Workplace safety is an important aspect to protect personnel against injury or serious accident.In case of animal cell culture safety takes a front seat due to nature of work i.e. handling of human cells and tissues, viruses with high potential to cause infections to humans and other adventitious micro organisms. This presentation presents various methods of safety to protect lab personnel from infectious biological agents.
Safety cabinets are intended to protect a laboratory worker from aerosols and airborne particles.
They will not protect the person from spillages and the consequences of mishandling and poor technique.
Aerosol particles of less than 5 µm in diameter and small droplets of 5–100 µm in diameter are not visible to the naked eye.
The laboratory worker is generally not aware that such particles are being generated and may be inhaled or may cross contaminate work surface materials.
BSCs, when properly used, have been shown to be highly effective in reducing laboratory-acquired infections and cross-contaminations of cultures due to aerosol exposures. BSCs also protect the environment.
Most BSCs use high efficiency particulate air (HEPA) filters in the exhaust and supply systems.
The exception is a Class I BSC, which does not have HEPA filtered supply air.
Biosafety is the application of safety precautions that reduce a Laboratory based risk of exposure to a potentially infectious material and limit contamination of the working and surrounding environment.
The primary principle of biosafety is “Containment”.
Containment
The action of keeping harmful things under control and within limits
Or
A series of safe methods for managing infectious bacteria in the laboratory.
Safety cabinets are intended to protect a laboratory worker from aerosols and airborne particles.
They will not protect the person from spillages and the consequences of mishandling and poor technique.
Aerosol particles of less than 5 µm in diameter and small droplets of 5–100 µm in diameter are not visible to the naked eye.
The laboratory worker is generally not aware that such particles are being generated and may be inhaled or may cross contaminate work surface materials.
BSCs, when properly used, have been shown to be highly effective in reducing laboratory-acquired infections and cross-contaminations of cultures due to aerosol exposures. BSCs also protect the environment.
Most BSCs use high efficiency particulate air (HEPA) filters in the exhaust and supply systems.
The exception is a Class I BSC, which does not have HEPA filtered supply air.
Biosafety is the application of safety precautions that reduce a Laboratory based risk of exposure to a potentially infectious material and limit contamination of the working and surrounding environment.
The primary principle of biosafety is “Containment”.
Containment
The action of keeping harmful things under control and within limits
Or
A series of safe methods for managing infectious bacteria in the laboratory.
deals with biosafety in medical labs. universal safety precautions included. Includes updated 8 categories and colour coding for BMW management. Being a budding microbiologist, kept it focused on microbiology lab
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
deals with biosafety in medical labs. universal safety precautions included. Includes updated 8 categories and colour coding for BMW management. Being a budding microbiologist, kept it focused on microbiology lab
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
Biohazards,Institutional Biosafety Committees and Cartagena Protocol:
Biohazards:
Biological hazards also known as biohazards, refer to biological substances that pose a threat to the health of living organisms, especially that of humans. For example: Viruses, bacteria ,fungi etc.
These hazards can be encountered anywhere in the environment. The biohazard symbol was developed in 1966 by Charles Baldwin, an environmental health engineer.
Types of Biological Hazards: Biological hazards can be put into different categories:
Bacteria: microscopic organisms that live in soil,water or the bodies of plants and animals and are characterized by lack of distinct nucleus and the inability to photosynthesize. Examples are E.coli, TB and Tetanus.
Viruses: These are a group of pathogens that consist mostly of nucleic acids and that lack cellular structure. Viruses are totally dependent on their hosts for replication. Examples: common cold, influenza, measles, SARS, Hantavirus and rabies.
Fungi: Major group of lower plants that lack chlorophyll and live on dead or other living organisms. Examples: mould,rust, mildew,smut,yeast and mushrooms.
Biohazard Classification: Conventional Agents
Recombinant DNA
Tissue Culture
Animal work
Anatomical Specimens
Unconventional Agents
What is Biosafety ? Biosafety is the application of safety precautions that reduce a laboratorians risk of exposure to a potentially infectious material and limit contamination of the work environment and ultimately the community (CDC).
Achieved through;
Administrative controls
Engineering controls
Personal protective equipment
Practices and procedures
Institutional Biosafety Committee (IBC): Under section 5 (1) of regulations
All organisations involved in research and development that deals with modern biotechnology shall establish an IBC.
IBC is a formal expert committee of an organisation undertaking modern biotechnology research and development which involves use of any LMO/rDNA materials.
IBCs are registered with the National Biosafety Board (NBB).
Its function is to monitor and ensure compliance to the biosafety act 2007 at the institutional level and safe handling of modern biotechnology activities.
IBC Members: Head of the organization or his designate as the chairperson.
Three or more scientists engaged in rDNA work or molecular biology with at least one outside expert in the relevant discipline.
A member with medical qualifications - Biosafety officer.
A nominee of DBT.
Cartagena Protocol: History: CBD opened for signature in 1992 and entered into force on 29 Dec 1993.
Cartagena Bio Safety Protocol (CBSP) negotiated from 1996-2000; entered into force in 11 Sept. 2003; over 170 Party Members; an international treaty.
This is a complementary agreement to the United Nations Convention on Biological Diversity (CBD).
Total parties to the cartagena protocol as of June 2021 are 173.
Objectives: The cartagena protocol on Biodiversity seeks to protect biodiversity from the potential risk
Biosaftey means the needs to protect human and animal health along with the environment from the possible adverse effects of the products of modern biotechnology. Biosafety defines the containment conditions under which infectious agents can be safely manipulated. Biosafety word is used to reduce and eliminate the potential risk regulating from the modern biotechnology and its products.
safety data sheet, an introduction to cell culture, safety equipment, safe laboratory practices, ascetic techniques, sterile work area, good personal hygiene, sterile reagents and media, sterile handling, planning of cell culture labs.
According to the Centre Of Disease Control and Prevention (CDC), Biosafety is the application of safety precautions that reduce a laboratorian’s risk of exposure to a potentially infectious material and limit contamination of the work environment and ultimately the community.
This presentation consists of topics related to oncogene, proto oncogene, Tumor suppresor gene, Ras gene family and structure and functions of tumor suppressor gene.
Stem cells are the cells which have the capability to differentiate into any cells of the body when provided with right stimulus and environment. This presentation teaches about stem cells, characteristics, types and cultivation of stem cells in artificial environment. Sample practice questions are also provided in the end to review the concept learned from this presentation.
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1. Animal cell science and Technology
7. Laboratory safety and Bio hazard
Shailendra singh Shera, Ph.D
2. 7. Laboratory safety and biohazard
Outline
1. Laboratory safety
2. General precautions
3. Risk associated with tissue culture laboratory
4. Safety Regulations
5. Biohazard
6. Bio-safety & Bio-safety level
7. Containment
8. Practice questions
3. Laboratory Safety
•Working safely to avoid personal as well as injury to other laboratory members working around you
is of utmost important.
•Every laboratory has their set of guidelines designed according to national and regulatory bodies
guidelines
•The most important element of safety in a cell culture laboratory is the strict adherence to standard
microbiological practices and techniques
Common safety risks in cell culture laboratory
•Electrical & Fire hazard
•Exposure to contaminating animal cells, viruses and pathogenic bacteria
•Exposure to toxic, corrosive, or mutagenic solvents and reagents.
•accidental punctures with syringe needles or other contaminated sharps
•inhalation exposures to infectious aerosols
4. Some of the general precautions for the safety of a tissue culture laboratory
•Strict adherence to recommendations of regulatory bodies.
•Periodical meetings and discussions of local safety committees.
•Regular monitoring of the laboratories.
•Periodical training of the personnel through seminars and workshops.
•Print and make the standard operating procedures (SOPs) available to all staff.
•Good record keeping.
•Limited access to the laboratory (only for the trained personnel and selected visitors).
•Appropriate waste disposal system for biohazards, radioactive wastes, toxins and corrosives.
5. Risk associated with tissue culture laboratory
Risk associated with tissue culture laboratory can be categorized in following category :
General Broken glass, Liquid nitrogen, cylinder , tubing
Burns Autoclaves, hot air ovens, hot plates
Fire Flaming particularly in association with alcohol
Radiological Radioisotopes in sterile cabinet, Irradiation of culture
Biological Importation of cell lines and biopsies, Genetic manipulation, Propagation of
viruses, position and maintenance of laminar flow hoods.
Safety Regulation
•Safety regulations are formulated by regulatory authorities to guide safe handling of equipments
and safety of personnel working in animal tissue culture laboratory.
•These guidelines are country specific and may not be applicable to all countries.
7. Biohazard
The accidents or the risks associated with the biological materials / agent of biological origin
capable of causing disease in humans are regarded as biohazards or biological hazards.
Examples:
•Pathogenic microorganisms,
• Toxins, allergens,
•Tissues samples
• Cultures with human pathogens
•Cells subjected to various genetic manipulations (genetically modified organisms),
•Developments of primary cultures,
•Cell lines.
Biohazard waste categories
•Solid Bio-hazrdous waste ( non-sharp)
•Solid collection, treatment & Disposal
•Liquid bio-hazrdous waste
•Sharps Symbol of Biohazard
8. Biohazards can be controlled to a large extent by strict adherence to the regulatory guidelines and
maintenance programmes. Some important aspects are listed.
•Microbiological safety cabinet or biohazard wood with pathogen trap filters have been developed.
•Vertical laminar-flow hood (instead of horizontal laminar-flow hood) reduces direct exposure of the
operator to the samples/processes.
•Pathogen containing samples are treated in separate rooms with separate facilities
•(centrifuge, incubator, cell counting etc.).
• Sterilization of all wastes, solid glassware etc. and their proper disposal.
•Facilities for change of clothing while entering and leaving the rooms.
•Strict adherence to the access of designated personnel to the culture rooms
Control of Biohazard
9. Disposal of biohazard waste
The disposal of biohazards should be as per the guidelines of local regulatory authority.
•Potentially bio-hazardous materials must be sterilized before disposal by autoclaving
•By immersion in a sterilizing agent such as hypochlorite
•Hypochlorite is often used at 300 ppm of available chlorine, but some authorities demand 2500
ppm (a 1:20 dilution of Chlorine).
10. Biosafety
Definition
•The discipline addressing the safe handling and containment of infectious microorganisms and
hazardous biological materials.
• Alternatively, it can also be defined as “The application of knowledge, techniques and equipment
to prevent personal, laboratory and environmental exposure to potentially infectious agents or
biohazards”.
•Biosafety defines the containment conditions under which infectious agents can be safely
manipulated.
•It describes the microbiological practices, safety equipment, and facility safeguards for the
corresponding level of
risk associated with handling a particular agent.
•Depending upon handling of risk associated with agents in tissue culture, there are four ascending
levels of containment, referred to as biosafety levels 1 through 4.
11. Containment principle
The action of keeping something harmful under control or within limits is called as containment.
Safety in the laboratory is achieved by application of layered, containment principles applied in
accordance with the risk assessment to prevent exposure of laboratory workers to a pathogen or
the inadvertent escape of a pathogen from the microbiological laboratory.
Safety layers in containment
Safety layers include primary and secondary containment.
Primary: Primary containment provides immediate protection to workers in the biological laboratory
from exposure to chemical and biological hazards.
•Primary barriers include biological safety cabinets, fume hoods and other engineering devices
used by laboratory technicians while working with a biological hazard.
Secondary Containment: Secondary containment is intended to protect the laboratory worker, the
community and the environment from unintended contamination with a biological hazard.
•Secondary containment consists of architectural and mechanical design elements of a facility that
prevent worker contamination and escape of pathogens from the laboratory into the environment.
12. Biosafety Levels
Biosafety Level 1 (BSL-1)
BSL-1 is the basic level of protection common to most research and clinical laboratories, and is appropriate for
agents that are not known to cause disease in normal, healthy humans. E.g. Saccharomyces cerevisiae, E.coli K-12
And non-infectious bacteria
Biosafety Level 2 (BSL-2)
BSL-2 is appropriate for moderate-risk agents known to cause human disease of varying severity by ingestion or
through percutaneous or mucous membrane exposure. Most cell culture labs should be at least BSL-2, but the
exact requirements depend upon the cell line used and the type of work conducted. E.g. Hepatitis A virus,
Salmonella, Streptoccoccus pyrogens.
Biosafety Level 3 (BSL-3)
BSL-3 is appropriate for indigenous or exotic agents with a known potential for aerosol transmission, and for
agents that may cause serious and potentially lethal infections. E.g. Hanta virus, Mycobacterium tuberculosis,
rabies virus, West Nile Virus, SARS, Corona Virus
Biosafety Level 4 (BSL-4)
BSL-4 is appropriate for exotic agents that pose a high individual risk of life-threatening disease by
infectious aerosols and for which no treatment is available. These agents are restricted to high
containment laboratories. E.g. Ebola virus, small pox virus
13. Practice questions : MCQs*
1. What are risk associated with tissue culture laboratory?
a. Fire Risk
b. Biological risk
c. Burns
d. All of the above
2. Containment is required to
a. Protect exposure of pathogenic microbes and virus to laboratory worker
b. To prevent advertent release of pathogen to environment
c. Both (a) and (b)
d. None of the above
3. Bio-safety describes the microbiological practices, safety equipment, and facility safeguards for the
corresponding level of risk associated with handling a particular agent. ( True / False)
4. Which of the following safety regulation describes the biological safety
a. CDC/OHS, 1999
b. CDC NIOS 2009
c. OSHA 2009
d. HSE-COSHH
* Questions adapted from : Practice and Learn Animal cell Science and Technology: Multiple choice question for
learning. Author: Shailendra Singh Shera . Publisher: Amazon Kindle.
14. References & Suggested Reading
1. https://www.chabsa.org/index.php?option=com_content&view=article&id=42&Itemid=218
2. https://biosafety.utk.edu/biosafety-program/biosafety-manual/2-principles-of-biosafety/
1. Watson, J.D., Gilman, M., Witowski J.and Zoller, M. Recombinant DNA, 2nd ed., Scientific
American Books, 1983
2. Glick, B.R. and Pasternack, J.J. Molecular Biotechnology, 3rd ed., ASM Press, 2003
3. Davis J.M. Basic Cell Culture: A Practical Approach, IRL Press, 1998
4. Freshney R.I. Animal Cell Culture a practical approach, 1987
Pratice MCQs Questions
1. Practice and Learn Animal cell Science and Technology: Multiple choice question for learning.
Author: Shailendra Singh Shera . Publisher: Amazon Kindle.
Further reading