General Biosafety Training Module

Biosafety Compliance Program

Office of the Vice Chancellor for Research and Graduate Education
 Eleanor Low
Research Compliance Officer
lowelean@hawaii.edu
956-8420 285-6367 (emergencies)
Stephen Case
Biosafety Specialist
stevec@hawaii.edu
956-8009

BIOSAFETY COMPLIANCE PROGRAM

uhpermit@hawaii.edu
http://manoa.hawaii.edu/ovcrge/research/biosafety_program/index.html
Learning Objectives:
• Describe the differences between the FOUR Biosafety
Laboratory Levels
• Explain what types of risks are involved when working
in a lab
• Understand how to minimize the potential risks when
working in a lab through RISK ASSESSMENT!
• Describe incident response procedures to a biological
spill
• Describe emergency response procedures
Acronyms and Abbreviations commonly used in Biosafety
• BSL - Biosafety Level, criteria defined in Biosafety in Microbiological and
Biomedical Laboratories, 5th edition. (BMBL)
• BSC - Biosafety Cabinet
• LAI - Laboratory Acquired Infection
• N95 - Filter respirator mask used to protect an individual from airborne
particles. Filters at least 95% of airborne particles. Not resistant to oil. Not
all N95’s are created equal.
• PPE - Personal Protective Equipment
Standard Terminology
• Biohazard - A biological substance which has or may have the ability to
pose a threat to the health of a living organism (human, animal or the
environment). Examples include microorganisms, toxins or allergens
• Decontamination - The process of removing, neutralizing or destroying a
biohazardous agent.
• Risk Group Classification - Risk group classifications are used in the
research lab as part of a biosafety Risk Assessment to determine the
proper containment, PPE used…
o http://www.absa.org/riskgroups/index.html
 Known as the "BMBL" . The standard established
guideline for best safe biosafety practices

Established to promote safe science practices

for research involving the use of or creation of
recombinant material.

Institutional Biosafety Committee’s comply with the NIH

Guidelines and use the BMBL as the reference standard
 2 3
Biological Safety
Levels?
BMBL 5th Ed.

1 4
• Biosafety Levels were developed to provide increasing
levels of protection to Personnel, Materials and the
Environment based on an agents risk level.
A combination of procedures, engineering controls, work practices,
and policies are utilized to develop these different biosafety levels.

Low Risk BSL-1

BSL-2
BSL-3
High Risk BSL-4
 Basic Biological RISK Groups
Risk Group 1 Risk Group 2 Risk Group 3 Risk Group 4
Agents are not Agents are Agents are Agents are likely
associated with associated with associated with to cause serious
disease in healthy human disease serious or lethal or lethal human
adult humans. which is rarely human disease disease for which
serious and for for which preventative or
which preventative or therapeutic
preventative or therapeutic interventions are
therapeutic interventions may usually not
interventions are be available. available.
often available.
 Basic Biosafety Levels
Risk Facilities,
Group BSL Level Examples Practices
Equipment
Basic Basic Teaching, Good None required;
Research Microbiological open benchwork,
1 BSL 1 Techniques (GMT) directional
airflow
Basic Primary health Level 1 plus Open bench plus
services, diagnostic, protective Biological Safety
2 BSL 2 teaching, public clothing, biohazard Cabinet (BSC) for
health signage aerosols
Containment Special diagnostic, Level 2 plus BSC and/or other
research special clothing, primary
3 BSL 3 controlled access containment
devices for all
activities
Maximum Dangerous pathogen Level 3 plus Class III BSC or
unit airlock entry, positive pressure
Containment
4 shower exit, suits, double
special waste door autoclave,
BSL 4 disposal filtered air
• No eating, drinking, chewing gum, smoking, handling
contact lenses or applying cosmetics
• No mouth pipetting.
• No open toe shoes in the lab
− No sandals
− Sandals with socks are still not acceptable!!
• Perform procedures carefully to minimize generation
of aerosols
• Sharps Handling Policy Do Not recap, re-use, bend
or break Needles Properly contain and dispose
• Limited/Restricted access when work is in
progress
• Decontaminate work surfaces before and after use
with an effective disinfectant.
• Decontaminate waste from all Infectious and potentially
infectious materials.
• Biohazard signage posted when infectious agents are
present or in use
• Training. PI/lab supervisor must ensure laboratory
personnel demonstrate proficiency (written
documentation) prior to working in lab;
− Standard and special microbiological
− Lab specific training
• Always remove gloves before touching clean surfaces;
phone, computers etc..
• Always wash hands with warm water and soap for 10-15
seconds after:
− Handling any viable material
− Removing gloves
− Before leaving lab
• Maintain a Pest Management program
MSU Lab Wars

Again remember one of the most effective means of

preventing disease spread is proper hand washing!!
http://www.cdc.gov/Features/HandWashing/
 BIOSAFETY CONTAINMENT
Basic Elements
Each element plays a role in protecting the
individual researcher, the environment, and the
material from contamination.
– Practices - Standard Microbiological Procedures,
SOP’s
– Personal Protective Equipment (PPE) - primary
barrier
– Facility Design - secondary barriers
– Containment Equipment - secondary barriers
(examples include BSC, HEPA filtered
centrifuges)
Clearly written Standard Operating Procedures are a
resource for working safely in the lab.

Steps in writing SOPs for the laboratory:

• Identify each task that will be performed
• Develop procedures based on risk assessment
• Incorporate safety measures (PPE, Equipment,
Personal Behavior, etc)
• Procedures for
− Mitigating aerosol generation
− Safe sharps use and handling
− Decontamination
− Equipment certification
 PPE- Protect the skin, body, mucous membranes,
respiratory system and clothing from potential
biohazards when working in the lab.

• Gloves- protect hands, act as a barrier. Must be removed

and disposed of when contaminated. Never re-use
contaminated gloves!
• Lab coats/solid-front gowns- protect street clothing and
skin
• Eye Protection- protect against splashes
• Additional PPE that may be required:
− Sleeve covers,
− Shoe Covers, booties
− Full face protection (Shields)
− Respiratory protection, such as N95, if needed/required
The Facility acts as a secondary barrier to minimize potential
contamination and prevent spread of disease.
Biosafety Level 1-4 Facilities
• Signage - Door sign - Restricted Access
• Hand washing sinks
• Eye wash
• Autoclave
• Biosafety Cabinet
• Communication outside the lab (phone)

Biosafety Level 3-4

• Anteroom
• Negative pressure gradient
• Airflow monitor
• Set Air changes per hour (10-15)
• Sealed penetrations, covered flooring
• Facility alarms/interlocks
• Biological Safety Cabinets (BSC); Class I, II, III
− BSC’s are NOT fume hoods. Fume hoods remove chemical
fumes and aerosols away from the working area. BSC’s provide
a clean work area for personnel and environmental protection
while working with a biological agent.
• Safety centrifuges; HEPA filtered, sealed
• Sealed sonicators, blenders, homogenizers
• Sealed tubes, transport carriers
• Safe sharps, needle boxes, medical waste bags, tongs,
forceps, etc.

Dedicated

Safety Needle Metal Sharps

Container

CDC
 Routes of Exposure

Discovery Channel, Coughing Robot 2010

AEROSOL generated from a simple cough!

Route Microbiological practices/accident
Inhalation
Procedures that produce aerosols:
Centrifugation
Mixing, sonication, vortexing, blending
Spills and splashes
Pouring/decanting culture fluids
Manipulation of inoculation loop
Inoculation Needle stick
Lacerations from sharp objects (e.g. blades, broken glass)
Ingestion Splashes to the mouth
Placing contaminated articles/fingers in mouth
Consumption of food in the laboratory
Mouth pipetting
Contamination of skin and Splashes
mucous membranes Contact with contaminated fomites
From: Sewell, DL. 2006, Clinical Micro Newsletter 28:1-6
• Aerosols are a serious hazard because they are common
in lab procedures and usually go undetected.
• Aerosols are the probable cause of many LAIs,
•
particularly in cases where there was no known accident.
• Lab procedures which could potentially produce aerosols:

− Homogenization, sonication, blending, mixing,

grinding, shaking, vortexing, spills, animals excreting
agent, etc.
− Opening vials; snap cap tubes, vials that have
developed pressure
− Pipetting
− Animal inoculations
− Use of a Flow Cytometer
• Pipetting:
− Cotton plugged pipettes
− Gentle mixing and expelling.
− NO MOUTH PIPETTING!!
− Mechanical pipetting devices
• Centrifugation: MSU lab wars

− Use O-ring screw caps, recommended speeds, plastic

tubes, open lid slowly, decontaminate regularly
• Mixing (sonication, homogenizers, etc) operations:
− Operate equipment in BSC, use O-ring caps, open tubes in
BSC, decontaminate after use
• Sharps/needles:
− Use in BSC, fill syringe slowly (avoid bubbles), extract
needle carefully from insertion points
− Never re-cap or un-cap a used needle!! Discard used
needles immediately
In the Lab
 What Are Sharps?
ANY OBJECT that can puncture or cut the skin
and cause injury…..
– Needles
– Any broken glass
– Microscope Slides
– Cover Slips
– Petri Dish
– Pipet tips
– Broken plastic ware
…..and ANY OBJECT from the lab that can puncture a
plastic trash bag should also be considered a SHARP!!
• Sharps? Needles, Scalpels or any object that can puncture or cut skin
and cause injury including microscope slides and cover slips
• Major Sharps Injuries:

− Skin puncture from needle

OUCH !

− Aerosols CDC

− Spills caused by leaking syringe/needle

− Poor work practices; tapping on syringe or recapping needle
− Inappropriate disposal such as overflowing sharps container(s)

OUCH !

CDC
• Adequate training prior to use of sharps.
• Use needle-locking syringes
• When working with animals use restraints when possible CDC

• Immediate collection/disposal of sharps, needles

• Do not recap or bend the needle!!

• Location of sharps container should be within easy reach

and proper height
• Do not allow sharp containers to overflow

• Eliminate/minimize use/ of Sharps. Use safe sharp devices

whenever possible
• Avoid
− glass Pasteur pipettes
− sharps use with infectious agents or pathogens
 Proper Disposal
• Contaminated Sharps:
− For Biological/Infectious contaminated; decontaminate
first (autoclave)
− Radioisotope contaminated- Contact Radiation Safety
− Chemical contaminated- Contact Chemical Safety
• Contact EHSO for disposal
− Use appropriate Sharps Container to contain sharps. Do
not overfill. Follow guidelines on container

• Non Contaminated Sharps:

− Use appropriate Sharps Container to dispose of used
sharps. Do not overfill. Follow guidelines on container
(usually the capacity is about 50 to 75% full)
− Follow appropriate guidelines for disposal
General Practices
• All contaminated wastes should be separated into sharps, liquid, solid
biological wastes.
• Waste should be placed in autoclave bags inside a durable container
(secondary container) that is foot operated to minimize contamination of
the lid area. (Non foot operated containers must have a lid/cover)
• Do not overfill the bags
• Autoclave when ¾ full

Lid/Cover

Foot Pedal Operation

MSU Lab Wars

1. STERILIZATION
• The destruction of ALL forms of microbial life. Kills all
microorganisms including spores.
• Method: Steam sterilization through the use of an autoclave
• Applications: Regulated waste and critical instruments or
media

2. DISINFECTION
• Eliminates NEARLY ALL pathogenic microorganisms, but
not all forms (e.g. bacterial spores). Less destructive than
sterilization
• Method: Use of chemical liquid germicides known as
disinfectants
• Applications: Biological spills, general decontamination of
lab surfaces and equipment
• Place in a sturdy secondary container – plastic or metal.
• Add small volume of water to the bag (10ml-250ml).
• Loosely close bag. Never tightly seal containers or bags.
• Record in autoclave log book.
• Monitor the temperature, pressure and time during the autoclave
cycle.
• Routinely run Biological Indicators (BI’s) (monthly, quarterly).
– May use indicator strips (i.e. Class 5 CI) to the load. (opp.)
• Use Caution when removing autoclaved material. Do not open
door quickly.
• Maintenance. Autoclaves should have a regular maintenance
check performed by certified technician.
Effectiveness of disinfection is determined by:

• Nature of microorganisms (presence of spores?)

• Number/Amount of microorganisms
• Amount of organic matter present (soil, feces,
blood?)
• Temperature
• Time allowed for disinfection activity
• Concentration of Disinfectant. (Higher
concentration is not necessarily more effective!!)
• Type of Disinfectant used
• Prions High level of resistance to
• Protozoan cysts germicidal chemicals
• Bacterial spores
• Mycobacteria
• Non-lipid or small viruses
• Fungal spores, fungi
• Vegetative bacteria
• Lipid or medium-size viruses

Low level of resistance to

germicidal chemicals

BMBL, 5th Edition

 Practical Requirements Inactivates
Disinfectants Category Use Contact Time (minutes) Vegetative Lipid Non lipid Bacterial
Dilution Bacteria * envelope Viruses Spores
Lipid Broad viruses
envelope Spectrum( all
viruses microbes)

LIQUID Quaternary 0.1%-2.0% 10 Not Effective + +

Ammonium
Compounds
Chlorine 500 ppm 10 30 + + + +
Compounds
Alcohol, Ethyl 70%-85% 10 Not Effective + + Variable results
dependent on
virus

Alcohol, 70%-85% 10 Not Effective + + Variable results

Isopropyl dependent on
virus

Adapted from Yale University

RISK ASSESSMENT?

Dean Potter
 RISK ASSESSMENT
A process used to identify the
hazardous characteristics of a known
infectious or potentially infectious agent
or material, the activities that can result
in a person’s exposure to an agent, the
likelihood that such exposure will cause
a LAI, and the probable consequences
of such an infection.
Definition from CDC
 WHY do a Risk Assessment?
• To better understand how a Lab Acquired
Infection may occur.

• To assist in determining safe procedures to

use to prevent or reduce potential exposure
and infection.

• A risk assessment is the backbone to

SAFE LAB PRACTICES
In Lab Setting:
• Microorganisms are grown, cultured, purified and used at
concentrations that are at much higher levels than in a natural
setting.
• Microorganisms are treated and used differently than in the
natural setting; inoculation studies, purifications, cell culture.

Immunocomprimised Individuals.
• Exposure can be lethal (Opportunistic Infection) An exposure
to a BSl-1 organism may be relatively safe for a normal healthy
individual.
− Staphylococcus aureus, Pseudomonas aeruginosa, JC
polyomavirus, Cytomegalovirus, Cryptococcus neofomans
Determine how you will minimize the Identified Risks:

• Review the SOP/Procedures and work practices.

− Are they adequate to reduce/prevent potential hazards or
accidents?
• What types of PPE should you use?
− Will gloves and lab coat be sufficient?
• Training
− Are you staff adequately trained to perform the research
activity?
• Facilities Design (Engineering Controls).
− Are you working in and at the correct Biosafety Lab level for the
microorganism and research activity?
• Equipment.
− Is the BSC certified?
− Are centrifuges, autoclaves functioning properly?
• Signs and Symptoms of Disease vary depending on
the microorganism, the persons age, if they are
Immunocompromised.
• As a lab worker, you should be aware of the
symptoms of disease, the transmission route and
hazards associated with working with infectious
agents

References:
• ABSA (American Biological Safety Association)
• ASM (American Society of Microbiology)
• Microbiology, Sherris (Text books)
• CDC
• Web
 Identify agent
hazards
Review
1
and perform
risk
initial
assessment risk
assessment

5
RISK ASSESSMENT
2
is always ongoing
In the working lab Identify
Evaluate
laboratory
staff
procedure
proficiencies
hazards

4 3
Determine
Required
PPE,
Equipment
 Identify agent
hazards
Review
1
and perform
risk
initial
assessment risk
assessment

5 Consider all 2
Potential Risks;
Actual and Identify
Evaluate
staff Perceived laboratory
procedure
proficiencies
hazards

4 3
Determine
Required
PPE,
Equipment
1997. A student working in a microbiology was tasked with the
cleaning and decontaminating lab ware used in the lab for
cultures of Shigella dysenteriae. The student was suppose to
perform this step in a dedicated sink for contaminated materials.
However the sink was dirty and the student went to the dedicated
hand washing sink to clean the materials
12 hours later, Six co-workers became ill and were diagnosed
with Shigella dysenteriae. Other lab workers were identified as
asymptomatic (exposed but at lower doses)
The procedure generated splash that contaminated the
hand washing sink and was spread via the handles as they
were turned on and off.
Lessons Learned:
• All procedures involving a biohazardous material must be
completed by trained personnel including the decontamination
process.
• All surfaces, equipment and areas that may have come in
contact with an infectious material must be decontaminated.
• Dedicated hand wash area must be respected
• Biohazard control procedures should be established and
implemented.

Risk Assessment:
• Training on:
− safe and proper handling of the organism including proper
decontamination
− Know the signs and symptoms of Shigella infection
• More experienced staff should do the work
• Dedicated room with restricted access
• Hands free sinks should have been installed.
 The most frequent types of lab
accidents:
• Inhalation (aerosols)
• Needle Sticks/Cuts
• Splashes/Spills
• Animal Bites/Scratches
• From mouth pipetting CDC

• Direct contact; broken skin cuts

Accidents occur when in a hurry, when tired, not paying attention.
Review all potential risks before performing the task. Think before
you Act

LAI’S can occur from any of the above and unknown routes of
transmission
BSL2 – LAI’s in the News

• October 2009 Boston University. Graduate Student became

infected with Neisseria meningitides while working in a lab.

• 2004 - 3 BU researchers become ill after working with what was

believed to be an avirulent strain of Francisella tularensis. Lab
sample later found to be contaminated with virulent wild type.

• 1987- Researcher becomes infected with HIV when working on HIV.

Found “hole in glove”

• Most common LAI species include Brucella, Mycobacterium

tuberculosis, Shigella, Salmonella.

Reports are voluntary and often are unreported because of

fear of reproach from reporting.
 Emergency Response

All Lab personnel should

Be able to identify the locations of the:

• Eye Wash Station
• Designated Hand Wash Station
• Phone
• Spill Kit

Find the:
• Emergency Contact Information
• Procedures on reporting an accident
Provide care to injured person first. Always treat injured.
Call 911, physician, Campus Security (66911) depending on
severity of injury.

Percutaneous Injury. Wash affected area with disinfectant/soap and

warm water. Apply dressing and seek medical attention. Notify your PI,
Biosafety Compliance Program.

Splashes to Face. Flush affected area in eyewash for 15 minutes.

Notify your PI, Biosafety Compliance Program.

Aerosol Exposure. Hold your breath, leave room where aerosol was
generated immediately. Carefully remove your PPE (turn inside out)
Wash hands with warm water and soap. Post a sign “Do Not Enter; Spill”
on door. Lab should be evacuated for a minimum of 30 minutes.
• Notify PI, Research Compliance Officer/Biosafety.
• Until PI/ Safety Officers gives permission, no one should enter lab.
 LAB ACCIDENTS-
INJURY and REPORTING
Less than 10 percent of Lab accidents are
reported in the Nation.

Fewer than 20% of those accidents have

a known cause of transmission. (Aerosol
is the plausible cause in most cases) NBBTP

By reporting you provide assistance to

those who have been injured and provide
invaluable help to prevent future similar
incidents
For all exposure and possible exposure incidents:
• Notify Principal Investigator, lab manager or supervisor to
initiate Incident report within 24 hours of accident
• PI/supervisor must report the accident to IBC (rDNA &
Infectious agents), IACUC (animals), Human Resources, Risk
Management

PI notification to Research Compliance Officer within 24

hours 956-8420, 285-6367
• Large spills (> 100-200 ml or when unable to contain)
• All BSL-3 spills
• All Select Agent spills
• rDNA that results in an environmental exposure or
injury
• Incidents resulting in personal injury
• Cover spill with absorbent material.
• Saturate with appropriate disinfectant. Starting from the
outside and working toward the center of spill.
• Allow disinfectant to soak (minimum 30 minutes).
• With gloved hands; pick up all wastes and place slowly
and carefully into biowaste container.
• Cover area again with disinfectant then clean up with
absorbent material
• Remove your gloves and dispose in the biowaste
container.
• Wash hands with warm water and soap.
• Put on new gloves and if necessary new lab coat.
• Spill Cleanup Protocol/instructions
• Absorbent material; paper towels or pads
• Disinfectant -Bleach or other appropriate disinfectant
• A bottle to make fresh diluted disinfectant
• Forceps, dust pan, plastic scoop, or other device for handling
sharps or broken glass
• Autoclavable biohazard bags to collect contaminated items
• Disposable nitrile gloves
• Durable Utility Gloves
• Appropriate PPE (lab coat or jumpsuit, safety goggles, shoe
covers, face mask, N95**).
**Lab personnel requiring N95 respirators must have a medical evaluation from a registered health care
professional prior to being fit tested in order to wear a respirator. (N95 fit testing is provided at UH
Manoa ekennedy@hawaii.edu 956-3204 )
 Outside of Biosafety Cabinet (BSC)
BSL-1 Spills, or Small Scale (<200 mL) BSL-2 Spills
Notify Notify others in the immediate area that a spill has occurred.
Treat Human Injury Tend to any injured personnel. Call 911 if necessary
Treat personal Place contaminated clothing in biohazardous bag. Wash skin thoroughly with
contamination soap and water.
Wear PPE Don lab coat, gloves, face protection.
Decontaminate spill Cover spill absorbent material. Saturate the paper towel with appropriate
disinfectant, starting with the edges of the spill and working towards the
center. Allow contact time of at least 30 minutes.
Clean up After the 30 minute contact time, with gloved hands pick up absorbent
material and place in biohazardous bag. Use appropriate equipment (scoop,
dustpan, or forceps) to pick up any broken glass. Place in sharps container.

Disinfect surrounding area Carefully spray area and surrounding areas with appropriate disinfectant wipe
up with absorbent material. Place all waste material in autoclave bag
Decontaminate material Autoclave contaminated material and PPE. Decontaminate any reusable
and PPE items with disinfectant.
Wash hands and exposed skin areas with soap and warm water
Document and reporting Notify PI or supervisor of large spills. If exposure occurred, submit exposure
incident report to Supervisor and Biosafety Compliance Proram
Principal Investigator Responsibilities

• Ensure Adequate Training

• Equipment Integrity
• Proper notification of incidents involving staff, students and
visitors of the University of Hawaii
• University of Hawaii Approvals
− ORS
− IACUC, IBC, CHS
• Federal and or State permits or licenses
• Compliance with all applicable University of Hawaii, State
of Hawaii, and Federal policies, guidelines and regulations
 Principal Investigator Responsibilities
(continued )

Develop Biosafety Manual (BMBL 5 th Ed. good reference)

Components:
− Lab personnel review and acknowledgment (date, name,
signature)
− General and Organism specific SOP’s /Procedures
− Daily Clean Up & General Decontamination
− Biological Spill Response Plan
− Waste Management Plan
− Sharps Injury Prevention Procedures
− Emergency response plans for lab
• Medical
• Fire
• Natural disaster
− Transportation and Storage of microorganisms
 Contact Information
University of Hawaii
• Eleanor Low, Research Compliance Officer, Biosafety (808)956-8420,(808)285-6367
• Stephen Case, Biosafety Specialist (808) 956-8009
• Hubert Olipares, Biosafety Officer (808) 956-3197
• Tim O'Callaghan, Hazardous Material Management Officer (808) 956-3198
• Emma Kennedy, Industrial Hygienist (808) 956-3204
• Irene Sakimoto, Radiation Safety Officer (808) 956-6475
UH HILO
• Ken Ikeda Environmental Health & Safety Officer (808)933-3300
Institutional Biosafety Committee (IBC)
http://manoa.hawaii.edu/ovcrge/research/ibc/index.html
• Eleanor Low @ uhibc@hawaii.edu
IACUC-Institutional Animal Care and Use Committee IACUC
http://www.hawaii.edu/ansc/IACUC/
• Mr. Norman Magno at nmagno@hawaii.edu
• Ms. Denise Yee at deniseye@hawaii.edu
Committee on Human Studies http://www.hawaii.edu/irb/index.htm
• uhirb@hawaii.edu
 UH Biosafety Compliance Program
PROMOTES SAFE LAB PRACTICES

Eleanor Low Stephen Case

QUESTIONS?