BIOSAFETY

Presenter: Dr. Serra Saji Moses

Moderator: Dr. Thomas. S. Kuruvilla
Overview

1. Introduction
2. Risk groups
3. Biosafety levels
4. Biosafety cabinets
5. General safety measures
6. References
Introduction

Defined as the application of safety precautions that:

• reduce a laboratorian’s risk of exposure to a

potentially infectious material

• limit contamination of the work environment

and ultimately the community
Need for biosafety:

1. Lab has hazards of processing infectious agents

2. Accidental threat to workers and environment

3. To have adherence with safety regulations while

dealing with highly infectious agents
Classification of microorganisms by risk group

Based on the following factors:

– Pathogenicity of the organism

– Mode of transmission and host range of the organism
– Local availability of effective preventive measures
– Local availability of effective treatment
WHO risk groups

Risk Group 1 (no or low individual and community risk)

Unlikely to cause human or animal disease

 Non pathogenic strain of E.coli

 Bacillus subtilis
 Canine hepatitis causing viruses
 Adeno-associated virus (AAV) types 1-4
 Naegleria gruberi
Risk Group 2 (moderate individual & low community risk)
• Can cause human or animal disease
• Effective treatment and preventive measures are available
• Limited risk of spread of infection
Herpes viruses Staphylococcus aureus Candida
Enterovirus Enterobacteriaceae
Adenoviruses Chlamydiae
HBV, HCV Borreliae
HIV MAC
Influenza A virus
Measles virus
Mumps virus
Slow viruses
Risk Group 3 (high individual risk, low community risk)
• Cause serious human or animal disease
• Effective treatment and preventive measures are available
HEV M. tuberculosis Blastomyces Echinococcus

Hantaviruses NTM Histoplasma Leishmania

JEV Yersinia pestis Coccidioides Naegleria fowleri

immitis
Rift valley fever virus Rickettesiaceae Paracoccidioides Plasmodium

Yellow fever virus Francisella Penicillium Tenia solium

tularensis marneffei
West nile fever virus Bacillus anthracis Trypanosoma

Rabies virus Brucella

Dengue virus Burkholderia
Risk Group 4 (high individual and community risk)
• Cause serious human or animal disease
• No effective treatment and preventive measures
• Can be readily transmitted from one individual to another,
directly or indirectly
Arenaviruses (Lassa, Junin, Machupo)

Filoviruses (Ebola & Marbug)

Smallpox virus

Crimean-Congo haemorrhagic fever virus

Nipah virus

Russian spring-summer encephalitis virus

Kyasanur forest disease virus

SARS CoV 2 virus

CDC Biosafety levels

High risk microbes

BSL 4

BSL 3

BSL 2

Low risk microbes BSL 1

Biological safety cabinet (BSC)

• An engineering control intended to protect laboratory

workers, laboratory environment and work materials from
exposure to infectious or biohazardous aerosols and splashes,
generated while manipulating materials containing infectious
agents
• Most effective & most commonly used primary
containment device when working with infectious agents

• Negative pressure

• 3 types: Classes I, II, and III

HEPA filter (High efficiency particulate air filter)
• A single sheet of borosilicate fibers treated with a wet-
strength water-repellent binder
• Pleated to increase the overall surface area, with pleats
being separated by corrugated aluminum tubes to prevent
collapse
• The filtering efficiency depends upon fiber diameter,
filter thickness and face velocity
• Fitted either in the exhaust or air supply system to
remove the most penetrating particle size (MPPS) of 0.3
μm with an efficiency of at least 99.97 %
• By 3 mechanisms: interception, impaction, diffusion
Class I Biological safety cabinet

• 1st designed and simple Biological Safety Cabinet (BSC)

• Provides personnel and environmental protection but not

product production as unsterilized room air is drawn over
the work surface

Requirements
• Open fronted with glass in upper portion
• HEPA filter
• Hard ducted
• Inward airflow with a minimum velocity of 75 LFM
Working mechanism
Unfiltered room air is drawn in  cabinet air enters the
exhaust plenum  drawn through HEPA filter
Purpose

• To enclose equipments

• For procedures with potential to generate aerosols

(BSL 2,3)
Class II Biological Safety Cabinet

• Vertical laminar flow BSCs

• Provides personnel, environmental and product protection

as HEPA filtered room air is drawn over the work surface

• 2 types: A & B; based on

1. Inlet flow velocity
2. Percent of air that is HEPA filtered and recirculated
Class IIA cabinet

• 70% of the air is recirculated

• 30% of the air exhausted through HEPA
• Not hard ducted
Type A1
Type A2
 Type A1 Type A2
Inward airflow 75 LFM 100 LFM
velocity

Contaminated air Positive pressure Negative pressure

Purpose • Biological agents without • Biological agents
volatile toxic chemicals and treated with trace
volatile radionuclides quantities of
radionuclides
• Not for sterile hazardous
pharmacy compounding • Minute quantities of
hazardous chemicals
Class IIB cabinet

• Designed and originated with the National cancer

institute type 212 ( later called Type B)
• Hard ducted
• Inflow velocity of 100 fpm
• Selected for radioisotopes, toxic chemicals, or carcinogens
Class II Type B1 BSC

• 30% of the air is recirculated

• 70% of the air exhausted through HEPA
• 1 extra HEPA filter for the recirculated air to pass
through after getting contaminated
Class II Type B2 BSC

• 100% Exhaust or Total Exhaust cabinets

• Filtered downflow air drawn from the lab or the outside

air (not recirculated from the cabinet exhaust)  filtration
through a HEPA filter  exhaust all inflow and downflow
air to the atmosphere without recirculation in the cabinet or
return to the lab
• Most hospital clinical microbiology laboratory scientists
use class IIA cabinets

• There is a limited need for Type B BSCs; only for

research

• Also, Type B BSCs require very specific installation and

operating conditions to function correctly
 exhaust 1200 cubic feet/min of room air
 expensive cabinet
 high cost of heavier gauge and higher capacity exhaust fan

• BSL 2,3,4
Class III Biological Safety Cabinet

• No exhaust through the general lab exhaust

• Totally enclosed
• Tested under pressure to ensure that no particles can leak
from it into the room
• BSL3,4
Requirements
• Non opening view window
• Passage of materials through a dunk tank
• Double door pass through box with autoclave
• Long heavy duty rubber gloves attached in a gas tight
manner to port in the cabinets
• Inward airflow velocity 75-100 lf/m

Working mechanism
HEPA-filtered supply air  into the cabinet 
exhausted air is discharged to atmosphere through
another HEPA filter
Precautions while working with BSCs

• Only the materials needed for work should be kept inside

• Maintain an open area of 3 feet from the cabinet during

operation of the air-circulating system  ensures that
infectious material is directed through the HEPA filter

• Access opening  8 inches

• Wait for minimum of 5 minutes to switch off the blowers

after the work is over
Quality Control of BSCs

 Certification:
• By a qualified technician:
• Initially
• Whenever moved more than 18 inches
• Annually thereafter
Airflow check: daily airflow indicator
monthly/ weekly  anemometer
 Recertification:
• By facility manager
• Annually
 HEPA filter usage and replacement:
• By facility manager

• Need for replacement of the

HEPA filter based on:
1. Downflow blower speed (if ≥90%)
2. Other indicators (e.g., age – 8 to 10 years)
3. HEPA filter leak test/ integrity test/ DOP test

• Once the HEPA filter is replaced, the BSC is recertified

and the certification is annotated by a Performance
Certification sticker
Decontamination

• Cleaned and disinfected at least quarterly

• Ethanol not used:
 Evaporates
 No proper contact time
Can be used as a rinsing agent

• Use of Ultraviolet lamps is not advisable

• Formaldehyde vapour sterilisation: to kill spores

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A) For cabinets with an external electric power supply:

25 ml formalin added to a vaporizer/ into a beaker on

a hotplate (cabinet with internal volume of 0.38cu.m)

Close the cabinet and ensure that the exhaust blow

back valve is closed

Boil away formalin

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 B)

35ml formalin in a 100ml beaker inside the cabinet +

10g potassium permanganate

Seal the cabinet

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• Leave the cabinet at least 5 hours, preferably overnight
and label DANGER - FUMIGATION IN PROGRESS

• Open next day and work after 30 min for residual

formaldehyde to exhaust
Relation of risk groups to biosafety levels,
practices and equipment

RG1  BSL1
• Easily cleanable lab with washing facilities
• Forbid activities that can lead to infection
• PPE: eye protection, gloves and a lab coat or gown
(standard PPE)
• Hands and benches disinfected after work
• Proper waste disposal

 Teaching purpose
RG2  BSL2
1+
• Negative pressure atmosphere
• Access limited to lab staff & other specified personnel
• Standard PPE + face shields as needed
• Autoclave

 BSC I/ II
 Teaching + diagnostic purpose
RG3  BSL3

1, 2 +
• In an area away from general circulation, sealable for
disinfection
• Continuous inflow of air, exhausted via HEPA
• Biohazard sign at entry
• Standard PPE + respirators + Solid-front
wraparound gowns, scrub suits or coveralls
• Own equipments
• BSC I/ II/ III
RG4  BSL4
1, 2, 3 +
• In an area away from general circulation, sealable
for disinfection
• Change clothing before entering, shower upon exiting
• Standard PPE + full body, air-supplied, positive
pressure suit
• Double ended autoclave
• Effluent treatment
• BSC II/ III
 BSL 1 BSL 2 BSL 3 BSL 4
Separate from _ _ + +
other activities
HEPA filter _ _ + +

Access only for _ + + +

authorised
personnel
Sealable for _ _ + +
disinfection
Negative _ + + +
pressure atm
Own _ _ + +
equipments
Effluent _ _ _ +
treatment
Shower _ _ _ +
Autoclave _ + + +
BSC _ I/ II I/ II/ III II/ III
Specimens for which there is limited information

– Universal precautions
– Basic containment with Biosafety Level 2
–National and/or international rules and regulations for
transport of specimens
–Some information may be available medical data,
epidemiological data, information on the geographical
origin of the specimen
General safety precautions

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Specimen transport
Universal/ Standard precautions
• 1987 – universal precautions
• 1996 – standard precautions
• Any blood and body fluids  considered potential
infectious agent

Include:
1. Do not eat, drink, smoke, or apply cosmetics
2. Do not insert or remove contact lenses
3. Do not bite nails or chew on pens
4. Do not mouth-pipette
5. Limit access to the laboratory to trained personnel only
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6. Barrier precautions to prevent skin and mucous
membrane exposure  gloves, masks, goggles, gowns,
aprons

7. Thoroughly wash hands and other skin surfaces after

removing gloves and immediately after any
contamination

8. Take special care to prevent injuries with sharp objects,

such as needles and scalpels
Personel Protective Equipments (PPE)
Includes:
• Impervious gowns
• Laboratory coats
• Disposable gloves
• Masks
• HEPA respirators
• Plastic shields or goggles
• Holders for glass bottles
• Trays to carry smaller hazardous items
• Handheld pipetting devices
• Safety carriers for centrifuges
• Disposal containers for sharp objects
Safe specimen handling

• Sample collection  sturdy self constructed containers

• Appropriate labels and signs

• Use personal safety measures  PPE

• Open injuries – covered with adhesive bandage

• Breakage or spillage  transfer as much of the specimen to a 2nd

sterile containers with adequately filled details

• Hand wash  with soap and water several times a day and after
processing
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Spill management
Cover spill with
Wear glove, absorbent material
Fragment of glass
mask ,gown and and concentrated
particles removed
shoe covers disinfectant
(10mts)

Clean the spill site

If specimen
Absorb with aqueous
aerosolised, leave
disinfectant solution of
for 30mts and
material detergent or 10%
proceed same
household bleach

Disposal all
Rinse with water material in
biohazard bags

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In BSL-3 spillage outside BSC  evacuate for 60mts
and notify authorities

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Centrifuge

• Remove broken glass

• Properly balanced machine

• Do not open the lid without the machine stops completely

• In case of spillage  turnoff and wait for 20mts & clean

the area with disinfectant

• Routine maintainence program

• 23/04/2024
Use closed carriers for potential infectious material 55
Chemical safety
• All hazardous chemicals in the workplace be identified
and clearly marked with a National Fire Protection
Association (NFPA) label stating the health risks &
hazard class
• Chemical hygiene plan:
 Proper labeling of chemical containers
 Manufacturers’ material safety data sheets (MSDSs)
 Written chemical safety training and retraining programs

• Hazardous chemicals inventoried annually

• File of every chemical they use with the corresponding

MSDS

• Manage spills

• Use nitrile gloves & fume hoods/ fume masks

MSDS:
• Substance name
• Name, address, and telephone number of manufacturer
• Hazardous ingredients
• Physical and chemical properties
• Fire and explosion data
• Toxicity
• Health effects and first aid
• Stability and reactivity
• Shipping data
• Spill, leak, and disposal procedures
• Personal protective equipment
• Handling and storage
Fire safety

• Fire evacuation plan

• Fire drills – quarterly/ annually
• Trained to use fire extinguishers – Type ABC
• In case of fire  RACE
1. Rescue any injured individuals
2. Activate the fire alarm
3. Contain (smother) the fire, if feasible
4. Extinguish the fire, if possible
Electrical safety

• Electrical cords checked regularly for fraying

• 3 prong, grounded type plugs
• Annually check all sockets for electrical grounding and
leakage
• Know the location of master switches and circuit breaker
board
• Do not repair equipments when plugged
• No extension cords

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Handling compressed gases
• Gas tanks should be properly chained
• Stored in well-ventilated areas
• Transported in special dollies
Waste disposal

• All materials contaminated with potentially infectious

agents must be decontaminated before disposal

• Infectious waste should be placed into 2 leak-proof,

plastic bags for sturdiness  double bagging

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• Pipettes, swabs, and other glass objects should be
placed into rigid cardboard containers before disposal
• Broken glasses are placed in thick boxes lined with
plastic biohazard bags

• Sharp objects placed in sharps

containers
Post exposure control

• All laboratory accidents and potential exposures 

reported to the supervisor and safety officer  immediately
send the individual to employee health or an outside
occupational health physician

• Immediate medical care

• Discuss the potential for disease transmission to family

members
• Assess follow up treatment

• Safety committee, or at least the laboratory director and

safety officer – review the events of the accident –
determine if it could have been avoided and to delineate
measures to prevent future accidents

• Investigation & corrective action – documented in an

incident report
References

1. WHO manual on laboratory biosafety – 3rd edition (2004)

2. CDC manual on biosafety – 5th edition (2009)
3. NSF International. (2008). Standard No.49 for Class II
(Laminar Flow) Biohazard Cabinetry. Ann Arbor, MI, USA
4. Labconco article on biosafety: july 2018
5. University of Delaware article on Biosafety: 2018
6. US EPA - Standard Operating Procedure for Use and
Maintenance of Biological Safety Cabinet: 2011
7. Diagnostic microbiology – Bailey and scott – 13th edition
8. Practical medical microbiology – Mackie and Mccartney –
14th edition
THANK YOU