Laboratory Biosafety

and
Good Laboratory Practices
Biol 107 Cell and Molecular Biology
Intended Learning Outcomes:
• Execute laboratory safety practices and SOPs
when doing laboratory exercises.
• Enumerate different protocols that have been
established at the international and national
levels to ensure biosafety in institutions
involved in biotechnology research and
development.
Introduction
GMT (good microbiological techniques)
will not suffice to provide safety during early
days of the development of rDNA technology

June 1976 after Asilomar Conference (Ensuring

Safety in Biotechnology)
NIH Guidelines for Research Involving
Recombinant Nucleic Acid Molecules
 UN WHO in 1983
published the Laboratory Biosafety Manual (aka
WHO Manual)
establishing basic concepts and practices in
the safe handling of pathogenic
microorganisms
encouraged countries to develop national
code of practice for implementation
Why it is important to adhere
to international standards?
To protect
plant, animal or human life
health of its citizen
trade in biotech products
Risk Categories of Microorganisms

NIH guidelines and WHO Manual, recognized 4

categories
Based on the risk of infection to:

• Laboratory workers
• Community in the event of an escape
from the lab
How to assign a microorganism
to a risk category?
Pathogenicity of the organism
Host range and mode of transmission of the
organism
Local availability of effective measures to prevent a
disease outbreak
Local availability of effective treatment
Risk Groups (RG) of Microorganisms:
RG 1: unlikely to cause human or animal diseases
pose little or no risk to individual and to the
community; generally regarded as safe
(GRAS)
RG 2: pathogenic, but unlikely to pose a serious
hazard to lab workers, livestock, community,
or environment as effective treatment and
preventive measures to limit the spread of
infection are available; moderate risk to
individual and low risk to the community
Risk Groups (RG) of Microorganisms:
RG 3: pathogenic and can cause serious human or
animal diseases, but are not contagious or have an
effective treatment and preventive measures to
limit the spread of infection are available; high risk
to individual, but low risk to the community

RG 4: causes serious diseases in humans and

animals and readily transmitted (direct or
indirect), effective treatment and preventive
measures are unavailable; high risk to individuals
and to the community
 BIOSAFETY LEVELS (BLs)
4 BLs for handling organisms corresponding to the
4 RGs
Relies on:
Standard practices of GMT
Physical barriers (procedures,
equipment, and lab installations
based on the estimated biohazard)
BLs for specific research
work depend on
the assessed RG of the organisms handled
professional judgement of risk associated with the
activity
Relation of RGs to BLs, Practices, and Equipment
 PHYSICAL CONTAINMENT

Strict adherence to good microbial practices

• all personnel directly or indirectly working with

recombinant or synthetic nucleic acids should
be trained in GMT
4 Levels of Physical Containment
BL1 to BL4 representing facilities in which
experiments ranging from low to high potential
hazard
Standard microbiological practices;
Special practices;
Containment equipment, and
Laboratory facilities
BIOLOGICAL CONTAINMENT
The growth and dissemination of organisms are
naturally limited due to:

The infectivity or host specificity of a vector or

virus
Its spread and survival in the environment
Example of biological containment
strategies:

Survival of the vector in its host outside the lab

Transmission of the vector from the propagation

host to other nonlaboratory hosts
Purpose of Physical and Biological
Containment for Reseach
to prevent unintentional transmission or release
of recombinant or synthetic nucleic acid molecule
GOOD LAB PRACTICES (GLP)
embodies a set of principles that provide a framework
within which laboratory studies are planned,
performed, monitored, recorded, reported, and
archived (Dolan, 2007)
What is the primary purpose
of GLP?
ensure uniformity, consistency, and reliability of
safety tests (nonclinical) for
pharmaceuticals, agrochemicals, aroma and color
food/feed additives, cosmetics, detergents, novel
foods, nutritional supplements for livestock, and
other chemicals.
GLP is mandatory to
evaluate safety or
toxicity of products
intended to undergo
clinical trials
References
Compliance of GLP requires:
• Tests conducted by qualified personnel
• Each study have Study Director
• The lab study and data are audited by a Quality
Assurance Unit
• All lab activities performed were according to SOPs
• All test articles and reagents must be ID,
characterized, and labeled
• Equipment must be maintained and calibrated
STANDARD OPERATING PROCEDURE
for GENERAL SAFETY in the LAB
• Carefully read the activity and systematically
plan the workflow before conducting
• Wear a lab gown while in the lab
• Clean your lab area & material/equipment with
disinfectant before and after use
• If you do not understand something, ask
• Any spillage must be immediately addressed
STANDARD OPERATING PROCEDURE
for BIOSAFETY CABINET Class II
• Decontaminate the BSC surface and the
materials to be placed inside (70% EtOH)
• Turn on the blower 10 mins before beginning
work, turn off UV lamps when the room is
occupied
• Heat sources are strictly prohibited inside the
BSCs (will disrupt the laminar flow of air)
• Keep the work area free of unnecessary
equipment or supplies
Biosafety Cabinet
A partially enclosed workspace
that has built in protection for
the worker, environment, and the
material inside.

Outside air is filtered (HEPA

High-efficiency particulate
absorber)
STANDARD OPERATING PROCEDURE
for LAMINAR AIR FLOW
• Close the sash and keep the UV on for 20 mins
• Turn on the blower 10 mins before beginning
work
• Surface sterilize (70% IPA v/v) all materials to
be placed inside
• Keep the work area free of unnecessary
equipment or supplies
 Laminar Floodhood
aka Tissue Culture Hood
enclosed bench designed to
prevent contamination of
biological samples
STANDARD OPERATING PROCEDURE
for WASTE DISPOSAL
• Decontaminate and waste should not be
stored for more than 1 week
• Used sharps should be placed in a safety box
(close when 3/4 full)
STANDARD OPERATING PROCEDURE
for COMPOUND MICROSCOPE
• Clean the lenses with cotton soaked in xylene
(before and after use)
• Observe with both eyes when using microscope
• Handle all the parts with care
• Turn off and cover the microscope when not in
use
STANDARD OPERATING PROCEDURE
for AUTOCLAVE MACHINE
• Sterilization time and temp are dependent on
the composition and nature of items (15 mins at
121C at 15 lbs pressure min)
• Small items should be bagged or wrapped with
label
• Sterile packs stored on open shelves are safe to
use up to 1 month (dry and intact)
Autoclave Machine
Sterilization refers to the
complete killing of all living
organisms (wet/dry heat,
chemicals, and radiation)
STANDARD OPERATING PROCEDURE
for PCR MACHINE
• Protect the outer sleeves from damage or sharp
objects

• Only the power cord of the machine supplied

with the workstation should be used to connect
PCR Machine
aka Thermocycler used
to amplify segments of
DNA

Has a thermal block with

holes where tubes can
be inserted
PERSONAL PROTECTIVE EQUIPMENT
To protect against potential chemical exposures

It is a supplement and not a substitute for feasible

engineering and administrative controls

Will vary with the nature of contaminant, route(s)

of exposure, and contaminant concentration
PERSONAL PROTECTIVE EQUIPMENT
RESPIRATORS may be required when engineering
controls are not sufficient to reduce the conc of
air contaminants below an acceptable level

TYPES ranging from filtering face pieces (dust

masks) to full face covering, depends on the conc
and form of contaminants (dusts, mists, fumes)
PERSONAL PROTECTIVE EQUIPMENT
EYE and FACE PROTECTION protective
eyewear/face shield combination may be needed
to prevent projectiles, airborne particles, thermal
burns, chemical splashes, vapors, mists, dusts, or
radiant energy from damaging the eyes and face
PERSONAL PROTECTIVE EQUIPMENT
HAND PROTECTION Gloves provide protection for
the hands from many types of hazards (chemical
absorption)

Butyl rubber gloves - chemicals except for alipathic

and aromatic hydrocarbons and halogenated
solvents

Natural (latex) rubber gloves - thin, elastic low barrier

protection against acids, alkaline, salts, ketones
PERSONAL PROTECTIVE EQUIPMENT
Neoprene gloves - similar in texture and appearance
to latex gloves but more resistant. Low barrier
protection against hydraulic fluids, gasoline,
alcohols, organic acids, and alkalis

Nitrile gloves - similar in texture and appearance to

latex or neoprene gloves. Made of copolymer and
provide protection from chlorinated solvents such as
trichloroethylene and perchloroethylene
PERSONAL PROTECTIVE EQUIPMENT
BODY PROTECTION - protective body apparel against
potential contact through spills or splashes.

In high-risk splash hazards, the body apparel should

fully cover the torso, arms, and legs.

Low hazard situations - lab coats made of cotton, or

flame-retardant material
 FIRE EMERGENCY
FIRE EXTINGUISHER TYPES:

CLASS A- trash, wood, paper involve ordinary

combustible materials (quenched by water or
multipurpose (ABC) dry chemicals agent)

CLASS B - liquid flammable liquid (quenched by dry

chemical and CO2)

CLASS C- electrical equipment (dry chemicals or CO2)

 FIRE EMERGENCY
FIRE EXTINGUISHER TYPES:

CLASS D- combustible metals like magnesium,

titanium, zirconium, and lithium (quenched by
sodium carbonate, graphite, bicarbonate, NaCl, and
salt based chemicals )

CLASS K fire - Cooking oil