Provides barrier against skin, mucous
membrane or respiratory exposure to
infectious agents during procedures
 Prevent spread of contamination
 Does not eliminate the hazard
 Integrity wanes with use (i.e., change gloves
frequently)

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
BSL 1: Work Practices and
Procedures

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
BSL 2: Work Practices and
Procedures







Increasing emphasis on safety
procedures and practices
Increasing need for staff training
Increasing need for competent
supervision
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
BSL 2: Work Practices and
Procedures


Aerosol generating procedures
performed in a biosafety cabinet:
 Homogenizing
 Vortexing
 Vigorous mixing
 Pipetting infectious liquids
 Sonication
 Pouring
If breach occurs:
 Evacuate lab, post spill sign
 With appropriate PPE and
disinfectant, decontaminate
centrifuge, buckets, other
items or areas
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.
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.
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.
Correct Use of Biosafety Cabinets



Moving/Installation
BSCs 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.
Decontamination
Decontamination is usually performed by certified
professionals.
Certification
All BSCs that are used for handling biohazardous materials
must be recertified annually. SDSU has contracted with a
specific contractor to provide a consistent level of
certification and maintenance service. Contact EHS at (619)
594-6778 to obtain contractor information.
Correct Use of Biosafety Cabinets
– Open Flames

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)
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
Load/unload centrifuge tubes, rotors and accessories in BSC
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
Safe Use of Blenders




Avoid use of glass blender jars, unless covered
with polypropylene jar
Place disinfectant-moistened towel over the top of
the blender during use
Before opening the blender jar, allow the unit to
rest at least 1 minute for aerosols to settle and then
open in a BSC
Decontaminate promptly after use
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
Careful Pipetting Techniques






Never blow out last drop in
pipette
Use pipetting aids with filters
Never mix by suction and
expulsion (mix by sonication)
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
doesn’t slide forward or
backward blocking air grill)
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
Vacuum System Protection

In-line filter and disinfectant in collection and
overflow flasks
Signs and Labels
Biohazard Signs



Completely filled out biohazard sign must be
displayed at entrances to laboratories where
biohazards are present and tissue culture rooms
Features fluorescent orange-red sign with lettering
“biohazard” and the international biohazard symbol
in contrasting color, name of biohazardous agent or
material, special entry requirement and PI and BSO
contact number
Available free of charge from EHS,
call (619) 594-2865
Biohazard Label



Must be attached to containers of biohazards or
biohazardous waste containers.
Features fluorescent orange-red label with
lettering “biohazard” and the international
biohazard symbol in contrasting color.
Red bags and sharps containers must also have the
generator address label affixed prior to use:
San Diego State University
5500 Campanile Drive
San Diego, CA 92182
(619) 594-6778
Laboratory Moves
It is important that the laboratory is safe:
 For custodians to clean
 For contractors to work in
 For the next group of laboratory personnel
to occupy
Disposal and Decontamination of
Biohazardous Waste
In preparation for moving, observe the following
guidelines:
 Label ALL biohazardous waste red bags and
sharps container
 Dispose of all biohazardous waste red bags and
sharps containers at approved accumulation sites
 EHS will not pick-up biohazardous waste except
in unusual situations
 Chemically disinfect biohazardous waste
 Autoclave liquid biohazardous waste and dispose
of down the drain
Moving the Biosafety Cabinet
Disinfect all BSC work surfaces prior to
moving the BSC to a new facility. BSCs
used for work with pathogenic organisms
may require paraformaldehyde
decontamination before being moved.
 Each BSC must be recertified for correct
airflow and filter integrity after it has been
moved and placed in its final location.

Moving or Disposing of
Refrigeration Units
Clear all materials stored inside the
refrigeration units.
 Disinfect all refrigeration units prior to
removal or disposal.
 Obtain clearance notification from EHS
prior to removal or disposal of refrigeration
units.

Shipment and Transportation
To Whom are the Shipping and
Transportation Regulations Applicable?
Under IATA, DGR apply to anyone who handles,
offers for transport, transports dangerous goods or
causes dangerous goods to be transported.
Under DOT, HMR apply to each person who
performs, or causes to be performed, functions
related to the transportation of hazardous materials
such as determination of, and compliance with,
basic conditions for offering; filling packages;
marking and labeling packages; preparing
shipping papers; handling, loading, securing and
segregating packages within a transport vehicle,
freight or cargo hold; and transporting hazardous
materials.
Emergency Response to
Biological Incidents
Response to Biological Spills in
the Laboratory
(Intentional or Accidental)
Exposure Management
For splash to eyes, mucous membranes, or
broken area of the skin
 Irrigate eyes with clean water, saline or
sterile irrigants
 Flush splashes to mouth, nose, and broken
area of skin with water
Exposure Management
For needlesticks or cuts with human blood, fluids,
infectious agents or antibiotic resistant organism
 Flush needlesticks and cuts with soap and water
 Get medical evaluation ASAP
 Inform PI, BSO and health professional (required
mandatory reporting of incident)
 Public Health Service has recommendations for
post-exposure follow-up
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
 One or two people can clean-up the spill
thoroughly in less than an hour
Note: Spill incident still needs to be reported to
BSO/EHS. If spill is in gallons or liters, call
BSO/EHS.
Biological Spill Clean-Up KitBasic
Nitrile gloves (double gloving), splash
goggles, shoe covers
 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

Spill Clean-Up for BSL 1-2
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.

Spill Clean-Up for BSL 1-2
If the spill is in the laboratory but outside the biosafety cabinet





Call the 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 germicid
action of disinfectant. Wipe up spill, working from the edges to the cente
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.
Spill Clean-Up for BSL 1-2
If the spill is outside the laboratory, in transit:
 To prevent a spill, transport labeled biohazardous
material in an unbreakable, well-sealed primary
container placed inside of a second unbreakable,
lidded container (cooler, plastic pan or pail).
 Should a spill occur in a public area, do not attempt
to clean it up without appropriate PPE.
 Secure the area, keeping all people clear of the
spill.
 Call the BSO to assist in the clean-up.
 Stand by during spill response and cleanup activity
to provide information and assistance.
Biosecurity
Biosecurity vs. Biosafety
Biosecurity refers to ensuring the security
of biological materials to prevent theft,
illicit use or release.
 Biosafety focuses on reducing exposure to
and release of biological materials.
 Integrating biosecurity and biosafety
programs is important for work with select
agents.

Inspection
Inspection Elements
Laboratory Identification
 Containment Facility/Equipment
 Work Practices
 Hazard Communication
 Biohazardous Waste Handling
 Laboratory Personnel
