Module 1: Safety in the
Microbiology Laboratory
Learning Objectives
At the end of this session participants should be able to:
Define the components (policies, practices & facilities) of biosafety
Compare & contrast the different biosafety levels
Identify level of biosafety needed for each procedure
Use protective equipment appropriately
Compare & contrast sterilization & disinfection
Apply basic laboratory safety practices & universal precautions in
compliance with international safety standards
Describe which methods of disinfection or sterilization are
appropriate for media sterilization, glass sterilization (test tubes,
Petri dishes etc.), & decontamination of infectious waste
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Content Overview
Objective of biosafety
Published reports of laboratory-associated
infections (LAI)
Biosafety levels (BSL)
Standard microbiological techniques & practices
Biosafety equipment & personal protective
equipment (PPE)
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Objective of Biosafety
To prevent transmission of biological agents to
lab workers, other persons, & the environment
A clinical laboratory needs to develop &
implement:
Administrative policies
Work practices
Safety equipment
Facility design
How important are these safety measures?
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Evidence Supporting Laboratory Exposure to
Infectious Agents
Technologists in clinical chemistry had a reported
incidence of hepatitis seven times higher than the
general population - Skinholdj, 1974 (Denmark)
Laboratory personnel had a five times increased risk of
acquiring tuberculosis compared with the general
population - Harrington & Shannon, 1976 (England)
Laboratory personnel had higher rates of shigellosis, &
hepatitis B than did the general population
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Other Examples of Laboratory Acquired Infections
Sulkin & Pike, 1949 222 viral infections were reported, 21 of
which were fatal
Cause:
Handling of infected animals & tissues
In 1951/1965/1976 surveys, brucellosis, tuberculosis,
tularemia, typhoid, streptococcal infections, hepatitis, &
encephalitis were reported
Cause:
Majority were related to mouth pipetting & the use of needles
& exposure to infectious aerosols
Infected person had “worked with the agent”
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Recent Hospital Laboratory Acquired Infections
2006: 3 laboratory acquired cases of tuberculosis
due to a leaking aerosol chamber in a BSL3 lab
2009: A case of tularemia in a BSL3 laboratory due
to a laboratory accident
2010: A researcher in a BSL4 laboratory contracted
Ebola virus after a needlestick injury
Most cases of laboratory acquired
infections go unreported!
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Principles of Biosafety
“Containment” a word used to describe safety measures employed
in the laboratory to limit exposure to infectious agents
Primary containment: Protection of laboratory environment &
laboratory personnel
Provided by both good microbiological technique & the use of
appropriate safety equipment
Secondary containment: Protection of the external
environment
Provided by a combination of facility design & operational
practices
The most important element of containment is strict
adherence to standard microbiological practices &
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techniques
Biosafety Levels, Practices, & Equipment
Biosafety Laboratory Type Laboratory Practices Safety Equipment
Level
Basic teaching, Good microbiological technique None, open bench work
research (GMT)
BSL - 1
Primary health GMT + protective clothing + Open bench + Class I or
services; diagnostic biohazard sign Class II Biosafety Cabinet
BSL – 2 services, research (BSC) or other primary
devices used for potential
aerosols
Special diagnostic As level 2 plus special clothing, Class I or Class II BSC &/or
services; research controlled access, directional other primary devices for all
BSL – 3 airflow activities
Dangerous pathogen As level 3 + airlock entry + Class III BSC or positive
units shower exit + special waste pressure suits in conjunction
BSL - 4 disposal with Class II BSC + double-
ended autoclave + filtered air
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Good Microbiological Techniques
Avoid ingestion of infectious materials
- No eating, drinking, gum chewing, etc.
- Use of pipetting aids
Avoid inhalation of infectious materials
- Avoid creation of aerosols
- Wear masks, safety shields, etc., when appropriate
- USE BIOSAFETY CABINET CORRECTLY
Avoid skin contact with infectious agents
- Wear laboratory coats & gloves
- WASH HANDS
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Other Sources of Laboratory Acquired Infections
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Standard Safety Practices in the Microbiology Laboratory (1)
Limit access to the laboratory
Wash hands after handling viable materials, after removing
gloves, & before leaving the laboratory, using appropriate
germicidal soap
Eating, drinking, smoking, handling contact lenses, applying
cosmetics, & storing food are not permitted in the work area
Do not wear laboratory coats outside the laboratory
Laboratory coats should be laundered on site
To prevent infection of family members, laboratory coats that
are taken home to be laundered should be placed inside a
sealed plastic bag until they are put directly into the wash with
detergent & bleach.
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Standard Safety Practices in the Microbiology Laboratory (2)
Avoid touching clean areas & answering telephones with
gloved hands
Do not use personal mobile phones while in the work
area
Decontaminate work areas daily after work & after any
spills, splashes, or other contamination by infectious
materials
Most common: Sodium hypochlorite (NaOCl), or other disinfectant.
Bleach is an aqueous solution of sodium hypochlorite.
A 1:10 dilution of household bleach is a good
disinfectant
- Must be made fresh everyday
- Can be used to disinfect surfaces & for disposal of
contaminated articles-swabs, applicator sticks, slides, etc.
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Standard Safety Practices in the Microbiology Laboratory (3)
All areas of the laboratory must be kept clean
& orderly (remove dirt, dust, or clutter)
Report all injuries or unusual accidents
immediately to the supervisor
Dispose of all contaminated materials
properly
Proper use of disinfection & sterilization
methods
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Sterilization & Disinfection
Sterilization
Complete destruction of all life forms
Disinfection
Removal of many organisms particularly
pathogens from inanimate objects – often
does not eliminate bacterial spores
Microbistatic vs. Microbicidal
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Microbistatic vs. Microbicidal
Control
Log number of viable bacteria
Disinfectant added
Microbistatic agent
Microbicidal agent
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Methods of Sterilization Used in Microbiology
Laboratories
Physical heat
- Moist heat: Steam autoclave
- 121ºC for 15-20 minutes (15 psi)
- Used for media & sterilization of contaminated materials
NOTE: Incineration can also be used to dispose of
contaminated materials
- Dry heat: Oven
- 160ºC for 2 hours
- Used for glassware & other material which may be
damaged by steam or moisture
Filtration
- Size of filter very important
- Used to sterilize heat labile additives that may be added to media
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How Can You Tell if Your Autoclave Works?
Indirect or non-biological indicators
- Chemical indicators that change colors when
sterilization temperatures are reached
- Does not ensure sterility
Direct or biological testing
- Use of spore strips
The use of spore strips is the
ONLY way to ensure that your
autoclave is functioning properly
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Biosafety Equipment
Equipment Hazards Corrected
BSC Aerosol & spatter
Splatter shield Splatter of chemicals
Ingestion of pathogens; inhalation
Pipetting aids
of aerosols
Loop microincinerators, disposable
Splatter from transfer loops
loops
Sharps disposal containers Puncture wounds
Screw-capped bottles Aerosols & spillage
Infectious materials made safe for
Autoclaves
disposal or re-use
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Personal Protective Equipment (PPE)
PPE Hazards Corrected
Laboratory coat Contamination of clothing
Footwear Impact & splash (closed-toe)
Goggles Impact & splash
Safety spectacles Impact (side-shield) & splash
Face-shields Impact & splash (shield entire face)
Respirators Inhalation of aerosols
Direct contact with microorganisms; cuts
Gloves
(hand protection)
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Example of a Microbiology Section Barrier Protection
Worksheet (1)
Disposable Lab
Task or Activity Personnel Involved Mask BSC Other
Gloves Gown
Specimen set-up (Routine Medical
√ √ √
culture) Technologist
Positive blood culture bottle Medical
√ √ √
subculture & smear preparation Technologist
Medical
Culture reading/ Work-up √
Technologist
Process
Brucella Culture Work-up Medical
√ √ √ √ inside
(ID/sensitivity)* Technologist
BSL-3 lab
Process
TB Culture Work-up Medical
√ √ √ √ inside
(ID/sensitivity) * Technologist
BSL-3 lab
Medical
Fungus culture manipulation ** √ √ √
Technologist
(√)
21 Required (R) Recommended (*) Disposable gown & N95 mask (**) Some fungi require a BSL3 lab
Example of a Microbiology Section Barrier Protection
Worksheet (2)
Disposable Lab
Task or Activity Personnel Involved Gloves Mask BSC Other
Gown
Parasitology Staining Medical Technologist R √
Routine Reagent Preparation Medical Technologist √ √
Refrigerator Cleaning Medical Technologist √ √
Centrifuge Cleaning Medical Technologist √ √ R
BSC/Incubator Cleaning Medical Technologist √ √ R
(√) Required (R) Recommended (*) Disposable gown & N95 mask (**) Some fungi require a BSL3 lab
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Biohazard Specimen Bags
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Examples of Biosafety Equipment & PPE
Safety
spectacles
Microincinerator
Biohazard
sign
Release
pipetting
Spatter shield N95 Particulate Pipet-Aid
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respirator
Face Shields
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Summary
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