This document provides information on laboratory safety practices. It discusses designating a safety officer, biosafety levels and practices, general safety rules and regulations, biosafety cabinets, and safe use of biosafety cabinets. Biosafety levels range from level 1 to level 4, with increasing containment practices required for higher levels due to more dangerous biological agents. Biosafety cabinets come in classes I-III and types A1, A2, B1, and B2, with each type providing different levels of protection for samples and/or personnel. Proper use of biosafety cabinets is important to maintain safe airflow.

1. LABORATORY
SAFETY

2. • Although it is the legal responsibility of hospital and laboratory
managers to provide for a safe working environment
• One person in the laboratory should be designated the safety
officer, whose duties are to see that safety standards and
guidelines are written and published, that employees are
informed of these standards through regularly scheduled
laboratory courses and in service briefings, and that a system
is in place to monitor compliance.

3. BIOSAFETY: Preventing lab-
acquired infections
• Bacteria
• Viruses
• Fungi
• Human blood, unfixed tissue
• Human cell lines

4. Why is Biosafety Important
• Laboratories recognize hazards of processing infectious agents
• Guidelines developed to protect workers in microbiological
and medical labs through engineering controls, management
policies, work practices

5. GENERALSAFETYRULESAND
REGULATIONS
• Whenever a person enters a laboratory he/she should remove
the footwear out of the laboratory.
• Dyes and chemicals are used in the laboratory due to which
our skin can get harmed as well as it can damage our clothes
so laboratory gown or apron should be used compulsory.
• Instruction given by the instruction should be noted and
followed carefully.

6. • Eating and drinking is not allowed in laboratory.
• Do not put anything in your mouth like pen, pencils, and
fingers etc.
• Hair should be packed with hair cap to avoid falling of hair on
face, working place as well as burning Bunsen burner

7. • Laboratory doors and windows should be kept closed during
experiments are going on.
• Extra material like bags ,cloths, purses and books should be
kept at proper places like drawers or shelf’s.

8. • The working area should be kept free from all these extra
material.
• Before starting your work on your work bench the bench top
should be disinfected by ethanol, phenol,Lysol or
cetylpyridinium chloride before and after each laboratory
work.
• Hands should be washed properly before and after each task.

9. • These above precaution may lessen chances of infection or
contamination due to cultures.
• Smoking is completely prohibited in microbial laboratory.
• Microbial cultures should be handled properly and should not
be taken out of laboratory.

10. • Mouth pipetting should be avoided and mechanical pipetting
devices should be used.
• All laboratory work should be carried out carefully to avoid
aerosol formation.
• Talking ,laughing should be avoided because it can result into
contamination.
• All contaminated material should placed into a proper
container.

11. • Culture plates, tubes should be discarded in basket.
• All microbial cultures should be either incubated or
refrigerated, Cultures should not be kept in drawers, cupboard
or desk.
• Pipettes, glass rods should be properly disinfected or
sterilized after each use.
• Before and after each use nichrome wireloop should be
sterilized and then used or kept.

12. • All laboratory instruments should be operated as per standard
operating procedure.
• Bunsen burner are very dangerous so the should be handled
with care and should be turned of when not in use.
• All electrical appliances such as oven, autoclave, centrifuge
and other such devices should be immediately turned off after
each use.

13. • All accidents, injuries or broken glassware should be reported
immediately.
• Alcohol should be kept away from flame.
• Waste paper, threads, broken glassware or used pH strips
should be discarded safely.
• The experiments that are permitted should only carried out.
• Keep the laboratory clean.

14. Biosafety Levels
• Precautions so people researching or trying to identify
organisms do not become infected
• While handling or testing clinical specimens, workers could
accidentally infect themselves or coworkers
• Labs must adhere to very specific safety regulations to work
with organisms that pose a threat to human health

15. Laboratories divided on basis of
nature of microbes
• Labs divided into 4 biosafety levels;
protective practices increase with each
• Biosafety Level 1 labs - work with least
dangerous agents, require fewest
precautions
• Biosafety Level 4 labs - have strictest
methods because dealing with agents that
are most dangerous to human health

16. Biosafety Level 1 Practices
• safety equipment and facilities appropriate for work with
defined and characterized strains of viable micro
organisms not known to cause disease in healthy adult
humans.
• The laboratory is not necessarily separated from the
general traffic patterns in the building.
• Work is generally conducted on open bench tops using
standard microbiological practices

17. • Special containment equipment or facility design is
neither required nor generally used.
• Laboratory personnel have specific training in the
procedures conducted in the laboratory and are
supervised by a scientist with general training in
microbiology or a related science.
• A biological safety cabinet is generally not required for
work involving these agents.
• Examples: Bacillus subtilis, Naegleria gruberi, Infectious
canine, hepatitis virus, E.coli

18. Biosafety Level 2 Practices
• safety equipment and facilities appropriate for work
done with a broad spectrum of indigenous moderate-risk
agents present in the community and associated with
human disease in varying severity. It differs from
biosafety level 1 in that:
a) laboratory personnel have specific training in handling
pathogenic agents and are directed by competent
scientists;
b) access to the laboratory is limited when work is being
conducted;

19. • c) extreme precautions are taken with contaminated
sharp items;
• d) certain procedures in which infectious aerosols or
splashes may be created are conducted in biological
safety cabinets or other physical containment
equipment.
• A Class I or Class II biological safety cabinet is highly
recommended for work involving these agents
• Examples : measles virus, salmonellae, Toxoplasma spp,
Hepatitis B Virus.

20. Biosafety Level 3 Practices
• safety equipment and facilities appropriate for work
done with indigenous or exotic agents with a potential
for respiratory transmission which may cause serious and
potentially lethal infection.
• More emphasis is placed on primary and secondary
barriers to protect personnel in the contagious area, the
community, and the environment from exposure to
potentially infectious aerosols.
• A Class I or Class II biological safety cabinet is required
for work involving these agents.
• examples: M. Tuberculosis, St. louis encephalitis virus,
Coxiella Burnetii, Bacillus anthracis (production level)

21. Biosafety Level 4 Practices
• safety equipment and facilities appropriate for work
done with dangerous and exotic agents which pose a
high risk of life threatening disease.
• May be transmitted via the aerosol route, and for which
there is no available vaccine or therapy. Members of the
laboratory staff have specific and thorough training in
handling extremely hazardous infectious agents and they
understand the primary and secondary containment
functions of the standard and special practices, the
containment equipment, and the laboratory design
characteristics

22. • . They are supervised by competent scientists who are
trained and experienced in working with these agents.
Access to the laboratory is strictly controlled by the
laboratory director.
• The facility is either in a separate building or in a
controlled area within a building, which is completely
isolated from all other areas of the building.
• A specific facility operations manual is prepared or
adopted.
• A Class III biological safety cabinet is required for work
involving these agents.
• Examples: Ebola zaire, Sin Nombre Virus, Rift Valley Fever

23. BIO SAFETY CABINETS
• The terms biological safety cabinet and biosafety cabinet
have been widely used to describe a variety of
containment devices equipped with HEPA filter(s),
designed to provide personnel or both personnel and
product protection from biohazardous materials.

24. Biosafety
cabinet
class
I
class
II
Class
3

25. class I Biological safety
cabinets
A. front opening
B. sash
C. exhaust HEPA
D. exhaust plenum

26. • A stream of inward air moving into the cabinet contains
aerosols generated during microbiological manipulations.
It then passes through a filtration system that traps all
airborne particles and contaminants. Finally, clean,
decontaminated air is exhausted from the cabinet.
• The filtration system usually consists of a pre-filter and a
HEPA (High Efficiency Particulate Air) filter.

27. • Although the Class I cabinet protects the operator and the
environment from exposure to biohazards, it does not prevent
samples being handled in the cabinet from coming into
contact with airborne contaminants that may be present in
room air.
• All Class I biological safety cabinets are suitable for work with
microbiological agents assigned to biosafety levels 1, 2 and 3.

28. class II Biological safety
cabinets
• 70% re circulated air; 30 % exhausted into
the room
• 75 fpm intake

29. • Like Class I safety cabinets, Class II cabinets have a stream of
inward air moving into the cabinet. This is known as the inflow
and it prevents the aerosol generated during microbiological
manipulations to escape through the front opening.
• However, unlike Class I cabinets, the inflow on Class II
cabinets flows through the front inlet grille, near the operator.
None of the unfiltered inflow air enters the work zone of the
cabinet, so the product inside the work zone is not
contaminated by the outside

30. • A feature unique to Class II cabinets is a vertical laminar
(unidirectional) HEPA-filtered air stream that descends
downward from the interior of the cabinet. This continuously
flushes the cabinet interior of airborne contaminants and
protects samples being handled within the cabinet from
contamination and is known as the downflow.

31. DIFFERENCEBETWEENVARIOUSCLASS2
BIOSAFETYCABINETS
• The differences between the various Class II cabinets available
lie primarily with the percentage of air exhausted to that of air
re-circulated from the common air plenum. In addition,
different Class II cabinets have different means of cabinet
exhaust. Some cabinets may exhaust air directly back to the
laboratory, while others may exhaust air through a dedicated
ductwork system to the external environment.

32. • Despite these differences, all Class II cabinets, like Class I
cabinets, protect both the operator and environment from
exposure to biohazards.
• In addition, Class II cabinets also protect product samples
from contamination during microbiological manipulations
within the cabinet interior and are all suitable for work with
agents assigned to biosafety levels 1, 2 and 3.

33. classII typeA (A1/A2) Biologicalsafety
cabinets
• 70% re circulated air; 30 % exhausted into
the room
• 75 fpm intake
Class II Type A1

34. Class II Type A2
•70% re circulated air; 30 % exhausted into
the room
• 100 fpm intake

35. • The Class II Type A biological safety cabinet is the most
common Class II cabinet. It is also the most common safety
cabinet of all the different types available. It has a common
plenum from which 30% of air is exhausted, and 70% re-
circulated to the work area as the downflow.
• Type A cabinets exhaust air directly back to the laboratory, and
they may contain positive pressure contaminated plenums.
When toxic chemicals must be employed as an adjunct to
microbiological processes, these cabinets should not be used.
Exhaust HEPA filtration only removes airborne aerosols
including biohazards, and not chemical fumes.

36. • The Class II Type A1 has the positively-pressurized
contaminated plenum bordering the ambient environment,
and therefore is less safe than the Class II Type A2 that has a
negative pressure surrounding the positively pressurized
contaminated plenum. In case there is a leakage on the
positive plenum, the leaking aerosol will be pulled by the
negative pressure back to the positive plenum, and it will not
leak out. Because of the safety issue, the Type A1 design is
now considered obsolete.

37. • In the A2 cabinet, about 70% of air from the positive plenum
is recirculated as downflow, and the remaining 30% is
discharged to the lab through the exhaust filter.

38. 38
class II typeB1 Biological safety cabinets
• Class II Type B1
• 40% re circulated air; 60 % exhausted
• 100 fpm intake

39. • Type B1 cabinets also have a dedicated exhaust feature that
eliminates re-circulation when work is performed towards the
back within the interior of the cabinet. Toxic chemicals
employed as an adjunct to microbiological processes should
only be employed if they do not interfere with work when re-
circulated in the downflow.

40. Class II BSC
• Class II Type B2
• 0% re circulated air; 100 % exhausted
• 100 fpm intake
40

41. • In the Class II Type B2 cabinet all inflow and downflow air is
exhausted after HEPA filtration to the external environment
without recirculation within the cabinet. Type B2 cabinets are
suitable for work with toxic chemicals employed as an adjunct
to microbiological processes under all circumstances since no
re-circulation occurs.

42. • In theory, Type B2 cabinets may be considered to be the safest
of all Class II biological safety cabinets since the total exhaust
feature acts as a fail-safe in the event that the downflow and /
or exhaust HEPA filtration systems cease to function normally.
However, Class II Type B2 cabinets are, in practice, difficult to
install, balance and maintain

43. 43
Class III BSC

44. • The Class III biological safety cabinet provides an absolute
level of safety, which cannot be attained with Class I and Class
II cabinets. All Class III cabinets are usually of welded metal
construction and are designed to be gas tight.
• Work is performed through glove ports in the front of the
cabinet. During routine operation, negative pressure relative
to the ambient environment is maintained within the cabinet.
This provides an additional fail-safe mechanism in case
physical containment is compromised.

45. • On all Class III cabinets, a supply of HEPA filtered air provides
product protection and prevents cross contamination of
samples. Exhaust air is usually HEPA filtered and incinerated.
Alternatively, double HEPA filtration with two filters in series
may be utilized.
• Materials are transferred into the cabinet using a pass-through
unit installed at the side of the work area. Class III cabinets
usually exhaust air back to the laboratory; however, air may
also be exhausted via a dedicated ductwork system to the
external environment. When a dedicated ductwork system is
employed, they are also suitable for work employing toxic
chemicals as an adjunct to microbiological processes.

46. • All Class III biological safety cabinets are suitable for work with
microbiological agents assigned to biosafety levels 1, 2, 3 and
4. They are frequently specified for work involving the most
lethal biological hazards.

47. HEPA & ULPA Filter
• The HEPA filter is the heart of the biological safety cabinet.
The HEPA filter is a disposable dry-type filter, constructed of
borosilicate microfibers cast into a thin sheet, much like a
piece of paper.
• • HEPA: high efficiency Particulate air: Modern “American-
convention” HEPA: 99.99% at 0.3 microns
• • ULPA: Ultra Low Penetration air: Modern “American-
convention” ULPA: 99.999% at 0.12 microns

48. • HEPA/ ULPA filters are designed to remove a broad range of
airborne contaminants, including:
• • Fine dust
• • Smoke
• • Bacteria (typical size: 500 to 0.3 microns)
• • Soot
• • Pollen
• • Radioactive particles

49. Safe use of biosafety cabinet in
laboratory
• 1. Know Your Airflow
• Maximize potential protection by making sure you know how
filtered and contaminated air, from both work surface and room,
passes through your cabinet.
• 2. Work at the Proper Sash Level
• Your BSC will be certified for safe operation at a certain window sash
height – most cabinets will indicate where the window should be
placed during work activities. It is crucial to work at the certified
window sash height to ensure that the balance between inflow and
down flow velocities is maintained. Excessive inflow can cause
contaminated air to enter the work area; excessive down flow can
cause contaminated air to enter the laboratory as well as creating
turbulent air in the work area, resulting in product contamination.

50. • 3. Never Block the Airflow Grill
• The airflow grill at the front of your cabinet performs the important
job of separating the clean air inside the cabinet from the
contaminated laboratory air outside. Take care to avoid covering the
air grill at the front of the cabinet with equipment, and even with
your arms, elbows or hands, as this can compromise airflow
integrity, resulting in contaminants entering the sterile work zone
inside the cabinet, or substances from inside the cabinet leaking out
into the laboratory.
• 4. Minimize Movement
• The best practice for working in your BSC is to move your arms in
and out slowly and perpendicular to the face of the opening of the
cabinet to minimize the disruption of airflow within the cabinet. It is
also important to organize the cabinet workspace so that minimal
arm movement is required; and make sure to take care when
walking near the cabinet or opening and closing doors – rapid
movement near the BSC may disrupt the cabinet air barrier.

51. • 5. Reduce Splatter
• Use good microbiological techniques to reduce the potential risk of
creating splatter or aerosol generation. Class II cabinets are designed so
that horizontally nebulized spores will be captured by the downward
flowing cabinet air within 14 inches of travel. As a general rule of
thumb, clean materials should be kept at least 12 inches away from
aerosol generating activities to prevent cross-contamination.
• 6. Know Your Work Area
• The middle third of the BSC work surface is the ideal area to be used for
your experiments and all operations should be performed at least 4
inches from the front grill. Materials placed in the cabinet can cause
disruption in airflow, resulting in turbulence, possible cross-
contamination, and/or breach of containment. It is therefore important
that only the materials required for immediate work are placed in the
cabinet work area, and that these are placed as far back as possible, but
still within reach. Bulky items such as biohazard bags should be placed
to one side in the interior of the cabinet, while aerosol-generating
equipment such as vortex mixers or centrifuges should be placed toward
the rear of the cabinet.

52. • 7. Work from Clean to Contaminated
• Active work should flow from a clean to a contaminated area
across the work surface to limit the movement of
contaminated equipment over clean items.
• 8. Working with Tubes & Bottles
• Open tubes and bottles should not be held in a vertical
position and their caps should not be placed on toweling. Caps
should be placed back on the tube or bottle as soon as
possible.

53. • 9. Working with Petri Dishes
• Then working with Petri dishes, the lid should be held above
the open sterile surface to minimize the direct impact of
downward air.
• 10. Working with Aspirator Bottles or Suction Flasks
• spirator bottles or suction flasks should be connected to an
overflow collection flask containing appropriate disinfectant
and an in-line HEPA or equivalent filter.

54. • 11. Dress for the Occasion
• Proper dress is also essential for your personal protection as well as
your product and working environment. Laboratory coats should be
worn buttoned over street clothing, protective eyewear should be
on at all times and latex or nitrile gloves are necessary when
handling culture, contaminated surfaces, or equipment.
• 12. Make Your Setup Ergonomic
• Optimize your setup to improve your safety. For example, use a good
laboratory chair and sit with 90-degree seating and knee angles to
reduce your risk of workplace injury. Also make sure to work with
straight wrists and arrange your work area properly so you do not
overextend your arms – accessories such as armrests and footrests
can be used to increase comfort and support.