1. LABORATORY ORGANIZATION AND
BIOLOGICAL SAFETY REQUIREMENTS OF
MICROBIOLOGY LABORATORY
By, Nissim Ghimire

2. WHY LAB SAFETY IS IMPORTANT?

4. WHAT IS BIOSAFETY?
CDC Definition:
Biosafetyisthe applicationof safety precautions that reduce a
laboratorian’sriskof exposureto apotentially infectiousmaterial and
limit contamination of the work environmentandultimately the
community

6. CLASSIFICATION OF INFECTIVE
MICROORGANISMS BY RISK GROUP
Risk Group 4 (high individual and community risk) :
A pathogen that usually causes serious human or animal disease / can be readily
transmitted from one individual to another, directly or indirectly/ Effective treatment
and preventive measures are not usually available.
Risk Group 1 (no or low individual and community risk):
A microorganism that is unlikely to cause human or animal disease.
Risk Group 2 (moderate individual risk, low community risk):
a pathogen that can cause human or animal disease/ unlikely to be a serious hazard to
laboratory workers, the community, livestock or the environment/ Laboratory exposures
may cause serious infection/ availability of effective treatment and preventive measures /
limited risk of spread of infection
Risk Group 3 (high individual risk, low community risk) :
A pathogen that usually causes serious human or animal disease but does not ordinarily
spread from one infected individual to another. Effective treatment and preventive
measures are available.

7. EXAMPLES
Risk Group 1 Risk Group 2 Risk Group 3 Risk Group 4
Examples E. coli K-
12, S. cerevisiae(ye
ast), Lactobacillus,
B. subtilis
Streptococcus,
Herpes virus,
Staphylococcus
Yersinia pestis (
black plague),
Hepatitis virus,
Mycobacterium
Ebola virus,
Marburg virus,
Lassa virus
Description they do not cause
disease in healthy
adult humans.
they can cause
disease in
humans, but
the disease is
treatable or
preventable.
They cause
serious disease
in humans.
Treatments and
vaccines for
these diseases
may exist.
cause deadly
disease in humans,
and they can easily
travel from one
person to another.
No treatments or
vaccines exist for
these diseases.

8. Laboratory facilities are designated as
• basic – Biosafety Level 1,
• basic – Biosafety Level 2,
• containment – Biosafety Level 3, and
• maximum containment – Biosafety Level 4.
Biosafety level designations are based on a composite of the design features,
construction, containment facilities, equipment, practices and operational
procedures required for working with agents from the various risk groups.

9. RELATION OF RISK GROUPS TO
BIOSAFETY LEVELS, PRACTICES AND
EQUIPMENT
Risk
group
Biosafety level Laboratory type Laboratory
practices
Safety equipments
1 Basic Biosafety
level-1
Basic teaching,
research
GMT None: Open bench
work
2 Basic Biosafety
level-2
Primary health
services, diagnostic
services, research
GMT plus protective
clothing, biohazard
sign
Open bench plus BSC
Biosafety; for potential
aerosols
3 Containment-
Biosafety level-3
Special diagnostic
services, research
As Level 2 plus
special clothing,
controlled access,
directional airflow
BSC and/or other
primary devices for all
activities
4 Maximum
Containment-
Biosafety level-4
Dangerous pathogen
units
As level 3 plus
airlock entry, shower
exit, special waste
disposal
Plus III BSC, or
positive pressure suits
in conjunction with
BSC II, double ended
autoclave, filtered air

10. Countries (regions) should draw up a national (regional) classification of
microorganisms, by risk group, taking into account:
1. Pathogenicity of the organism.
2. Mode of transmission and host range of the organism. These may be
influenced by existing levels of immunity in the local population, density
and movement of the host population, presence of appropriate vectors, and
standards of environmental hygiene.
3. Local availability of effective preventive measures. These may include:
prophylaxis by immunization or administration of antisera (passive
immunization); sanitary measures, e.g. food and water hygiene; control of
animal reservoirs or arthropod vectors.
4. Local availability of effective treatment. This includes passive
immunization, postexposure vaccination and use of antimicrobials,
antivirals and chemotherapeutic agents, and should take into consideration
the possibility of the emergence of drug-resistant strains.

11. Biosafety levels
1 2 3 4
Isolation of laboratory No No Yes Yes
Room sealable for decontamination No No Yes Yes
Ventilation:
Inward air flow
Controlled ventilating system
HEPA- filtered air exhaust
No Desirable
Yes Yes
No Desirable Yes Yes
No No Yes/No Yes
Double-door entry No No Yes Yes
Airlock No No Yes Yes
Airlock with shower No No Yes Yes
Anteroom No No Yes Yes
Anteroom with shower No No Yes/No Yes
Effluent treatment No No Yes/No Yes
Biological safety cabinets No Desirable Yes Yes
Personnel safety monitoring capability No No Desirable Yes
Autoclave:
•On site
•In laboratory room
Double- ended
No Desirable Yes Yes
No No Desirable Yes
No No Desirable Yes
Summary Of Biosafety Level
Requirements

13. LABORATORY DESIGN AND FACILITIES
FOR BSL 1 & 2.
1. Formation of aerosols
2. Work with large volumes and/or high concentrations of microorganisms
3. Overcrowding and too much equipment
4. Infestation with rodents and arthropods
5. Unauthorized entrance
6. Workflow: use of specific samples and reagents.

14. BIOSAFETY LEVEL 1 (BSL-1)
• Is suitable for work involving agents of no known or
minimal potential hazard to laboratory personnel and the
environment. The laboratory may be integral to general traffic
patterns in the building.
• Work may be conducted on open bench tops.
• Special containment equipment is neither required nor
generally used.
• Laboratory personnel shall have specific training in
procedures conducted in the laboratory.

15. A typical Biosafety Level 1 Laboratory

16. BIOSAFETY LEVEL 2 BSL 2
Biosafety Level 2 is similar to Level 1 and is suitable for work involving agents
of moderate potential hazard to personnel and the environment.
It differs in that
o laboratory personnel are specifically trained to handle pathogenic agents
and are directed by scientists who are experienced in working with these
agents,
o access to the laboratory is limited when work is being conducted,
o extreme precautions are taken with contaminated sharp items, and
o certain procedures that may result in the creation of infectious aerosols or
splashes are conducted in biological safety cabinets or other physical
containment equipment.

17. A typical Biosafety Level 2 Laboratory

18. A typical Biosafety Level 2 Laboratory

19. BIOSAFETY LEVEL 3
•Applicable to clinical, diagnostic, teaching, research, or production facilities in which
work is done with indigenous or exotic agents which may cause serious or potentially lethal
disease as a result of exposure by the inhalation route.
• Laboratory personnel have specific training in handling pathogenic and potentially lethal
agents, and are supervised by competent scientists who are experienced in working with
these agents.
•All procedures involving the manipulation of infectious materials are conducted within
biological safety cabinet.

20. The containment laboratory – Biosafety Level 3 is designed and provided for work
with Risk Group 3 microorganisms and with large volumes or high concentrations of
Risk Group 2 microorganisms that pose an increased risk of aerosol spread.
A typical Biosafety Level 3 Laboratory

21. It is recognized, however, that some existing facilities may not have all the facility features
recommended for Biosafety Level 3 (i.e., double-door access zone and sealed penetrations).
In this circumstance, an acceptable level of safety for the conduct of routine procedures,
(e.g., diagnostic procedures involving the propagation of an agent for identification, typing,
susceptibility testing, etc.), may be achieved in a Biosafety Level 2 facility, providing
1) the exhaust air from the laboratory room is discharged to the outdoors,
2) the ventilation to the laboratory is balanced to provide directional airflow into the room,
3) access to the laboratory is restricted when work is in progress, and
4) the recommended Standard Microbiological Practices, Special Practices, and Safety
Equipment for Biosafety Level 3 are rigorously followed.

22. 1. The international biohazard warning symbol
and sign must be displayed on the doors of the
rooms where microorganisms of Risk Group
2 or higher risk groups are handled.
2. Only authorized persons should be allowed to
enter the laboratory working areas.
3. Laboratory doors should be kept closed.
4. Children should not be authorized or allowed
to enter laboratory working areas.
5. Access to animal houses should be specially
authorized.
6. No animals should be admitted other than
those involved in the work of the laboratory.
CODE OF PRACTICE

23. • is required for work with dangerous and exotic agents that pose a high individual
risk of aerosol-transmitted laboratory infections and life-threatening disease.
•Agents with a close or identical antigenic relationship to Biosafety Level 4 agents
are handled at this level until sufficient data are obtained either to confirm
continued work at this level, or to work with them at a lower level.
• 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. 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.
BIOSAFETY LEVEL 4
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.

24. Scientist
working on
EBOLA virus in
Bsl 4 laboratory
Within work areas of the facility, all activities are confined to Class III biological
safety cabinets, or Class II biological safety cabinets used with one-piece positive
pressure personnel suits ventilated by a life support system. The Biosafety Level 4
laboratory has special engineering and design features to prevent microorganisms
from being disseminated into the environment.

25. Bsl 4 laboratory

26. BIOLOGICAL SAFETY CABINETS- BSC
In varying degrees, a laminar flow biological safety cabinet is designed to provide
three basic types of protection:
•Personnel protection from harmful agents inside the cabinet.
•Product protection to avoid contamination of the work, experiment, or
process.
A biosafety
cabinet (BSC)—also
called a biological
safety cabinet or
microbiological
safety cabinet—is an
enclosed, ventilated
laboratory workspace
for safely working with
materials contaminated
with (or potentially
contaminated with)
pathogens requiring a
defined biosafety level.

28. • BSCsrequire consistent
useof
– good
microbiological
practices
– primary
containment
equipment
– primary
containment facility
design
Tobeprecise“BSCs
are designed to
provide personnel,
environmental and
product
protection when
appropriate
practices and
procedures are
followed”

29. 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
bio hazardous materials. The terms should only be applied
to those devices that meet the requirements of Class I, II, or
III specifications, based on their construction, airflow
velocities and patterns, and their exhaust systems.
Therefore,

30. HEPA FILTER
• HEPA–Highefficiency particulate air filter
• It removesthe most penetrating particle size(MPPS) of 0.3 μ m with an
efficiency of at least99.97 %
• The typical HEPA filter isa singlesheetof borosilicate fibers treated with
a wet-strength water-repellant binder
• Thefilter medium ispleated to increasetheoverall surfacearea,with
pleatsbeingseparatedby corrugated aluminumtubes
• Thisseparationismainly to preventcollapse
• It removesparticulate matter bythree mechanisms interception,
impaction,diffusion
• Thefiltering efficiency dependsupon fiber diameter, filter thicknessand
facevelocity
• Thesefilters arefitted either in the exhaustor air supplysystemto remove
particulatematter

32. NOTE:
laminar flow clean benches are NOT biosafety cabinets.
•They discharge HEPA-filtered air across the work surface and toward the
user.
•These devices only provide product protection.
•They can be used for certain clean activities, such as the dust-free assembly of
sterile equipment or electronic devices.
•Should never be used when handling cell culture materials or drug
formulations, or when manipulating potentially infectious materials.
•Workers can be exposed to materials being manipulated on the clean bench.
DO NOT GET CONFUSED BETWEEN BSCs AND LAMINAR AIRFLOW CABINET
laminar flow clean air benches should never
be used as a substitute for a biological safety
cabinet in research, biomedical or veterinary
laboratories and/or applications.

33. CLASSIFICATION:
BSCs are classified into 3 classes and these classes and types of BSCs within them are
distinguished in two ways:
• The level of personnel and environmental protection provided and
• The level of product protection provided
BSCs
Class I
Class II
A1
A2
B1
B2
Class III

34. BSC-CLASS I; Personnel and Environmental Protection
Only
•does not protect the product from
contamination : dirty room air constantly
enters the cabinet front to flow across the
work surface.
•As a partial containment unit, it is suitable
for work involving low to moderate risk
agents (biosafety levels 1,2 and 3): there is a
need for containment, but not for product
protection.
• the HEPA filter in the Class I cabinet
protects the environment by filtering air
before it is exhausted.
•Personnel protection is made possible by
constant movement of air into the cabinet and
away from the user.

35. PRINCIPLE
A: Front opening
B: Sash
C : Exhaust HEPA filter
D: Exhaust plenum
NOTE:
THE cabinet needs to be
hard connected to the
building exhaust system if
toxic vapors are to be used

36. BSC Class II: Product, Personnel and Environmental Protection
The Class II biological safety cabinet has three key features:
•A front access opening with carefully maintained inward airflow.
•HEPA-filtered, vertical, unidirectional airflow within the work area.
•HEPA-filtered exhaust air to the room or exhaust to a facility exhaust system.
Vertical, unidirectional airflow and a front access opening are common to most Class
II cabinets. But, because Class II designs permit different airflow patterns, velocities,
HEPA air filter position, ventilation rates and exhaust methods, a sub-classification of
Type is needed to differentiate Class II BSC designs.
Type A2 cabinets recirculate 70%
Type B1 recirculate less than 50%
Type B2 cabinets recirculate 0% (total exhaust)
The differences between the various Class II cabinets available lie primarily with the
percentage of air exhausted and 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.

37. Class II Type A (A1/A2) Biological Safety Cabinets
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.
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.
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. CLASSIIA1

39. CLASSIIA2

41. CLASS II TYPE B- BSC
The main difference between Type A and Type B cabinet is:
•Type B cabinets must be operated with an external blower and it exhausts air to the
external environment via a dedicated ductwork system.
•Without the external blower, the cabinets internal blower will blow the air (and
microbiological agents) inside the work zone through the front opening, towards the
operators face, creating a dangerous situation.
•This cabinet is not self-balancing, in the sense that its own blower can only create
downflow, and the cabinet relies on the external blower to create inflow.

42. CLASS II TYPE B1 BIOLOGICAL SAFETY
CABINETS
The Class II Type B1 biological safety cabinet was originally specified by the American
National Cancer Institute. It has a common plenum from which 70% of air is exhausted,
and 30% re-circulated to the work area as the downflow.
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.

43. CLASSIIB1

44. 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.
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.
Class II Type B2 cabinets are, in practice but difficult to install, balance and maintain.
CLASS II TYPE B2 BIOLOGICAL SAFETY CABINETS

45. CLASSIIB2

46. CLASS III BIOLOGICAL SAFETY CABINETS
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.

47. 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.

48. Figure 8. The Class III BSC
(A) glove ports with O-ring for attaching arm-lengthgloves to cabinet;
(B) sash; (C) exhaust HEPA filter;
(D) supply HEPA filter; (E) double-ended autoclave or pass-through box.
Note: A chemical dunk tank may be installed which would be located beneath the work surface of the BSC
with access from above. The cabinet exhaust needs to be hard connected to an exhaust system where the
fanis generally separate from the exhaust fans of the facility ventilation system. The exhaust air must be
double HEPA-filtered

49. Class III BSC

51. SUMMARY
BSC
class
Inflow
velocity
Air flow pattern applications
Nonvolatile Toxic
Chemicals and
Radionuclides
Volatile Toxic
Chemicals and
Radionuclid es
I 75 lf/m In at front through
HEPAto the outside or
into the room through
HEPA
yes When exhausted
out doors
IIA1 75 lf/m 70% recirculated to the
cabinet work area
through HEPA; 30%
balance can be
exhausted through
HEPAback into the
room or to outside
through a canopy unit
YES NO

52. BSC
class
Inflow
velocity
Air flow pattern applications
Nonvolatile
Toxic
Chemicals and
Radionuclides
Volatile Toxic
Chemicals and
Radionuclid es
II,A2 100 lfm Similar to II,A1, but has 100
lfm intake air velocity and
plenums are under negative
pressure to room; exhaust
air can be ducted to the
outside through a canopy
unit
YES When exhausted
outdoors
(Formerly
“B3”) (minute
amounts)
II,B1 100 lfm 30% re circulated, 70%
exhausted. Exhaust cabinet
air must pass through a
dedicated duct to the outside
through a HEPAfilter
YES YES

53. BSC
class
Inflow
velocity
Air flow pattern applications
Nonvolatile
Toxic
Chemicals and
Radionuclides
Volatile Toxic
Chemicals and
Radionuclid es
II,B2 100 No recirculation; total
exhaust to the outside
through a HEPAfilter
YES YES
III Not applicable Supply air is HEPA filtered.
Exhaust air passes through
two HEPAfilters in series
and is exhausted to the
outside via a hard
connection
YES YES

54. PLACEMENT OF BSC
30 cm to 1 foot space between its sides and the wall or any large
obstruction
2m to 7 feet distance between its front aperture and the wall or any large
obstruction
1.5m to 5 feet of space when its against laboratory furniture or a bench
3 m to 10 feet space when its against lab equipments or another BSC
 1.5m to 6 feet away from opening doors
1m to 3 feet away from any foot traffic

55. A typical layout for working “clean to dirty” within a Class II BSC. Clean cultures
(left) can be inoculated (center); contaminated pipettes can be discarded in the
shallow pan and other contaminated materials can be placed in the biohazard
bag (right). This arrangement is reversed for left- handed persons.

56. Workpracticesandprocedures
• Checklistof materialsandworkactivity
protocol
• Slow andun-intersectedArmmovement
• Minimum persons
• Labcoatsbuttoned fully
• ProperStoolheight • Dailycheckof airflow byairflow
indicatorand monthly or weeklywith
an anemometer
• Idealair flow –0.7to 1m/s
• All proceduresshouldbedoneatleast four inchesin from the front
grille
• Onlythe materialsneededfor work shouldbe keptinside
• Waitforminimumof fourminutesto switch off theblowersafterthe
workisover

57. DECONTAMINATION
• Disinfectantselection : EPA registration number in the label andlist of
infectious agentsthat the disinfectant iseffective
• BSC–ethanol not usedasdecontaminant asit evaporates– no proper
contact time – ethanol canbeusedasa rinsing agent
• Formaldehyde vapour sterilisation to bedoneto kill spores

58. DISINFECTION METHODA
• Cabinets with an internal electricpower
supply
• Place25 ml formalin(cabinet with internal
volume of 0.38cu.m) toavaporizer, or into a
beaker on ahotplate
• Closethe cabinet and ensure that theexhause
blow back valve isclosed
• Boil awayformalin

59. DISINFECTION METHODB
• 35ml formalin in a100ml beakerinside the
cabinet add 10gpotassiumpermanganate
sealthe cabinet
• Leavethe cabinet at least 5 hours ,preferably
overnight and label DANGER–FUMIGATION IN
PROGRESS
• Opennext dayand work after 30minfor
residual formaldehyde to exhaust