BIOSAFETY MEASURES IN

MICROBIOLOGY LAB

Dr. Shallu athuria,

Ph.D (Medical Microbiology)
 CONTENTS

 DEFINITION
 EPIDEMIOLOGY
 GOAL
 RISK GROUPS
 SAFETY LEVELS
 PERSONAL PROTECTION
 SAFETY EQUIPMENTS
 EXPOSURE TO INFECTIVE MATERIAL
 DEFINITION
BIOSAFETY:
The application of knowledge, techniques
and equipments to prevent personal, laboratory and
environmental exposure to potentially infectious agents
or biohazards.

Biosafety defines the containment conditions under

which infectious agents can be safely manipulated.
 EPIDEMIOLOGY

 The first known laboratory acquired infection, was

Mediterranean Fever, reported in 1899. First systematic
study was done by Sulkin and Pike (1951) who sent
questionnaires to laboratories in the USA and identified
1342 cases of laboratory acquired infections, of which
39 were fatal.

 The results of four surveys performed in the United

kingdom between 1971 and 1991 indicated that within
the clinical facilities, the most infections were identified
among workers in microbiology laboratory followed by
those in autopsy services.
 WHY IS BIOSAFETY IMPORTANT

Laboratorians recognize hazards of processing

infectious agents. Hence guidelines were developed
to protect workers in microbiological and medical labs
through control programs, management policies, and
work practices.
 Define barriers and procedures used by laboratories
to protect workers and others from lab acquired
infections.
 Apply the four biosafety levels and the protective
measures used by each type of laboratory when
handling infectious materials.
 Provide examples of the types of biological agents
handled in each type of laboratory.
 RISK GROUP

Risk groups are classified into four groups according

to following factors:
 Pathogenicity
 Infectious dose
 Mode of transmission
 Host range
 Availability of effective preventive measure and
treatment.
Risk Pathogenicity Hazards Spread to Effective
groups for human to worker communit prophylaxis or
y treatment
1. Unlikely Unlikely Unlikely Available

2. Can cause May be Unlikely Usually

disease available
3. Can cause May be May Usually
severe disease serious spread available
4. Cause human Serious Likekly Usually not
disease available
 RISK GROUPS
Bacterium(a) Viruses Fungus(i) Parsasite
Group

1. •No clinical organisms

2. •Actinobacillus sp. •Adenovirus •Aspergillussp. •Acanthoaem

•Bacteroides sp. •Arenoviridae •Cryptococcus oebae

•Clostridium sp. •Norwalk sp. sp. •Ancylostoma

•Corynebacterium sp. •BK and JC virus •Candida sp.

sp.
•Trichophyton •Ascaris sp.
•Enterobacteriaceae •Human

•Mycobacteria sp. metapneumonovi sp. •Brugia sp.

•Haemophillus sp.
rus
•Influenza virus
•Neisseria sp.
Group Bacterium(a) Virus Fungus(i) Parasite

3. •Bacillus anthracis •Dengue virus •Coccidiodes

•Brucella sp. •Hanta virus immitis

•Chlamydia psittaci •Japanese B •Blastomyces

•Coxiella burnetti encephalitis dermatidis

•Lymphocytic •Histoplasma
•Mycobacteria
tuberculosis virus capsulatum
•Nairovirus •Paracoccidioi
•Rickettsiae
•JE virus
des
•Yersinia sp.
braziliensis
•SARS virus

•HIV
Group Bacterium(a) Virus Fungus(i) Parasite

4. •Lassavirus
•Marburg virus

•Ebola virus

•Herpes simiae
virus
•KFD virus
MICROBIOLOGICAL SAFETY LEVELS

 The levels are designed by degree of protection

provided to personnel, the environment, and the
community.
Four types:
Safety Level 1
Safety Level 2
Safety Level 3
Safety Level 4
 BIOSAFETY LEVEL-1

 Agents not known to cause disease in healthy adults

 Some organisms may cause disease in
immunocompromised individuals.
 Agents include Bacillus subtilis, infectious canine
hepatitis virus, non-pathogenic E. coli species etc.
 Standard practices required:
 Frequent hand-washing.
 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 or
after any spills.
 Standard practices (continued):
 Use of mechanical pipettes only (no mouth
pipetting)
 Maintenance of insect/rodent control program.

 Use of personal protective equipments (lab coats,

latex gloves, eye protection or face shields)
 BIOSAFETY LEVEL-2

 Agents which are 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, Clostridium botulinum, hepatitis
virus etc.
 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.
 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.
 Proper disposal of contaminated waste
 Secondary barriers: BSL-1 barriers plus autoclave for
glassware.
 BIOSAFETY LEVEL-3

 Agents with potential for respiratory transmission, may

cause serious and potentially lethal infection
 May be studied at BSL-2 for diagnosis

 Agents include Mycobacterium tuberculosis, St. Louis

encephalitis virus, Leishmania sp. , Bacillus anthracis
etc.
Standard practices include BSL-2 plus:
 Strictly controlled access to the lab
 Specific training for lab personnel in handling
potentially lethal agents
 Changing contaminated
n protective lab clothing,
decontaminating lab clothing before laundering
 Institutional policies regarding specimen
collection and storage from workers to establish
exposure
 The laboratory must be a separate building or isolated
zone, with double-door entry, directional inward airflow.
PROHIBITED AREA
 BIOSAFETY LEVEL-4
 Dangerous and exotic agents with high risk of life-
threatening disease, aerosol-transmitted and risk to
community.
 Represents an isolated unit that is completely self-
contained to function independently. Secured with an
air lock for entry and exit, Class III biological safety
cabinets, and a separate ventilation system with full
controls to contain contamination.
 Personnel must receive specialized training in handling
extremely dangerous infectious agents, containment
equipments and functions.
 Access to lab is restricted. Immuno-compromised
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
 PERSONAL PROTECTION

Safety equipments also include items for

personal protection, such as gloves, coats,
gowns, shoe covers, boots, respirators, face
shields, safety glasses, or goggles.
DON’T EAT
WEAR MASK, GLOVE,
OR DRINK IN HEAD CAP AND GOWN
LAB
WASH WEAR
HANDS GOGGLES
 DISINFECTANTS

 Disinfectants are needed in the laboratory for the skin,

work surfaces, discards jars and spillages.

Skin and Work surfaces: Can be decontaminated

with 70% ethanol. These alcohols will kill vegetative
bacteria and some viruses.
General laboratory use: Use hypochlorite for general
purpose, but not for tubercle bacilli, on metals or in the
presence of much organic matter, which inactivate it.
e.g.
• 10% sodium hypochlorite for virology and spilled blood,
• 1% sodium hypochlorite for routine surface
disinfections.
• For Mycobacterium tuberculosis-5% phenol is most
effective.
 DISPOSAL OF HAZARDOUS WASTE
 All material contaminated with potentially infectious
agents must be decontaminated before disposal.
e.g. unused portion of patient specimens, patient
cultures, stock cultures of microorganisms etc.
 Infectious waste may be decontaminated by use of an
autoclave, incinerator etc.
 In 1986, EPA (Environmental protection agency)
published a guide to hazardous waste reduction.
These are following :
 Substitute less hazardous chemicals when possible
e.g. Substitution of ethyl acetate for ether in ova and
parasite concentration.
 Segregating infectious waste from uncontaminated
trash.
 Substituting miniaturized systems for identification and
antimicrobial susceptibility testing of potentials
pathogens.
THE FACILITY AS BARRIER
 Facility design and
construction contribute
to the laboratory
workers' protection,
provide a barrier to
protect persons outside
the laboratory, and
protect people and
animals in the
community from
infectious agents which
may be accidentally
released from the
laboratory.
 Primary barriers:
 Similar to BSL-2 personal protective equipment
 Respiratory equipment if risk of infection through
inhalation
 Secondary 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 is not re-circulated in building.
 THE ESSENTIALS OF UNIVERSAL
PRECAUTIONS AND SAFE
LABORATORY WORK PRACTICE

 Do not eat or drink, smoke, or apply cosmetics

(including lip balm)
 Do not insert or remove contact lenses.
 Do not bite nails or chew on pen.
 Do not mouth pipette.
 Limit access to the laboratory to trained personnel only.
 Assume all patients are infectious for HIV or other
blood borne pathogens.
 Use appropriate barrier precautions to prevent skin and
mucous membrane exposure as wear gloves and masks,
goggles, gowns, or aprons.
 Thoroughly wash hands and other skin surfaces after
gloves are removed and immediately after any
contamination.
 Take special care to avoid injuries with sharp objects
such as needles and scalpels.
 Safety Practices for laboratory Workers

•Placement of specimens in leakproof, sealed containers for transport and avoiding

contamination of specimen container surfaces.

•Changing gloves and washing hands after specimen processing

•Limiting the use of syringes and needles.

•Thorough decontamination of any equipment that has been contaminated with

any blood or any body fluid before it is transported.
 SAFETY EQUIPMENTS

 Splashguards
 Microbiological safety cabinets
 Air shower
 Chemical fume protection
 Centrifuges
 Sharps protection
 Other
• fire safety
• Electrical safety
 SPLASHGUARDS

 Should be made of cleanable, clear glass or

plastic and should be large enough to protect
worker from gross splashes.
 Splashguards can be effective barriers provided
that they are appropriately placed with respect to
both workflow and worker’s height.
MICROBIOLOGICAL SAFETY CABINETS

 Three classes of cabinets are defined, the class requiring

being determined by the degree of hazard and
protection.
Types are
Class 1 Cabinet
Class 2 cabinet
Class 3 Cabinet
 CLASS 1 CABINET

 Open fronted
 An inward airflow of 0.7-1.0m/s provides
a protection factor of at least 1.5x106
particles.
 The airborne particles are contained with
in the cabinet and filtered from the
exhaust air through a HEPA filter
(removes particles larger than 0.3um in
diameter) which sterilize the air to be
exhausted.
 CLASS 2 CABINET
 Open fronted
 Sterilize air that flows over
the infectious material as
well as air to be exhausted.
 Designed to prevent the
airborne contamination of
the work material and
reduce exposure of operator
to particles dispersed with in
the cabinet.
 Re-circulate filtered air over
the work area and maintain
an inflow of air.
 Air is exhausted through a
HEPA filter.
Type IIA cabinets have a fixed opening
Type IIB cabinets have a variable sash opening through which
the operator gains access to the work surface
 CLASS 3 CABINET
 Totally enclosed cabinets
with negative pressure.
 Separate the operator
from the work by an
airtight barrier.
 Leak free boxes with the
gloves sealing the hand
holes.
 Scavenged by air entering
and leaving through the
HEPA filter.
 AIR SHOWER

Air showers are self

contained chambers install
at entrance to cleanrooms
and other controlled
environment.They minimize
particulate matter entering
or exiting the clean place.
 CHEMICAL FUME PROTECTION

 A fume hood is a mechanically

ventilated partially enclosed
workspace where harmful volatile
chemicals and reagents can be
handled safely.

 Chemical fume hood differ from

biosafety cabinets in that they are
usually ducted, must be constructed
of noncombustible materials, and
must be explosion proof.
 CENTRIFUGES

Centrifuges must be
maintained and used
properly.
 SHARP PROTECTIONS
Sharp containers should be used. They should be
leak proof and puncture resistant and should not
degrade in autoclaves.
 EXPOSURE TO INFECTIVE MATERIAL

 Report all mishaps to the safety officer or appropriate

member of staff, who should record them in accidental
books.
 Encourage cuts and puncture wounds to bleed and
then wash with soap and water.
 If eye is splashed, rinse with tap water.
 If the skin is soiled with infective material, rinse with
70% alcohol or dilute hypochlorite solution, and then
with soap and water.
Spillage:
o if small spillage- disinfection and cleaning up

o if there is gross spillage-evacuate the room for atleast

an hour to allow possible aerosol to be dispersed.
then for group 2 organisms disinfect and cleanup and if
there are exposure to group 3 organisms, fumigate the
room.
  In the event of possible exposure to blood borne
pathogen lab attendant should act promptly to carry out
steps listed below:

1.Decontaminate the Area of the Exposure

• Needle stick
o Remove gloves immediately, if present
o Wash area with soap and water
o Avoid squeezing or milking the wound
o Do not use caustic agents, such as bleach
• Blood splash on skin
o Wash area with soap and water
• Blood/body fluid splash to mucous membranes
o Wash/irrigate area copiously with water or sterile saline
2. Contact designated person or exposure control
team and source patient
3.Evaluation of exposure and follow up
 HIV PEP FOR PERCUTANEOUS INJURIES: SOURCE
WITH KNOWN HIV INFECTION
EXPOSURE TYPE HIV HIV INFECTED
INFECTED CLASS 2
CLASS 1
Less severe Recommend Recommend expand 3
Solid needle basic 2 drug drug PEP
Superficial injury PEP
More Severe Recommend Recommend expand 3
-Large bore hollow needle expand 3 drug drug PEP
Deep puncture PEP
Visible blood on device
Needle used in patient artery
or vein
HIV PEP FOR PERCUTANEOUS INJURIES:
SOURCE- UNKNOWN HIV STATUS , HIV
NEGATIVE OR NOT TRACEBALE
Exposure Type Source of Unknown Unknown HIV Negative
HIV Status Source

Less Severe Generally no PEP Generally no No PEP

– Solid needle warranted; Consider PEP warranted
–Superficial injury basic 2-drug PEP for warranted;
source with HIV risk factor Consider
basic 2-drug
PEP in
settings where
exposure to
HIV infected
persons is likely
Exposure Source of unknown Unknown HIV
Type HIV Status Source negative
More Severe Generally no PEP Generally no No PEP
– Large-bore, warranted; Consider PEP warranted
hollow basic 2-drug PEP for warranted;
needle source with HIV risk factor Consider
– Deep puncture basic 2-drug
– Visible blood PEP in
on settings where
device exposure to
– Needle used in HIV infected
patient's artery or persons is likely
vein
HIV PEP FOR MUCOUS MEMBRANE AND NON-
INTACT SKIN EXPOSURE

Exposure HIV infected class HIV infected Source of unknown

Type 1 class 2 HIV status

Small volume Consider basic 2 Recommend basic Generally no PEP

e.g. few drops drug PEP 2 drug PEP warranted, consider basic
2 drug PEP for source
with HIV risk factor
Large volume Recommend basic 2 Recommend Generally no
drug PEP extended 3 drug PEP warranted;
PEP Consider basic
2-drug PEP for
source with HIV
risk factors
UNIVERSAL PRECAUTIONS

 Applied for handling blood and body fluids, including

all secretions and excretions (e.g. serum, semen, all
sterile body fluids, saliva from dental procedures and
vaginal secretions).
 Are not applied for feces, nasal secretions, saliva
(except in dental procedures), sputum, sweat, tears,
urine or vomitus unless they are grossly bloody.
 When collecting specimens outside the laboratory,
individuals should follow these guidelines:

• Wear gloves and a laboratory apron.

• Deal carefully with needles and lancets.
• Discard sharp in an appropriate puncture resistant
container.
• Never recap needles by hand if necessory special
devices should be available for resheathing needles.