Laboratory safety protocols, how to use biosafety cabinets, and what are the biosafety levels

1. BS MLT-408
Practical 1(a)
General safety rules
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2. COURSE CONTENT
Practical’s
1. General safety rules/BSCs/BSLs
2. Genomic DNA isolation using CTAB
3. Agarose gel electrophoresis.
4. Spectrophotometric quantification of nucleic acids.
5. Melting kinetics of DNA.
6. Extraction of proteins from various sources, estimation and separation by native
PAGE/SDS-PAGE.
7. RNA extraction, estimation and separation by denaturing agarose gel
electrophoresis.
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3. Instructions
• Practical copies
• Index on the first page of practical copies
• Lab coats must
• Quiz
• Viva
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4. Index pattern
Sr. No Experiment Name Date Sign
1 General safety rules 28-02-2024
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5. General safety rules
1. Upon entering the laboratory, place coats, books, and other
things in specified locations never on bench tops.
2. Keep doors and windows closed during the laboratory session
to prevent contamination from air currents.
3. At the beginning and termination of each laboratory session,
wipe bench tops with a disinfectant solution.
4. Do not place contaminated instruments, such as inoculating
loops, needles, and pipettes, on bench tops.
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6. 5. On completion of the laboratory session, place all cultures and
materials in the disposal area.
6. Avoid skin and eye contact with all chemicals
7. Do not taste or sniff the chemicals
8. A laboratory coat may be necessary while working in the
laboratory. Lab coats protect clothing from contamination or
accidental discoloration by staining solutions
9. Wash your hands with liquid detergent upon entering and prior
to leaving the laboratory.
10. You may be required to wear gloves while performing the lab
exercises.
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7. 11. Masks and safety goggles may be required to prevent materials
from coming in contact with your eyes.
12. Always use the appropriate safety equipment as determined by
your instructor.
13. Never apply cosmetics or insert contact lenses in the laboratory.
14. Do not smoke, eat, or drink in the laboratory. These activities
are absolutely prohibited
15. Report accidental cuts or burns to the instructor immediately.
16. Speak quietly and avoid unnecessary movement around the
laboratory to prevent distractions that may cause accidents
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8. Hazard Symbols
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9. 9

10. Activity
Other than signs mentioned above or discussed in class, draw 6
laboratory safety symbols and understand their meaning.
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11. Practical 1(b)
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12. Biosafety cabinets
Biosafety cabinets also known as biological safety cabinets (BSCs) are
an enclosed, ventilated hood or workspace that allow for the safe
handling of pathogens, contaminants or other potentially hazardous
materials.
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13. 13

14. Purpose of using
• To protect the operator and
• The surrounding environment from biological contaminants and other
hazardous materials.
Design of BSCs
• These cabinets are designed to provide various levels of protection.
There are various classes of biological safety cabinets, each defined by
the required level of biosafety and containment.
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15. Classes of biosafety cabinets
Class Ι: This older class of biosafety cabinet is rarely used.
➢ Provision of Protection
• The Class I biosafety cabinet provides personnel and environmental
protection, but no product protection.
➢ Design
• It has a HEPA filter in the exhaust system to provide containment and
environmental protection
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16. 16

17. Class ΙΙ:
➢ Provision of Protection
• The Class II biosafety cabinet provides protection to the user, the
experimental material and the environment.
➢ Design
• Air flow is drawn from the room around the operator into the front grille
of the cabinet, which provides personnel protection.
• In addition, the downward laminar flow of HEPA-filtered air provides
protection for experimental material inside the cabinet.
• Because cabinet air has passed through the exhaust HEPA filter, it is
contaminant-free, providing environmental protection.
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18. Sub types
• Class II Type A: HEPA filtered air is recirculated back into the
laboratory.
• Class II Type B: HEPA filtered air is ducted out of the building.
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19. 19

20. Class ΙΙΙ:
It is most suitable for work with bio-hazardous agents requiring high contain
(biosafety level 3 or 4).
➢ Provision of Protection
• It offers the highest degree of personnel and environmental protection from
infectious aerosols.
➢ Design
• The Class III cabinet is completely enclosed, HEPA filter-ventilated cabinet
fitted with glove ports and decontamination capabilities for entry and exit of
material
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21. 21

22. CLOSE VENTILATED STRUCTURES
OTHER THAN BSCs
Clean bench & Fume Hood: Both are not Biological Safety Cabinets.
➢ Fume Hood
• Fume hoods are ventilated enclosures that remove hazardous chemical
fumes and volatile vapors from the laboratory, providing personnel
protection only
• Hence should not be used for biohazard substances.
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23. 23
Fume Hood

24. ➢ Clean Bench
• Clean bench provides a space to work with a product or specimen where it
will be protected from contamination by particulates such as
microorganisms.
• This is accomplished by the laminar flow of clean air from a HEPA filter,
which is blown across the workspace and out toward the user and the lab.
• Thus, clean benches should not be used when working with potentially
infectious materials, chemical hazards or radioactivity.
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25. 25
Clean bench

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27. ACTIVITY
• What is another name for clean bench?
• Differentiate between Fume hood and Clean Bench
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28. Practical 1(c)
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29. Biosafety Levels
A biosafety level (BSL) is a set of bio-containment precautions required
to isolate dangerous biological agents in an enclosed laboratory facility.
Biosafety levels
Biosafety Levels 1-4 are combinations of laboratory practices and
techniques, safety equipment and facilities appropriate for the biohazard
posed by the infectious agents used in research and experiments.
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30. Biosafety level 1
• The lowest level applies to work with agents that usually pose a minimal
potential threat to laboratory workers and the environment and do not
consistently cause disease in healthy adults.
• An example of a microbe that is typically worked with at a BSL-1 is a
nonpathogenic strain of Escherichia. coli.
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31. Precautions of biosafety level 1
1) Normal laboratory personal protective equipment is generally worn,
consisting of eye protection, gloves and a lab coat or gown.
2) Standard microbiological practices also require attention to personal
hygiene, i.e., hand washing and a prohibition on eating, drinking or
smoking in the lab.
3) Research with these agents is generally performed on standard open
laboratory benches without the use of special containment equipment.
4) Labs are not usually isolated from the general building.
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32. 5) Standard microbiology practices are usually enough for protection such
as mechanical pipetting only (no mouth pipetting allowed), safe sharps
handling, avoidance of splashes or aerosols, and decontamination of all
work surfaces when work is complete.
6) Decontamination of spills is done immediately, and all potentially
infectious materials are decontaminated prior to disposal, generally by
autoclaving.
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33. Biosafety level 2
It deals with agents associated with human disease, in other words,
pathogenic or infectious organisms posing a moderate hazard. Example:
Dealing with HIV when performing routine diagnostic procedures or
work with clinical specimens
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34. Precautions of biosafety level 2
1) Appropriate personal protective equipment (PPE) must be worn,
including lab coats and gloves. Eye protection and face shields can also
be worn, as needed.
2) Laboratory personnel have specific training in handling pathogenic
agents.
3) Access to the laboratory is limited when work is being conducted and
the lab has self-closing, lockable doors.
4) Extreme precautions are taken with contaminated sharp items.
5) Class II biological safety cabinet is highly recommended for work
involving these agents
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35. Biosafety level 3
It is suitable for work with infectious agents which may cause serious or
potentially lethal diseases as a result of exposure by the inhalation route.
Example: Mycobacterium tuberculosis.
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36. Precautions of biosafety level 3
1) Solid-front wraparound gowns, scrub suits or coveralls are often
required.
2) BSL-3 laboratories are located in a unique high containment building
that also houses the BSL-4 laboratory that have a double door entry.
3) Exhaust air is not recirculated to other rooms.
4) Standard microbiological practices are the same as for BSL-1 and
BSL-2 laboratories.
5) Class ΙΙ biological safety cabinets are suitable in BSL-3 laboratories.
6) Additional personnel protective devices may be worn, such as
respirators.
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37. Biosafety level 4
It deals with extremely dangerous agents and pose a high risk of life
threatening disease.
Examples are the Ebola virus, Lassa virus, and any agent with unknown
risks of pathogenicity and transmission.
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38. Precautions of biosafety level 4
1) To the BSL 3 practices, we add requirements for complete clothing
change before entry, a shower on exit and decontamination of all materials
prior to leaving the facility.
2) Personnel must wear appropriate personal protective equipment from
prior BSL levels, as well as a full body, air-supplied, positive pressure
suit.
3) The BSL 4 laboratory contains a class III biological safety cabinet.
4) BSL 4 laboratories are in separate buildings or a totally isolated zone
with dedicated supply and exhaust ventilation.
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39. Activity
• Enlist two examples of organisms or biological agents that we can
study in each biosafety level.
• Which level of lab is suitable for studying of corona virus? Can it be
studied in BSL-II? If yes, then why?
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40. THANK YOU
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