The document provides a detailed overview on the basic principles of operating a biotech or micro laboratory along with basic techniques with which to handle organisms, chemicals &equipment and ensuring your own, your colleagues and your environment's safety.

1. BIO-SAFETY GUIDELINES

2. Contents:
1. Introduction
2. History
3. Background
4. Purpose
5. Basic Principles
6. Biological Waste & Its Disposal
7. Risk Groups
8. Bio-Safety Levels
9. Risk Assessment
10.Conclusion

3. Abstract
These guidelines for bio-safety laboratory
competency outline the essential skills, knowledge,
and abilities required for working with biologic
agents at the four bio-safety levels (BSLs) (levels 1,
2, 3, and 4). This article comprises all the basic
guidelines necessary to carry out any type of
research being carried out on biological organisms
carefully and without harming anyone i.e. the
researcher, workers and co-workers and most
importantly our environment and also it carries
guidelines to handle bio-hazardous materials and
dispose them off according to the protocol.
For a person with no scientific background bio-hazardous
symbols have also been discussed so
that when they see those symbols they would be
able to protect themselves accordingly.
In the end a conclusion has been given which
consists the essence of the discussion of the whole
research paper.

4. 1. Introduction:
Bio-safety is the prevention of large
scale loss of biological integrity, focusing both on
ecology and human health. These prevention
mechanisms include conduction of regular reviews
of the bio-safety in laboratory settings, as well as
strict guidelines to follow. Bio-safety is used to
protect us from harmful incidents. High security
facilities are necessary when working
with Synthetic Biology as there are possibilities of
bioterrorism acts or release of harmful chemicals
and or organisms into the environment. A
complete understanding of experimental risks
associated with synthetic biology is helping to
enforce the knowledge and effectiveness of bio-safety.
1
Despite a greater awareness of bio-safety
and bio-containment practices, handling infectious
microorganisms and other organisms remains a
source of infection, and even mortality, among
laboratory workers. Incidents of secondary
transmission of disease to the public at large,
which may be due to possible contamination of the
environment or personnel, are also occurring.
Laboratory workers can minimize the risks
associated with work involving these infectious
agents through the application of appropriate bio-safety
and containment principles and practices.
Bio-safety laboratories must ensure
adequate safety conditions to avoid potential
hazards associated with the handling of biologic
materials, the manipulation of genomes, the
creation of synthetic organisms, and the spread of
multidrug-resistant bacteria, and threats of biologic
terrorism. These guidelines define the essential
competencies needed by laboratory personnel to

5. work safely with biologic materials and other
hazards that might be found in a biologic
laboratory (e.g. those related to research animals,
chemicals, radiologic materials, and the physical
environment).
A successful laboratory safety program
encompasses a continuous process of hazard
recognition, risk assessment, and hazard
mitigation. The risk for exposures, laboratory
acquired infections, and the unintended release of
research or clinical materials to the environment
should ultimately be reduced by ensuring the
competency of laboratories at all levels.
Based on this above discussion bio-safety
can be defined as “procedures or measures
designed to protect the population against harmful
biological or biochemical substances”.2
2. History:
On 18 April 1955 the first biological safety
conference took place at Camp Detrick and
involved members of the military representing
Camp Detrick, Pine Bluff Arsenal, Arkansas (PBA),
and Dug way Proving Grounds, Utah (DPG). In that
conference role of Safety in the Biological Warfare,
chemical, radiological and industrial safety issues
were discussed. Later in the US, the Centre for
Disease Control (CDC) specified 4 different levels of
bio-containment which ranges from bio-safety level
1 to bio-safety level 4. 3 4
3. Background:
The Laboratory bio-safety guidelines were
initially developed to guide government, industry,
university, hospital, and other public health and
microbiological laboratories in their development

6. of bio-safety policies and programs.
The Guidelines also serve as a technical document
providing information and recommendations on
the design, construction and commissioning of
containment facilities.5 Several other
circumstances led to the development of bio-safety
competencies for practicing laboratorians. In 2006,
PAHPA called for the assessment, development,
delivery, and evaluation of competency-based
training for bio-safety in high containment
laboratories. In 2008, CDC convened the Blue
Ribbon Panel for issues of Clinical Laboratory Safety
to address incidence of laboratory-acquired
infection. Also in 2008, a Trans-Federal Task Force
was convened for federal agencies with
laboratories to address bio-safety and bio-security
in working with agents that pose a significant
public health threat, whether they arise from
nature, accidental exposure, or deliberate terrorist
attacks. These efforts underscored the need to
develop competency guidelines for laboratory bio-safety.
4. Purpose:
The primary purpose of these guidelines
is to establish the behaviors and knowledge that
laboratory workers at all levels should have to work
safely with biologic materials. Other key issues are:
the need for a well-designed workspace,
knowledge of specific biologic agents and toxins,
quality laboratory management practices, and an
overall safety culture.
5. Basic Principles:
No regulation or guideline can ensure
safe practices. Individual and organizational
attitudes regarding safety will influence all aspects

7. of safe practice, including willingness to report
concerns, response to incidents, and
communication of risk. Each organization should
strive to develop a culture of safety that is open
and nonpunitive, encourages questions, and is
willing to be self-critical. Persons and organizations
must be committed to safety, be aware of risks,
behave in ways that enhance safety, and be
adaptable. Scientists understand that practices
should be refined as observations are made,
hypotheses tested, findings published, and
technical progress achieved. The same holds true
for safety in the laboratory, which should evolve as
experience is gained and as laboratory activities
change. As laboratorians gain more knowledge
over time concerning how to recognize and control
hazards, the level of risk that is considered
acceptable should become smaller, with the goal of
moving continuously to eliminate or reduce risk to
the lowest reasonably achievable level.
Laboratorians have both the responsibility to
report concerns to management and the right to
express concerns without fear of reprisal. Similarly,
management has the responsibility to address
concerns raised from any direction. A continuous
process of hazard recognition, risk assessment, and
hazard mitigation practices ensures that
management and laboratory workers alike are
aware of the issues and work together to maintain
the highest standard of safety.
6. Bio-Hazardous Waste & Its Disposal:
Bio-hazardous waste:-
Bio-hazardous waste is defined as materials
containing:
 Infectious agents (to human, plants, animals)

8.  Biological toxins
 Materials derived from humans and primates
(blood, body fluids, tissues)
 Human and primate cell lines (including
recombinant)
 Recombinant animal cell lines
 Recombinant microorganisms
 Transgenic animals (vertebrate and
invertebrate)
 Materials derived from transgenic animals
(body fluids, tissues)
 Transgenic plants
 Recombinant materials such as plasmids,
DNA/RNA, synthetic DNA
Disposal Of Bio-Hazardous Waste:-
Liquid Waste:
Bio-hazardous liquid wastes are liquids
containing bio-hazardous waste.
Disposal Method: Decontaminate by treating with
an appropriate disinfectant. The amount of contact
time will depend on the chemical used and the
material decontaminated. For specific procedures,
refer to your approved protocol or contact EHS.
Solid Waste:
Bio-hazardous solid wastes are solids that
contain bio-hazardous materials or lab waste that
has come in contact with bio-hazardous materials.
These include:
 Culture media
 Personal Protective Equipment (contaminated)
 Plastic ware including pipette tips

9.  Transgenic plants including soil
Disposal Methods: Place material in an autoclave
bag, close bag loosely; attach a strip of autoclave
tape and autoclave. Apply “treated” label and place
in black garbage bag for disposal in trash. Record
treatment on autoclave waste treatment record
form.
Autoclave bag
Autoclave bag with “treated” label attached.
Treated bag placed in black trash bag for disposal.
Or;
Place in biohazard waste container and contact EHS
for pickup.

10. Sharps: Dispose into sharps container
Glassware: Decontaminate for re-use or dispose in
sharps container. Do not use glass trash boxes for
glassware contaminated with biological materials.
Transgenic Animals and Materials:
Transgenic animals and materials
includes animals (vertebrate and invertebrate) and
materials (tissues and infected bedding) from
transgenic animals.
Transgenic insects:
Freeze, then autoclave or place in
biohazard container
Transgenic animal carcasses/tissues:
Place in 2X biohazard bags and place in
freezer
Animal bedding from animals shedding
pathogens:
Place in biohazard waste container.
Disposal Method: Place in appropriate container.
Request EHS pickup.

11. Non-transgenic Animals/Tissues:
Non-transgenic animals or tissues
include animal carcasses/tissues not infected with
infectious agents.
Disposal Method: Place in double bags and place
material in freezer and request EHS pickup.
Used Animal Bedding:
Used animal bedding includes bedding
from animals that is free of pathogens or biological
toxins
Disposal Method: Can be disposed as conventional
trash. Containers should be secured.6
7. Risk Groups:
Classification of organisms according to
risk group has traditionally been used to categorize
the relative hazards of infective organisms. The
factors used to determine which risk group an
organism falls into is based upon the particular
characteristics of the organism, such as
 pathogenicity
 infectious dose
 mode of transmission
 host range
 availability of effective preventive measures
 availability of effective treatment
These classifications presume ordinary
circumstances in the research laboratory or growth
in small volumes for diagnostic and experimental
purposes. Four levels of risk have been defined as
follows.
Risk Group 1 (low individual and community risk):
Any biological agent that is unlikely to cause
disease in healthy workers or animals.

12. Risk Group 2 (moderate individual risk, low
community risk):
Any pathogen that can cause human
disease but, under normal circumstances, is
unlikely to be a serious hazard to laboratory
workers, the community, livestock or the
environment. Laboratory exposures rarely cause
infection leading to serious disease; effective
treatment and preventive measures are available,
and the risk of spread is limited.
Risk Group 3 (high individual risk, low community
risk):
Any pathogen that usually causes serious
human disease or can result in serious economic
consequences but does not ordinarily spread by
casual contact from one individual to another, or
that causes diseases treatable by antimicrobial or
anti-parasitic agents.
Risk Group 4 (high individual risk, high community
risk):
Any pathogen that usually produces very
serious human disease, often untreatable, and may
be readily transmitted from one individual to
another, or from animal to human or vice-versa,
directly or indirectly, or by casual contact.7
8. Bio-Safety Levels:
There are 4 types of bio-safety levels
according to the risk factors involved depending on
the nature of pathogen being handled.

13. Bio-Safety Level 1 (BSL 1):
This applies to the basic
laboratory that handles agents requiring bio-safety
level 1. BSL 1 requires no special design features
beyond those suitable for a well-designed and
functional laboratory. Biological safety cabinets
(BSCs) are not required. Work may be done on an
open bench top, and containment is achieved
through the use of practices normally employed in
a basic microbiology laboratory.
Bio-Safety Level 2 (BSL 2):
This applies to the
laboratory that handles agents requiring bio-safety
level 2. The primary exposure hazards associated
with organisms requiring BSL 2 are through the
ingestion, inoculation and mucous membrane
route. Agents requiring BSL 2 facilities are not
generally transmitted by airborne routes, but care
must be taken to avoid the generation of aerosols
(aerosols can settle on bench tops and become an
ingestion hazard through contamination of the
hands) or splashes. Primary containment devices
such as BSCs and centrifuges with sealed rotors or
safety cups are to be used as well as appropriate
personal protective equipment (i.e., gloves,
laboratory coats, protective eyewear). As well,
environmental contamination must be minimized
by the use of hand washing sinks and
decontamination facilities (autoclaves).
Bio-Safety Level 3 (BSL 3):
This applies to the
laboratory that handles agents requiring bio-safety
level 3. These agents may be transmitted by the
airborne route, often have a low infectious dose to

14. produce effects and can cause serious or life-threatening
disease. BSL3 emphasizes additional
primary and secondary barriers to minimize the
release of infectious organisms into the immediate
laboratory and the environment. Additional
features to prevent transmission of BSL3 organisms
are appropriate respiratory protection, HEPA
filtration of exhausted laboratory air and strictly
controlled laboratory access.
Bio-Safety Level 4 (BSL 4):
This is the maximum
containment available and is suitable for facilities
manipulating agents requiring bio-safety level 4.
These agents have the potential for aerosol
transmission, often have a low infectious dose and
produce very serious and often fatal disease; there
is generally no treatment or vaccine available. This
level of containment represents an isolated unit,
functionally and, when necessary, structurally
independent of other areas. BSL 4 emphasizes
maximum containment of the infectious agent by
complete sealing of the facility perimeter with
confirmation by pressure decay testing; isolation of
the researcher from the pathogen by his or her
containment in a positive pressure suit or
containment of the pathogen in a Class III BSC line;
and decontamination of air and other effluents
produced in the facility.
9. Risk Assessment:
Risk assessment is a
critical step in the selection of an appropriate bio-safety
level for the microbiological work to be
carried out. A detailed local risk assessment should
be conducted to determine whether work requires
containment level 1, 2, 3 or 4 facilities and
operational practices. Individuals with varying

15. expertise and responsibilities should be included in
the risk assessment process and can include,
among others, the facility director, laboratory
supervisor, principal investigator, senior
microbiologist, bio-safety officer and bio-safety
committee.
Available information can be used as a starting
point to assist in the identification of risk factors,
including the recommended Risk Group of the
organism. In addition to the Risk Group
classifications, which are based on the risk factors
inherent to the organism, the following factors
associated with the laboratory operation should
also be examined:
 potential for aerosol generation
 quantity
 concentration
 agent stability in the environment (inherent
biological decay rate)
 type of work proposed (e.g., in vitro , in vivo ,
aerosol challenge studies)
 use of recombinant organisms (e.g., gene
coding for virulence factors or toxins; host
range alteration; oncogenicity; replication
capacity; capability to revert to wild type)
10. Conclusion:
These guidelines outline the essential
expectations for behaviors and knowledge of
laboratory workers necessary to work safely with
biologic materials at all levels of the profession in
the life sciences. The development of these
guidelines is a first step toward defining
comprehensive safety competencies in biologic
laboratories. These guidelines reflect a range of
past experiences and will be reviewed periodically
and refined as additional experience is gained. The
guidelines can be used as a resource to develop

16. educational goals, training standards, safety
assessments, professional development, and
certification.
Every organization using these competencies
should regularly review and improve its practices
and documents with an eye toward continual
reduction of the risks involved in working with
biologic and other hazardous laboratory materials.
Training is not limited to the initial instruction
received at the start of a laboratory worker’s
employment but is continuous and refreshed
periodically. Some professions or organizations
that were contributors to these guidelines,
including AALAS and the Council of State and
Territorial Epidemiologists, also have addressed
biologic safety practices. 8
1 http://en.wikipedia.org/wiki/Biosafety
2 https://www.google.com.pk/?gws_rd=cr&ei=y2PcU5rOG6mh0QX4s4CQBg#q=define+biosecurity
3 http://www.slideshare.net/neharachankar/neha-biosafety-levels-ppt
4 http://fas.org/pubs/pir/2011fall/2011fall-bioagents.pdf
5 http://www.phac-aspc.gc.ca/publicat/lbg-ldmbl -04/ch1-eng.php
6 http://www.utexas.edu/safety/ehs/biosafety/waste_guidelines.html
7 http://www.phac-aspc.gc.ca/publicat/lbg-ldmbl -04/ch2-eng.php
8 https://www.aaalac.org/accreditation/RefResources/guide_for_biosaf_comp.pdf