BIOSAFETY

IN
MICROBIOLOGY LABORATORY
Dr.T.V.Rao MD

DR.T.V.RAO MD 1
 BIOSAFETY: PREVENTING
LAB-ACQUIRED INFECTIONS
• Bacteria
• Viruses
• Fungi
• Human blood, unfixed
tissue
• Human cell lines
• Recombinant DNA

DR.T.V.RAO MD 2
 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
DR.T.V.RAO MD 3
 STANDARD MICROBIOLOGICAL
PRACTICES
• NOT permitted in
laboratories:
 Eating
 Drinking

 Smoking

 Handling contact lenses

 Pipetting by mouth
 Storing food and drink

DR.T.V.RAO MD 4
 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
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 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

DR.T.V.RAO MD 6
 BARRIERS - PRIMARY BARRIERS
• Primary barriers:
physical barriers or
personal protective
equipment between
lab worker and
pathogen
• Gloves, masks,
special breathing
apparatuses

DR.T.V.RAO MD 7
 BARRIERS SECONDARY BARRIERS:
• Secondary barriers:
structural aspects of the
laboratory that make
working environment
safer against infection
• Sinks for handwashing,
special containment
areas, special air
ventilation patterns

DR.T.V.RAO MD 8
UNIVERSAL PRECAUTIONS
• Include hand hygiene, gloves,
gown, masks, eye protection,
face shields, safe injection
practices
• Require that all equipment or
contaminated items are
handled to prevent
transmission of infectious
agents
• Special circumstances may
require additional precautions
• Protective clothing, special
site decontamination

DR.T.V.RAO MD 9
 RISK GROUPS, BIOSAFETY LEVELS, PRACTICES AND
EQUIPMENT
BSL Laboratory type Laboratory Safety equipment
practices
1 Basic teaching, Good microbiological None
research techniques Open bench work
2 Primary health Good microbiological Open bench PLUS
services; diagnostic techniques, biological safety cabinet for potential
services, research protective clothing, aerosols
biohazard sign

3 Special diagnostic As BSL 2 PLUS Biological safety cabinet and/or other

services, research special clothing, primary devices for all activities
controlled access,
directional airflow

4 Dangerous As BSL 3 PLUS Class III biological safety cabinet,

pathogen units airlock entry, shower positive pressure suits, double ended
exit, special waste autoclave (through the wall), filtered air
CDC/NIH: BIOSAFETY LEVEL 1 AND LEVEL 2.

DR.T.V.RAO MD 11
CDC/NIH: BIOSAFETY LEVEL 3 AND LEVEL 4.

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 LEVELS OF CONTAINMENT
• BL1 - microorganisms
that don’t consistently
cause disease in healthy
adults
• E. coli K12, S.
cerevisiae, polyomaviru
s
• Basic laboratory
• Standard
Microbiological
Practices
DR.T.V.RAO MD 13
 BIOSAFETY LEVEL 1 (BSL-1)
• Agents not known to cause
disease in healthy adults
• Some organisms may
cause disease in
immunocompromised
individuals
• Agents include Bacillus
subtilis, Naegleria
gruberi, infectious canine
hepatitis virus, non-
pathogenic E. coli species

DR.T.V.RAO MD 14
 BIOSAFETY LEVEL 1 (BSL-1)
• Standard practices required:
• frequent handwashing
• door that can be kept closed when working;
• limits on access to the lab space when working;
• no smoking, eating, drinking, storage of food in laboratory;
• care to minimize splashes and actions that may create
aerosols (tiny droplets);
• decontamination of work surfaces after every use after any
spills;

DR.T.V.RAO MD 15
 BIOSAFETY LEVEL 1 (BSL-1)
• Standard practices (continued):
• decontamination of laboratory wastes;
• use of mechanical pipettes only (no mouth pipetting);
• "sharps" precautions, including special containers for
disposing of needles and other sharp objects;
• maintenance of insect/rodent control program;
• use of personal protective equipment (lab coats, latex
gloves, eye protection or face shields)
• Open bench top sink for hand washing

DR.T.V.RAO MD 16
 BIOSAFETY LEVEL 2 (BSL-2)
• Agents associated with human
disease
• Generally required for any
human-derived
blood, bodily fluids, tissues
in which infectious agent
may be unknown
• Agents include
measles
virus, Salmonella
species, pathogenic
Toxoplasma, Clostridiu
m botulinum, hepatitis
B virus

DR.T.V.RAO MD 17
 LEVELS OF CONTAINMENT
• BL2 - microorganisms of
moderate potential
hazard, transmitted by
contact, ingestion, punctu
re
• Salmonella, herpesvir
us, human blood
• Basic laboratory
• Standard Practices
PLUS

DR.T.V.RAO MD 18
 BIOSAFETY LEVEL 2 (BSL-2)
• Primary hazards:
• accidental needle sticks
• exposure to eyes and nose (mucous membranes)
• ingestion of infectious materials
• Agents do not cause lethal infections, are not transmissible via
airborne route
• (do not cause infection if tiny droplets become airborne and are inhaled,
which might occur if the material were spattered)
• Agents are pathogens for which immunization or antibiotic
treatment is available
• Extreme care should be taken with contaminated needles and
sharp lab instruments

DR.T.V.RAO MD 19
 RISK GROUP 2
Pathogenic for humans
Unlikely a serious
hazard
Treatment and
preventive measures
available
Limited risk of spread
of infection
 BIOSAFETY LEVEL 2 (BSL-2)
• Standard practices include BSL-1 plus:
• policies to restrict access to lab;
• biohazard warning signs posted outside lab;
• surveillance of laboratory personnel with appropriate
immunizations offered;
• biosafety manual with definitions of needed waste
decontamination or medical surveillance policies;
• supervisory staff who have experience working with
infectious agents and specific training for laboratory
personnel in handling these agents

DR.T.V.RAO MD 21
 BIOSAFETY LEVEL 2 (BSL-2)
• Primary barriers: biosafety cabinets or other approved
containment devices
• Personal protective equipment: lab coats, gloves, face
protection as needed
• Protective clothing removed when personnel leave
laboratory area
• Cabinets thoroughly decontaminated daily and
monitored for radiation for personal protection
• Secondary barriers: BSL-1 barriers plus autoclave for
glassware
DR.T.V.RAO MD 22
 LEVELS OF CONTAINMENT
• BL3 - microorganisms that
cause serious disease,
transmitted by inhalation
• M. tuberculosis, yellow
fever virus, hantavirus,
Y. pestis (plague)
• Containment lab:
double door entry;
directional airflow; all
work in biosafety
cabinet

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 BIOSAFETY LEVEL 3 (BSL-3)
• Agents with potential for respiratory
transmission, may cause serious and
potentially lethal infection
• May be studied at BSL-2 for diagnosis
 Mycobacterium
Agents include
tuberculosis, St. Louis encephalitis virus,
Francisella tularensis, Coxiella burnetii
()
DR.T.V.RAO MD 24
 TB DIAGNOSTICS AND LABORATORY
STRENGTHENING

• Care of patients with tuberculosis starts with a quality assured diagnosis,

obtained by growing and identifying Mycobacterium tuberculosis from clinical
specimens and conducting DST of the organism to confirm or exclude
resistance. Uptake of TB diagnostic technologies requires appropriate
laboratory infrastructure and adequate policy reform at country level to
enable their effective use in TB screening and diagnostic algorithms
• Laboratory infrastructure, appropriate biosafety measures and maintenance
Equipment validation and maintenance Specimen transport and referral
mechanisms Management of laboratory commodities and supplies
Laboratory information and data management systems Laboratory quality
management system are a priority.

DR.T.V.RAO MD 25
 MDR – TB , XMDR-TB AND BIOSAFETY
• With growing incidences of
MDR-TB and XMDR-TB it is
highly essential all
Microbiology laboratories
must install Grade 3
Biosafety cabinets to
prevent exposure to
Infection. It necessary
precaution's are not taken a
fraction of Medical and
Technical personal will be
infected with grave
consequences.
DR.T.V.RAO MD 26
 RISK GROUP 3
Pathogenic, cause
serious disease
Effective treatment
and preventive
measures usually
available
Little person-to-
person spread
DR.T.V.RAO MD 27
 BIOSAFETY LEVEL 3 (BSL-3)
• Standard practices include BSL-2 plus:
• strictly controlled access to the lab;
• specific training for lab personnel in handling potentially
lethal agents;
• decontaminating all waste;
• changing contaminated protective lab clothing,
decontaminating lab clothing before laundering;
• institutional policies regarding specimen collection and
storage from workers to establish exposure

DR.T.V.RAO MD 28
 BIOSAFETY LEVEL 3 (BSL-3)
• Primary barriers:
• Similar to BSL-2 personal protective equipment
• Respiratory equipment if risk of infection through inhalation
• Secondary barriers:
• All BSL-2 barriers
• Corridors separated from direct access to lab
• Access through self-closing double doors
• Air handling systems to ensure negative air flow (air flows into the lab)
• Air pumped into lab not re-circulated in building

DR.T.V.RAO MD 29
 LEVELS OF CONTAINMENT
• BL4 - microorganisms
that cause lethal
disease, with no known
treatment or vaccine
Ebola virus, Marburg
virus
Maximum
containment lab;
positive pressure
ventilated suits
(moon suits)

DR.T.V.RAO MD 30
 RISK GROUP 4
Lethal, pathogenic
agent
Readily transmittable
• direct, indirect
Effective treatment and
preventive measures
not usually available
 BIOSAFETY LEVEL 4 (BSL-4)

• Dangerous and exotic agents with high risk of life-

threatening disease, aerosol-transmitted
• Related agents with unknown risk of transmission

 Agents (all viruses) include Marburg

virus, Ebola virus, viruses that cause
Congo-Crimean hemorrhagic fever, Lassa
fever
DR.T.V.RAO MD 32
 BIOSAFETY LEVEL 4 (BSL-4)
• Primary hazards:
• respiratory exposure to infectious aerosols
• mucous membrane exposure to infectious droplets
• accidental sticks with needles or other sharp objects contaminated
with infectious material
• For example
• In late 1960s, 25 laboratory-acquired Marburg infections, including
5 deaths
• Workers studying infected monkeys from Uganda
• First documented naturally-occurring human case occurred in 1975
DR.T.V.RAO MD 33
 BIOSAFETY LEVEL 4 (BSL-4)
• Personnel must receive specialized training in
handling extremely dangerous infectious
agents, containment equipment and functions
• Access to lab is restricted: immunocompromised
persons are never allowed to enter the lab
• Standard practices include BSL-3 plus:
• strictly controlled access to the laboratory;
• changing clothing before entering and exiting lab
(showering upon exiting recommended);
• decontaminating all material exiting facility
DR.T.V.RAO MD 34
 BIOSAFETY LEVEL 4 (BSL-4)
• Primary barriers:
• Biosafety cabinets used at other biosafety levels
• Full-body, air-supplied, positive pressure personnel suit
• Secondary barriers:
• All physical barriers at BSL-3
• isolated zone or a separate building;
• dedicated supply and exhaust, vacuum, decontamination
systems;
• a recommended absence of windows (or sealed
and resistant to breakage)
DR.T.V.RAO MD 35
 ACTIVITY SPECTRUM OF
SELECT
DETERGENTS AND DISINFECTANTS

Myco
BG+ BG- Spores Yeast Virus Prions
B
Alcohol 70° ++ ++ ++ 0 + + 0
Aldéhydes +++ +++ ++ + +++ ++ 0
Ammonium IV +++ + 0 0 + + 0
Anilides + 0 NP NP 0 NP 0
Chlorhexidine +++ ++ 0 0 + + 0
Cl compounds +++ +++ ++ ++ ++ ++ + (a)
Iodine (+ der.) +++ +++ ++ ++ ++ ++ 0
Hg compounds ++ ++ 0 0 + 0 ou + 0
Phénols : Variable activity depending on components (b)
Hexachlorophène +++ + 0 0 + 0 0
(a) Bleach (6%) during 60 min at 20°C ; (b) discussion on efficacy of phénol on prions
 LABORATORY LOCATIONS
• BSL-1: high schools, community colleges, municipal drinking water
treatment facilities
• BSL-2: local health departments, universities, state laboratories, private
laboratories (hospitals, health care systems), industrial laboratories
(clinical diagnostic companies)
• BSL-3: state health departments, universities, private
companies, industry, federal government (NIH, CDC)
• BSL-4: only 15 facilities in the US
• 9 federal (CDC, NIH), 4 university (Georgia State University, University of Texas
Medical Branch), 1 state, 1 private
• Renovations underway at several labs, new facilities proposed at additional sites

DR.T.V.RAO MD 37
 STANDARD MICROBIOLOGICAL
PRACTICES
• NEVER
• recap, bend, or
break needles
• discard needles or
sharps into
biological waste
bags
• discard needles
into regular trash

DR.T.V.RAO MD 38
 BIOSAFETY IS EVERYONE'S CONCERN
• Laboratorians have long recognized hazards of processing
infectious agents
• Biosafety guidelines developed to protect workers in
microbiological and medical labs through a combination of
safeguards including engineering controls, management
policies and work practices.
• Issue described differences between biosafety levels
• Help you understand process labs may have to undertake to
identify microorganism, why every lab cannot test for every
organism
DR.T.V.RAO MD 39
REFERENCES
• UNC School of Public Health Laboratory Safety Levels
• WHO guidelines on laboratory training for Field Epidemiologists

DR.T.V.RAO MD 40
 • Programme created by Dr.T.V.Rao MD for
Medical ,Technical and Health care workers
in the Developing World
• Email
• doctortvrao@gmail.com

DR.T.V.RAO MD 41